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Crypto investors are closing out 2025 with one eye on sentiment and the other on opportunity. Liquidity has been creeping back into the market since early Q4, and the rebound has arrived at the perfect moment: historically, December has been one of the most active months for capital rotation as traders position ahead of January inflows. The current macro environment isn’t euphoric, but it is constructive. Bitcoin has held key support for weeks, Ethereum gas fees are stabilizing at multi-month lows, and on-chain activity across altcoins suggests that retail participation is picking up again. This is the market behaviour that tends to precede year-end rallies. Analysts often describe December as the ‘setup month’ for early-cycle runs. When liquidity enters the market gradually, the first recipients are high-conviction presales and low-cap altcoins because they offer asymmetric upside with less capital. The best altcoins in December 2025 won’t necessarily be the largest but they’ll be the ones with strong narratives, tokenomics that encourage retention rather than speculation and ecosystems that reward long term participation. That leads to an important shift in the way traders are building their December 2025 crypto watchlists. Rather than chasing coins that have already pumped this quarter, the smarter strategy has been identifying projects at the beginning of their adoption curves. Three presales have become central to that conversation — Bitcoin Hyper, Maxi Doge and Pepenode — positioned for different narratives but unified by strong market traction and early-cycle advantages. Bitcoin Hyper sits at the top of many December lists because its model appeals to users who want sustainable returns rather than short term hype. The token is still in presale at $0.013355 and has already raised more than $28M. That liquidity base indicates real market confidence rather than low-float artificial scarcity. The project is built around an ecosystem that rewards activity, not speculation; users are incentivized to stay engaged rather than sell as soon as the token goes live. A 40% staking reward supports that behavior, making it structurally harder for early holders to abandon the network during dips. The long-term advantage of Bitcoin Hyper is that its growth model doesn’t depend on a perfect macro environment. Investors are drawn to assets that can thrive during both consolidation and expansion, and Bitcoin Hyper positions itself as an ecosystem for day-to-day usage and utility, not just trading. If the broader crypto market continues trending upward into January — historically one of the strongest months for altcoins — projects with liquidity, staking depth and active user infrastructure typically outperform. For early-cycle traders, getting exposure while $HYPER is still in presale aligns timing with growth momentum. Discover Bitcoin Hyper. Maxi Doge brings a meme coin identity but underpinned by mechanics that solve one of the biggest flaws of traditional meme tokens: fast pumps followed by brutal selloffs. Its staking model, offering 73% rewards, gives holders a reason to stay rather than flip at the first sign of profit. That has already translated into strong early adoption, with more than $4.2M raised during presale. The current price of $0.0002705 keeps it accessible to small buyers, which historically matters when meme coin seasons heat up. Where Maxi Doge becomes particularly interesting for December 2025 is the macro trend. Meme coins tend to shine when confidence returns to the markets but capital hasn’t yet become aggressive. That’s exactly the stage the market is in now. The narrative is shifting from “who survives the bear” to “what could run next,” and Maxi Doge matches the criteria traders usually favor: strong branding, sticky staking incentives, and a community-driven identity rather than purely opportunistic marketing. If meme coins retest momentum during the year-end rally which has been a recurring pattern in past cycles, Maxi Doge is positioned to benefit. Explore Maxi Doge. Pepenode is the most experimental of the three, and that’s exactly why it has been gaining attention. It treats every interaction including referrals, challenges, streaks, and even social engagement, as an economic driver inside its network. The result is a hyper-gamified social layer built around activity rather than passive speculation. The token price is currently $0.0011731 and the presale has passed $2.2M, signaling strong community interest in its model. The 579% staking reward is eye-catching, but it is backed by a tokenomics system that ties emissions to participation rather than time-based inflation. The market has repeatedly rewarded platforms that merge social interaction with value flow, and Pepenode pushes that narrative forward by enabling pseudonymous crypto identities with actual economic weight. This is the kind of ecosystem that historically performs well during rallies driven by culture rather than pure fundamentals. If December’s year-end rotation spills into January — particularly with retail participation rising — projects that integrate social logic will have an advantage over those relying exclusively on hype. The upside here is both narrative-driven and model-driven, which is why Pepenode continues to show strong presale momentum. Join Pepenode. Recap: December 2025 is shaping up to be a strategic month for altcoin positioning rather than reactionary trading. Bitcoin Hyper emphasizes sustainable utility, Maxi Doge refines the meme coin formula with long-term retention and Pepenode builds a crypto-native social engine designed for engagement. All three projects have gained early traction ahead of the potential year-end rally.
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By CryptoNinjas Editorial Team Market momentum is beginning to shift again, and traders are watching closely for the top crypto to buy before the next round of movement. Dogecoin’s latest price prediction highlights a tightening wedge structure that could trigger a breakout, while Stellar’s (XLM) price continues to slip under a stubborn resistance level as open interest fades and momentum weakens. At the same time, Zero Knowledge Proof (ZKP) is entering the conversation on a whole new level. While DOGE and XLM wait for confirmation signals, ZKP’s presale auction is already live, its Proof Pods are ready to be ordered, and its entire ecosystem is active now. This operational readiness is why many analysts now list ZKP crypto among the top crypto to buy as the cycle accelerates. The current Dogecoin price prediction centers on a technical formation that has started to attract stronger attention. DOGE continues to trade above the $0.15 support zone, where a falling wedge pattern and tightly compressed EMAs are shaping a controlled buildup in momentum. These structures frequently appear before directional breakouts, and analysts are watching the $0.18 to $0.20 region as the first meaningful resistance cluster. A sustained push through that range could open a path toward $0.27 to $0.29 if volume expands. Additional signals reinforce the constructive tone, with an RSI divergence suggesting underlying strength, and a firm MACD crossover supports the case for continuation, consistent with earlier Dogecoin price prediction trends. A close below $0.13 would weaken this outlook, but traders remain focused on a move above the $0.16 pivot. As speculation grows around a broader market rebound, DOGE stays on top crypto to buy watchlists. Stellar’s (XLM) price continues to struggle beneath a firm ceiling that has held its progress in place for several weeks. The XLM price sits just under $0.25, meeting consistent rejection in the $0.27 to $0.29 range, which has become the defining barrier of this consolidation phase. Open interest has flattened near 75 million, signaling that traders are hesitant to commit, while RSI nears 40, and a negative MACD reinforces the picture of restrained momentum rather than active selling pressure. The key level to watch sits near $0.22, a zone that has repeatedly steadied Stellar’s (XLM) price during periods of uncertainty. A rebound from that support with stronger volume could trigger a sentiment shift, but Stellar must clear resistance before confidence fully returns. In top crypto to buy discussions, Stellar currently represents a project in waiting, driven by technical compression rather than decisive market direction. While DOGE and XLM wait for structure to develop, Zero Knowledge Proof has launched its Initial Coin Auction with every component of the ecosystem already active. Buyers are entering a fully operational network, not a speculative roadmap. The team invested more than $100M to build the infrastructure before allowing the public to participate, including a complete $20M four-layer stack and a $17M inventory of Proof Pods ready for immediate shipment. The Proof Pods have become one of the biggest drivers of demand. Each unit costs $249 and ships globally within 5 days. They begin earning between $1 and $300 per day by validating compute tasks from the moment they are activated. The buying guides, activation walkthroughs, and earning dashboards are all live, giving participants real utility right away. By comparison, no other project offering presale crypto coins has launched with hardware already working. ZKP’s ICA model is also reshaping expectations. Instead of fixed rounds or insider allocations, each day, Zero Knowledge Proof (ZKP) releases 200M tokens, and buyers collectively determine the price through their contributions. Everything is recorded on chain, and a strict $50K per wallet cap prevents whales from dominating early supply. This has created a competitive but fair environment that analysts now estimate could surpass $7B in participation. With the presale auction now live and surging demand for Proof Pods, ZKP is positioning itself as the top crypto to buy in this cycle. That is why joining the presale auction now is becoming the practical choice rather than a rushed one. Dogecoin’s wedge structure keeps traders focused on a potential upside push, while the Stellar (XLM) price continues to battle resistance with weakening momentum. Both remain important market signals, but neither delivers the level of readiness now seen in Zero Knowledge Proof. ZKP crypto is not waiting for technical confirmation or narrative shifts; its ICA is live, its Proof Pods are already in demand, and its entire infrastructure is now active. This is why analysts increasingly classify Zero Knowledge Proof (ZKP) as the potential crypto to buy while the auction window is still open. For anyone tracking early phase opportunities with real utility, entering the presale auction now offers the strongest position before demand accelerates further. Join the Presale Auction Now: Website:zkp.com Read more: Please be advised that all information, including our ratings, advices and reviews, is for educational purposes only. Crypto investing carries high risks, and CryptoNinjas is not responsible for any losses incurred. Always do your own research and determine your risk tolerance level; it will help you make informed trading decisions Tags CryptoNinjas Editorial Team Expert Crypto and Blockchain Analysts The CryptoNinjas Editorial Team is a dynamic group of crypto analysts with over five years of collective experience in cryptocurrency and blockchain analysis. Renowned for their high-success-rate fundamental analysis, the team has been featured in CoinDesk and CoinTelegraph, delivering trusted insights to traders and investors. Specializing in decentralized finance (DeFi), tokenomics, and blockchain applications across sectors like finance, supply chain, and healthcare, they provide actionable crypto trading insights and cryptocurrency market analysis. From evaluating exchange platforms to uncovering market trends, the team empowers enthusiasts with clear, authoritative content. The latest news, articles, and resources, sent to your inbox weekly. News Exchange Crypto News News Crypto Referral Code News News Crypto News News CryptoNinjas is a global news and research portal that supplies market and industry information on the cryptocurrency space, bitcoin, blockchains. CryptoNinjas aims to expand knowledge and understanding of the cryptocurrency and blockchain space. The latest news, articles, and resources, sent to your inbox weekly.
The Real World Assets market is bouncing back after a gloomy November. With institutional stablecoin experiments and promising technical setups, three RWA tokens stand out. Charts reveal clear upward momentum for some projects while others position for a potential technical rebound. Written by Simon Dumoulin Translated on December 1, 2025 at 08:07 by Simon Dumoulin Among the RWAtokens to watch this month, Stellar (XLM) stands out as the reference blockchain infrastructure for cross-border payments used by genuine financial sector players. November was challenging for XLM with an 18.9% correction, but recent sessions show an encouraging technical rebound with a 4.9% gain over seven days. This recovery is far from insignificant. It coincides with the announcement of stablecoin tests by the American bank on the Stellar network, as well as a significant increase in AUDD activity (tokenized Australian dollar). These developments are drawing institutional attention to the network’s real capabilities in terms of tokenized assets. Recent price action shows an attempt to reclaim support levels that have become resistance. Trading volumes are increasing alongside these institutional experiments, which could signal a shift in market sentiment for XLM in the coming weeks. Quant (QNT) represents the positive anomaly among RWA tokens currently. While the majority of tokenized asset projects have suffered selling pressure in November, QNT has exploded with a 32% surge for the month and approximately 37% over the last seven days. The past 24 hours have even added an additional 12% to its valuation. This exceptional performance positions QNT as one of the strongest charts in the RWA segment. The token benefits from clear bullish momentum with growing volumes confirming the strength of the move. Resistance levels are being breached successively without major retracement, a sign of probable institutional accumulation. Quant’s blockchain interoperability technology particularly appeals to financial players looking to connect different networks for their tokenized asset operations. This gradual enterprise adoption partly explains QNT’s remarkable resilience in the face of unfavorable market conditions. Ondo (ONDO) occupies an intriguing position in this RWA token selection. The project shows a 9.3% gain over seven days but remains among the worst performers over 30 days with a 25% decline. This dichotomy creates an interesting technical setup for traders seeking reversal opportunities. Interest in ONDO intensified this week following announcements suggesting that Ondo Finance could extend the tokenization of U.S. stocks and ETFs to Europe. This geographic expansion represents a major catalyst for protocol adoption and could justify a rebalancing of valuations. The current technical support appears to be holding firmly despite the significant monthly correction. Buy volumes are starting to return gradually, which could indicate that sellers are capitulating while opportunistic buyers are positioning themselves. The price action in upcoming sessions will be decisive in confirming or invalidating this rebound scenario. Secure your crypto on a Ledger now by taking advantage of the exceptional limited-time Black Friday offer. 50% discount and up to $90 free! Related articles: Passionate about cryptocurrencies since 2019, I cover the latest news through clear and accessible articles. My goal is to make crypto understandable for everyone, with reliable and well-researched content.
This is one of a series of guest blogs by people who have enrolled in MyLymeData. By Ruben Lee Sims I have had Lyme disease for 34 years and began treatment for the first time last month. I was active duty Air Force when I contracted Lyme disease in Southern California in 1982. I know exactly when, where, and how I was infected. 1982 was the same year Dr. Willie Burgdorfer identified the spirochete that causes Lyme. The experts claimed that Lyme only existed in endemic areas of the northeastern United States. Although it was published as fact, there is no real scientific basis for this statement. It seems purely arbitrary and subjective. However, this false statement has caused California Lymies and Lymies outside so-called endemic areas enormous grief and suffering; because many doctors still believe you cannot get Lyme disease in California and other non-endemic areas. As a military man, I was punished under the Uniform Code of Military Justice with two Article 15s and nearly court martialed twice, because doctors said no disease caused my many symptoms. I was committed to secure psychiatric wards three times as a hopeless hypochondriac. I was told I would stay on the psychiatric ward until I got better. After congressional investigation, I was finally discharged from the Air Force after 14 years of highly recognized and awarded military service that ended in shame because doctors did not understand Lyme disease. I stood before a military hearing where attorneys laughed and joked as they decided I was no longer fit for duty and discharged me from the Air Force without benefits, pay, or health insurance. I was essentially dumped on the streets homeless, disabled, and unable to work, with a wife and a child to provide for. Three decades after my discharge, the VA still cannot diagnose Lyme disease based upon symptoms. Therefore, I learned I had Lyme disease from a May 2015 Costco magazine article that encouraged me to review my medical records and study Lyme disease. That review taught me that the same rash that started three decades ago was the Acrodermatitis Chronica Atrophicans (ACA) rash that is caused by untreated Lyme disease. Nonetheless, it took six months of constant confrontation with the VA to get them to bring in someone that understood Lyme disease. I am seronegative by ELISA and Western Blot. So, doctors’ over reliance on serology test nearly left me blind from severe sensitivity to light. However, in December 2015, the VA finally brought in a Lyme expert from the Hospital of Special Surgeries in New York. Unknown to me, the VA had already confirmed my ACA rash. The expert reviewed my documents and found the confirming skin biopsy in my records. Therefore, even though I was seronegative, the ACA rash confirmed my Lyme disease 34 years after my infection. After less than a month of treatment, my photophobia is nearly gone and the ACA rash is gradually fading. I no longer take daily naps and I can sleep 7-8 hours several days a week. I no longer feel totally wasted and out of things every day. I am encouraged by treatment and look forward to setting my records straight and helping others. It is important to know, the VA did not recognize Lyme disease until the mid-1990s. That means that veterans who had Lyme disease before then were misdiagnosed and some are perhaps homeless and disabled. However, our government is no wiser today than before they recognized Lyme disease. We are still being misdiagnosed and perhaps dying of other Lyme-related diseases. I cannot sit by and do nothing to change this. I was a management/cost/ financial analyst for over 30 years. I know that data is a valuable resource. Thus, one of the ways I’m going to help bring about change is by participating in MyLymeData. This important project gives Lyme patients a chance to make a real difference. By pooling our information, we can help build up the knowledge base of how real people experience Lyme disease. This will help direct researchers in a more useful direction. No one in the military—or anybody else in this country—should have to suffer so long without proper medical care. Click here for more information about MyLymeData. Ruben Lee Sims lives in Southern California. Thank you for sharing your story. My son is active duty Air Force and although he does not have Lyme, his lovely wife does. There are glimmers of hope. His current flight surgeon believes in chronic Lyme, although his hands are tied by the military (read government) when it comes to treatment. Thank you for your service and I am heartbroken that you were so poorly treated by the military in a time of pain and need. Thank you for your story!keeping awareness gets us closer to getting the word out!i have chronic LYME since 2001 and know where when to but Drs told me i was negative for the disease as well!treated me for MS!God Bless you keep fighting! Complete denial of chronic Lyme baby every doctor I have been to. Painful and debilitated unless I can somehow get a hold of a couple of weeks worth of doxycycline. Symptoms go away 80% for a month or so. Plus heat pads…antiinflammatories, turmeric, and several pigs of Clarita. Which does seem to help. My sister was diagnosed with Lyme via serology testing positive 3 years ago by a Missouri doctor who could only prescribe oral antibiotics not IV . After a year he gave up on her so she found another doctor who gave her IV antibiotics for 18 months but can now no long practice medicine and stopped all meds cold turkey. The joint pain and inflammation is totally diabilitating. Now she’s in limbo land. That is so wrong. Thank you for your story, it provides hope for those of us, my 12 year old son that is still suffering. We also had a Positive ELISA with 2/10 Western Blot positive at HSS and were told ‘Negative’ ….and he didn’t get treatment until I had his blood sent to Igenix, which was PCR positive. Thanks for telling us your story. It’s ironic that you were in the military and this was your biggest battle. Agree with you that there is no scientific basis for saying Lyme is only in endemic areas. I was bit by a known tick in Big Sur, CA in 1981. There have been plenty of people bitten even earlier than that in CA. This is for you, Ruben – please look at this site – it’s a list of US military bases rated low/med/high for tick infection risk btwn 1983-1996. You will see that the Vandenberg base was rated high risk, for example. http://lymeblog.com/LDRA-USARMY83-96/lyme.htm I can tell you’re going to be a good advocate! ruben, thank you for your heartfelt, tear-jerker story of how our military DOESN’T diagnose, treat, or recoginize lyme/vector-borne diseases. thank you for all the years you spent serving us; then to be treated shamelessly, is unacceptable! were you able to get SSDI early on? will you be able to get BACK PAY for this “unrecoginized/UNDIAGNOSED” lyme disease diagnosis? thank you for sharing your story; glad dorothy asked you to write your blog here! i just wrote and submitted mine and my late husband, jack’s, lyme story of 82 yrs. of chronic lyme; too long. it will be out sometime in future. hugs/prayers to you/family, and glad that treatment has helped your photosensitivity! lights, glare, & reflection, noise, and chemical smells are the worst things i go thru daily. bettyg, iowa activist 47 yrs. chronic lyme 35 yrs. MISDIAGNOSED by 40-50 drs. UNACCEPTABLE! Mr. Sims, I am deeply moved by your story. I wish you all the best in your recovery, and I thank you for fighting against the systemic neglect and mistreatment of Lyme patients. Dear Ruben, Do all of us a big favor and sue the pants off of them. They deprived you and put your family at risk for so very long. Sue them. Any of us would help in your battle if you sue them. witnesses, whatever. I wish some young lawyers would get involved, and start helping Lyme patients sue.When you do serve, you should be treated well, and obviously!!! you were not. Unacceptable. Sue them, please. Even one case of successfully suing wold help all of us. May God Bless all of those suffering from lymes disease. Read about US stating was a biological warfare agent. Plum Island New York Ruben, your story took me back in time as I re-lived my past with nearly every word you wrote. I also contracted Lyme while active duty (US Army) during the 70’s. I remember suffering through a worse than flu illness/fever during basic training at Ft. Leonardwood, MO. Went on sick-call, then was promptly put to work sweeping, mopping and waxing floors in the officer’s work area; could barely stand up. I later developed Achilles tendon pains, then knee joint pain, constant dizziness, inability to stay awake during the day, unable to sleep at night. Went on sick-call again, no help of course. Was threatened with article 15 for sleeping past time to get a haircut, couldn’t stay awake. Had to sleep, hidden behind equipment racks while on duty to avoid article 15’s. It was horrible having to hide from military prosecution because of an unknown illness. 39 years later, I figured out it was Lyme disease, had a Western Blot to confirm. Working on my own methods of treatment now. So, so very sorry!!! I live in Louisiana. I was diagnosed in Dec 2015 with Lyme. I have had 7 knee surgeries and a bunch of other orthopedic and neurologic issues. I was started on Doxy and within a few months I am taking 5 different antibiotics.They have bedridden me. I live alone and cannot just lay in bed everyday. I was going down hill fast. I am 58 yrs. I cut all the antibiotics except doxy. Within days I felt so much better. I have researched Lyme since. They don’t want to recognize that Louisiana has alot of people here have Lyme. Louisiana is known that we have so many bugs here and yes there is alot of tics here. I just wanted to get it out that there is Lyme besides in the north. We believe you! Montana is another state where people are thought not to get Lyme, but they very much do. More than one person in my family has contracted it there and one of them more than once. I was a military wife, am a physician and my husband was also. I contracted Lyme in the Black Forest when we were stationed in Germany. It was so long ago (I think 1986) that nobody knew what it was. We were back in the US 8 months later, when my husband saw an illustration of the same rash I had had posted in the American Journal of Family practice. he called CDC and they said for me to take oral antibiotics for a month. I felt much better and assumed I was OK.. 3 years later, we were back in Germany and I began having weird problems..from polymyalgia rheumatica to open chancre-like sores on- my fingers. My husband died of a heart attack I returned home, and developed more odd symptoms: knee swelling, nephrotic syndrome, along with depression and fatigue and sleep disturbances. I lived in a remote area of Northern California, but a family friend and physician decided we would assume that these disorders were due to chronic Lyme, although I was seronegative.to Elisha and Western Blot. I had a pic line installed and for 6 weeks administered 2 grams of antibiotic (Rocephin) with a pump every night.. All symptoms subsided over time, but by them, my knees ere shot, so I had them replaced. The military insurance I had from my husband paid for everything without a problem in coverage or expense. I am now 81 years old,my 16-year-old knee replacements are doing fine, and I am healthy other than the usual problems that go with advanced age. I now live in Arizona with my son and family..I have been a supporter of Lyme Disease.org for many years. Keep up the fight and the good work you do. Joyce C. Bradley, M.D. Thank you for your service. You’ve not only served your country, but you have continued serve as an inspiration to your fellow patients living with invisible and misdiagnosed chronic illness. Like you, I wonder about the prevalence of LD among our homeless veterans. I’m so glad you persevered in your quest for answers. May you find lasting health after Lyme. To all who still suffer or those who may discover they have Lyme disease, i highly recommend reading the book “Healing Lyme” by Stephen Harrod Buhner. The second edition was published in 2015. His knowledge of the subject and his Protocol for treating it is followed by MANY Lyme Aware Physicians so why not by we lay people? You can get a copy through Amazon.com The Protocol in this book is what my doctor used to treat my knee, along with other procedures that I would describe as “outside the box” but I can walk with only a slight limp where before treatment I was on crutches and in much pain. Oh yes, I submitted my information to the survey. I have to say, it took some truly whopping doses of some of these herbals to move the needle, but for sure Artemesinin and Cryptolepis sanguinolenta caused Herx reactions and improvements in my case. My diagnosing naturopath and my expert LLMD both recommended a dropperful of Cryptolepis twice a day. I did that for a couple years and it did absolutely zero for me. So, I assumed it just didn’t work… until I accidentally took way too much one day and Herxed so badly I missed work the next day and so by accident I discovered that it actually does help at 3x the dose. Thank you for sharing your experience in such a thoughtful way with remarkably measured rhetoric given your experience. Thank you too for your service to our country in the Air Force and again to the Lyme community and our country at large, including all those not yet infected, undiagnosed and diagnosed who will ultimately benefit from stories such as yours. Reading your story reminds me of those Gulf War veterans who suffer with Gulf War Syndrome. So often, I have wondered if they had a borrelia infection as their symptoms were remarkably similar to my Lyme symptoms. Wishing you continued hard won now good health, Kari May 2010 Bulls Eye Rash – told by family physician no Lyme in Indiana May 2012 Late stage disseminated Lyme-Negative according to CDC standard tests June 2015 Weaned off antibiotics after 3 years after treatment for Lyme and Bartonella. Sir, I salute you as a fellow Air Force Security Police Veteran (7/69-11/75) who spent a year in Thailand (bitten by everything imaginable) and two years at Pease AFB NH (endemic) only to be told in 2010 at the Salt Lake VA hospital I couldn’t have Lyme or co-infection as I was living in Idaho. Never mind the fact that at museums in SE Idaho have furs and pelts dating back to the 1800’s that still had (dead) ticks testing positive for Lyme. Never mind that I didn’t only live in Idaho. VA doctors are ignorant out of choice and the VA does not want to unleash another “snow storm” of claims like what is currently happening with herbicides exposure. Keep up the fight my friend. Thank You so much for sharing your story, It takes courage. When I contracted Lyme in Southern California 10 years ago, I had almost every symptom listed. I suffered greatly like so many, both physically and financially. After 10 doctors, several misdiagnoses and 3 years, I finally found info on the internet and gave it to my 10th doctor to read. He believed I had Lyme but didn’t want to write it down as a diagnosis. Antibiotics only made me worse, and I finally used a sound wave treatment in Las Vegas called Medsonix for 16 months to get rid of most of my symptoms. I never did get all my energy back and I have breathing problems that have never stopped, recently it has become severe. Over the years I have convinced myself I don’t have Lyme, but thanks to your brave letter Rubin, I now can face the truth. I bet there are many of us who don’t want to admit we have Lyme, even when we do test positive, because so many doctors want to ignore it, and we just don’t want to admit we’re sick. Maybe by sharing our experiences we really can make a difference. I sure hope so. Thank You Rubin Lee Sims! Sheila L. Stewart Dear Ruben, Thank you for sharing and being a great spokesperson and advocate. I was also misdiagnosed in California, but for merely nine years, haha, and have done various treatments off and on for the last four years. I also have a cousin who had Gulf War Syndrome, and now I’m wondering about that diagnosis. I am writing to encourage you to find an agent who could get you a nice contract with a movie producer. Lord knows Hollywood could use a good story, and yours is POWERFUL! Good luck with your treatment, and with your outreach to veterans groups. (There is such a large homeless population here in the Bay Area…I wish we could test them all.) Blessings to you and to your family who believed in you! Emily Dearest Ruben, What a story! First and foremost, THANK YOU for your service and your commitment to the lyme community. Ruben, I am asking for YOUR HELP if at all possible, perhaps by emailing me so we can discuss (chaendler@comcast.net). I have been diagnosed with late stage lyme by a lyme MD…in June 2015 I developed a skin condition/rash that I STILL HAVE 9 MONTHS later…multiple biopses and MD consults showed contact dermatitis. I was tested for that and came up with allergens. However, all the items I am allergic to, I have not used for many years. Naturally, all my regular MDs think I am crazy when I ask about ACA as they do not even consider lyme…even my lyme MD thinks that may not be what is wrong with my skin as she states ACA only found in Europe. COULD I HAVE ACA? How do I get it diagnosed…special biopsy?? Also, have had photophobia for many years, worsened since rash began June 2015 and getting worse. The reason I am so DESPERATE is that this skin condition has prevented me from going outside!! I need not say more. It is a skin eruption that just burns and burns, ears ooze…I never know when it will flair. When it is bad all i can do is sit with Manuka honey and ice to try and reduce the burn. I am trying my hardest to live this extremely restricted life but this skin thing has knocked me down so far i can no longer fight. If you are able, please email me so I can understand ACA better and see if that is what i have. My sincere thanks in advance for your time and expertise. I, also, live in northern CA. Here’s an article by an ID doc who found ACA in Georgia: https://www.lymedisease.org/lyme-disease-georgia-pugliese/ thanks so much! someone had sent that to me many months ago…brought to my MDs and they still do not believe! My MDs agree something is very wrong with me and they can clearly see the skin eruptions but they haven’t a clue as to what to do. I have tried about 10 steroid creams (i know, lyme and steroids) to no avail….those creams seem to make it worse! I just do not know who to turn to for any help with the further workup of this. If this is contact dermatitis, it has to be the worst case EVER! My friend committed suicide in 1991 after her disbelieving docs kept up their disbelief despite my friend’s continued downhill course (diagnosed back then with CFS as was I). I am at the stage my friend was many years ago…where do i turn for relief? Can not go to yet one more MDs consult and be further disheartened after the visit. Recommend you be evaluated by a Lyme-literate MD, affiliated with ILADS. I am seeing and have been seeing a LLMD who is just too unfamiliar with ANY LATE STAGE SKIN PROBLEMS! You can get LLMD referrals at http://www.lymenet.org in the Seeking a Doctor section. cindy, you can also go here for 24/7 answers. hope this helps your relentless search; been there, done that for 35 yrs. before i was CORRECTLY diagnosed after seeing 40-50 drs! https://sites.google.com/site/lymedoctors/home bettyg, iowa lyme activist Betty, so appreciate your recommendation – thanks, too for being an activist for those of us who can not currently. Hi Cindy – seeing all these comments coming in, just read through what you wrote and that you’re in CA. I’m going to email you about doctors. Cindy, Morgellon’s disease is a relatively rare variation of Lyme disease with unusual skin manifestations which is not yet well recognized. Here’s a link to the best website about Morgellon’s disease, where you will find photos of the various skin rashes associated with it, plus tons of other information about treatment for it. It is often more difficult to treat than ordinary Lyme disease, but it is definitely treatable once it has been properly diagnosed. http://www.thecehf.org/ cindy, perhaps this link of info may help you too about ACA…. http://www.mdjunction.com/forums/lyme-disease-support-forums/general-support/3695166-acaskin-manifestations-of-lyme-disease bettyg I am so pleased your condition has been acknowledged by the VA. As a Lyme’s Disease survivor now in remission) and husband of a current chronic Lyme’s sufferer, I wish you luck in you recovery. God bless you for sharing your story. I am very sorry for the years and years of feeling left behind. Again, it is heartbreaking hearing our Lyme family suffer not just the disease and co-infections but the LACK OF UNDERSTANDING AND HELP! I have late stage neuro lyme but continue to fight one day at a time. Antibiotics, picc line and now IV treatments. We must all stick together and YES RAISE OUR VOICES! My children and I wrote a little book to encourage families, that ALL is possible with Jesus! Check us out http://www.lymeonedayatatime.com. God bless you and THANK YOU FOR YOUR SERVICE!! First of all, thank you for your service and I am very sorry you are going through Lyme hell too. I’ve had Lyme since July 2002. Was bitten in Livingston, Texas. Was misdiagnosed at the Liberty, Texas ER July 25, 2002, with a big bullseye, very severe flu like symptoms and 104 fever. Was misdiagnosed by maybe 7 Drs between 2002 & 2005 when finally got a Lyme positive diagnosis at the Houston medical center. Was treated from that point until 2010 with basically 5 years of antibiotics. Doxycycline (year at a time. 6 months at a time) picc line (intravenous) with 2 grams of rocephin a day for 6 weeks. Months on end of riphampin. 6 straight months of malaron (malaria antibiotics) paired with azythromycin & flagyl (flagyl twice a week all 6 months). That was the last time I took antibiotics. And I ALWAYS felt better for a little while and could walk and function again after antibiotics. But we all know we can’t live the rest of our lives on antibiotics. I also tried every homeopathic “cure” out there. Colloidal silver for a year. Food grade hydrogen peroxide (drinking it in water) for a year. I’m not typing anymore….point is, if it could be cured, I would be well. It’s been 14 years and I have been bedridden, except to get to the bathroom with a walker most of the time, for 3 1/2 straight weeks right now. It’s incurable. ? Good luck to you sir. I hope you find a way to get well. And please share with us all if you do. God bless you and your family and again, thank you for your service. (My brother retired Air Force) Diana, Hello, fellow Texan. As co-moderator of our TxLyme Yahoo forum, we are encouraging all of our members to get a copy of the newest (Feb., 2017) book by Dr. Richard Horowitz, MD (“How Can I Get Well?”) and to read Chap. 4 because that chapter contains all of the very newest research about persistence of pathogens associated with chronic Lyne disease, plus the very newest combinations of antibiotic protocols which are working when other older antibiotic protocols have failed. The book is available on Amazon. So please don’t accept that Lyme is “incurable” because as long as you still have breath left in your body, then Lyme is not incurable. https://www.amazon.com/How-Can-Get-Better-Resistant/dp/1250070546/ref=sr_1_1?s=books&ie=UTF8&qid=1490121195&sr=1-1&keywords=Horowitz+%2B+Lyme You might also want to join our TxLyme Yahoo forum (link below) so that you can obtain information about our ILADS doctors and other “Lyme-friendly” doctors in Texas. These messages with info about our LLMDs are all sent privately though, according to our forum rules, because we want to protect our LLMDs from persecution by IDSA doctors, whose national IDSA headquarters is based here in San Antonio, TX. Also, we did succeed in passing protective legislation for our Lyme doctors a few years ago, based on a clause in our 150 yo Texas Constitution, so our state is a wee bit more Lyme-friendly than in many other states in the US. https://groups.yahoo.com/neo/groups/txlyme/info Elizabeth N. God bless you, Ruben Sims! I was an Air Force dependent for 12 years and suffered with Lyme Disease for the past 40 years. No one caught it. I insisted on testing, but I was told I was a negative result. For the past two years I have finally been diagnosed (in the civilian world) and still am in the midst of treatment. I feel for you, buddy. I know how the military doctors and bureaucrats behave. We must be tough old birds to go thru this hell and retain our sanity in the end. Love You! I’m sorry you had to go through all that. But fight the good fight, Ruben! And thanks for the article. Yes I have went undiagnosed, ms miagnosed and not believed. I as actually admitted to a hospital with testing done that showed I had bilirubin, kidney issues, high platelets, high neutriphills with severe confusion and my speech was impaired. They released me and sent me to a Psychiatric hospital instead where they kept me without any court appointed attorney for 4 month shoving forced antipsychotics down my throat . After I was released I tested positive for several tick born infections, Malaria, Leishmania, Meningitis, Pneumonia, Cytomegalovirus and EBV and other Viruses as well. Then was still refused treatment. Now I have Lymphatic Cancer from all of those untreated Viruses and Parasitic Infections infiltrating my Lymphatic System. It’s beyond me how people can actually believe that we have the World Best Health Care when you can’t be properly diagnosed and then refused treatment and then those involved cannot be held responsible! All of these are registered along with treatment on the CDC and World Health Organization for available information. It’s highly unfortunate that we as tax paying citizens are money is going towards research however denied treatment and respect. And now I cannot even be taken seriously because It is now in my Medical History of being diagnosed with a Mental Disorder which they refuse to correct even when I went back to the Hospitals with my lab tests. I am tired of being treated with disrespect, ignorance and undermined by those in the Medical field are the very ones who are disinformed, ignorant themselves. And then when you have proof still refuse to treat? It’s a Crime and should be punishable! I was just reading an article just how bad our Medical System is because of a faulty system designed by Specialties that send you in circles and all they want to do is find your health under their specialty and if they can’t they don’t offer you any further help. Then when you do go to a proper specialty they are not trained nor knowledgeable nor do they understand tat Viruses and bacterial infections left untreated create further disease in the body that leads to cancer. It’s easily understood if you understand Microbiology and how the blood works as well as all systems of the body interplay to function as a whole. And it’s a crime when Disease Becomes the arena of Politics ! Dear Ruben, Thank you for your service and for sharing your story. I think the way our vets are treated is a disgrace, but your treatment was horrific! Hold your head high and proud! Don’t let them bring you down! We have to stick together! A sad story, to bad it’s not a story at all it sound just my life only the VA is still out to lunch. All I have to ad is I have had to have a defibrillator put in my chest because of the damage to my heart for a tick I had living under my LBE line for at least 36 – 48 hours back in 1987. A Neurologist diagnosed my advance stage of Lymes in 2017. Looking back to 13 years of military medical records shows a rash and every 3 to 4 weeks going to sick call for flu like symptoms. Who gets flu like symptoms every month for 2 years? Then it went dormant or not as bad well till 2013 when like a ton bricks on the head my heart started to fail. LymeDisease.org is a non-profit 501(c)(3) that serves the patient community through advocacy, education and research. Federal EIN-68-0214101 Support LymeDisease.org Become a member. Comments?Questions? Ask your questions here. LymeDisease.org PO Box 16009 | San Diego, CA 92176
XRP, Stellar, and Algorand officially get integrated into Europe’s unified digital ledger. Renowned crypto analyst SMQKE confirms XRP is officially listed on the European Ledger, Europe’s emerging unified digital financial infrastructure. Joining XRP, blockchain leaders Algorand (ALGO) and Stellar (XLM) are also verified, marking a major milestone in integrating digital assets into Europe’s financial ecosystem.
"The European Ledger aims to revolutionize finance by uniting tokenized central bank money, commercial bank money, and other digital assets on a single, programmable platform," said fintech expert Mr. Cipollone. "This shared infrastructure could redefine how Europe manages and transacts digital value." The initiative transforms T2S, the centralized European securities settlement system, into a cutting-edge DLT-based infrastructure. Central banks provide the foundational 'rails,' while market participants operate the 'trains,' enabling seamless liquidity and asset flows across this next-generation ecosystem. Verification of XRP, Algorand, and Stellar on the European Ledger marks a key step toward institutional recognition and mainstream adoption in regulated markets. XRP’s payment-focused utility positions it for pivotal cross-border settlements, while Algorand’s scalable blockchain and Stellar’s fast, low-cost payments advance the ledger’s mission of interoperability and efficiency. Industry experts highlight Europe’s shift toward a DLT-based single market as a reflection of global trends in programmable money and tokenized assets. By combining regulatory oversight with blockchain efficiency, the European Ledger could become a blueprint for integrating traditional finance with emerging digital ecosystems. Banks, fintech innovators, and other market participants stand to gain from faster, more transparent, and cost-effective settlement processes. The initiative underscores Europe’s commitment to fostering financial innovation while upholding strict regulatory standards, a balance key to the continent’s future financial resilience. With XRP, Algorand, and Stellar officially verified, the European Ledger takes a decisive leap toward a unified, DLT-powered financial system, turning tokenized assets and programmable money from theory into integral, operational pillars of Europe’s economy. Don’t miss out and join our newsletter to get the latest, well-curated news from the crypto world!
Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Advertisement Scientific Reportsvolume 7, Article number: 1498 (2017) Cite this article 3794 Accesses 32 Citations 1 Altmetric Metrics details Dysregulated mitochondrial dynamics and biogenesis have been associated with various pathological conditions including cancers. Here, we assessed the therapeutic effect of cryptolepine, a pharmacologically active alkaloid derived from the roots of Cryptolepis sanguinolenta, on melanoma cell growth. Treatment of human melanoma cell lines (A375, Hs294t, SK-Mel28 and SK-Mel119) with cryptolepine (1.0, 2.5, 5.0 and 7.5 μM) for 24 and 48 h significantly (P < 0.001) inhibited the growth of melanoma cells but not normal melanocytes. The inhibitory effect of cryptolepine was associated with loss of mitochondrial membrane potential and reduced protein expression of Mfn1, Mfn2, Opa1 and p-Drp1 leading to disruption of mitochondrial dynamics. A decrease in the levels of ATP and mitochondrial mass were associated with activation of the metabolic tumor suppressor AMPKα1/2-LKB1, and a reduction in mTOR signaling. Decreased expression of SDH-A and COX-I demonstrated that cryptolepine treatment reduced mitochondrial biogenesis. In vivo treatment of A375 xenograft-bearing nude mice with cryptolepine (10 mg/Kg body weight, i.p.) resulted in significant inhibition of tumor growth, which was associated with disruption of mitochondrial dynamics and a reduction in mitochondrial biogenesis. Our study suggests that low toxicity phytochemicals like cryptolepine may be tested for the treatment of melanoma. The mitochondrion is the most important cellular organelle involved in the processes of bioenergetics and biosynthesis and plays a key role in cellular signaling. Vital cellular functions such as regulation of energy production, maintenance of redox status and cell growth are controlled by mitochondria1,2,3. Shifts in mitochondrial function may lead to impaired biosynthetic pathways, dysregulated cellular signal transduction pathways, and modulation of gene transcription as well as chromatin structure4, 5. Alterations in mitochondrial mass or functions influence cellular functions, proliferation, and survival, and have been shown to affect metastasis as well as the clinical outcome of patients with cancer2, 6. Mitochondrial biogenesis in cancer is influenced by the tumor type, tumor microenvironment and the metabolic state2, 7. The process of mitochondrial biogenesis is controlled by various genes expressed in both the nuclear genome and the mitochondrial genome2, 8. It has been suggested that the transcription factor c-Myc that regulates cell proliferation, cell cycle, metabolism and apoptosis is a central regulator of mitochondrial biogenesis as loss of c-Myc reduces mitochondrial biogenesis and, conversely, gain of c-Myc increases mitochondrial biogenesis9. Expression of Sirtuins (SIRT1 and SIRT3) also is related to enhanced tumorigenesis and metastasis through their regulation of mitochondrial biogenesis and activation of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α)10, 11. AMP-activated protein kinase (AMPK), which is a metabolic tumor suppressor, acts as an energy sensor12, 13 and maintains energy homeostasis as well as regulating various processes associated with tumor development such as cell growth, cell survival, cell cycle progression and apoptosis13, 14. Cutaneous melanoma is the most lethal form of skin cancer. The American Cancer Society estimated that in the United States in the year 2016 approximately 76,380 new cases of cutaneous melanoma were diagnosed and approximately 10,130 individuals were died of this disease15. Melanoma develops from neoplastic melanocytes which are constantly exposed to sunlight specifically in fair skin individuals. A growing body of evidence indicates that increased mitochondrial biogenesis in melanoma cells results in enhanced tumorigenesis and metastasis as well as drug resistance to inhibitors of the mitogen-activated protein kinases (MAPK)3, 16, 17. Thus, inhibition of mitochondrial biogenesis can be considered as a potentially novel approach to inhibition of melanoma development and progression. Cryptolepine, a pharmacologically active plant alkaloid, is isolated from the roots of the shrub Cryptolepis sanguinolenta (Lindl.) commonly found in the Central and Western regions of the African continent18. In various biochemical and pharmacological assays, it has been demonstrated that cryptolepine has significant potential as an anti-malarial, anti-bacterial, and anti-hyperglycemic agent under different in vitro and in vivo conditions19,20,21. Some studies have reported a significant anti-inflammatory potential. Both inflammatory mediators, such as COX-2/PGE2 signaling, and promoters of inflammation, including TNFα and iNOS, have been identified as potential targets of the anti-inflammatory action of cryptolepine22,23,24. The anticancer effects of cryptolepine have been reported as due to its direct interactions with DNA, inhibition of DNA synthesis and inhibition of topoisomerase functions25,26,27. However, the molecular mechanisms underlying the potential cytotoxicity against cancer cells and in particular melanoma are not known and have not been explored. Therefore, we investigated the anti-cancer potential of cryptolepine using human melanoma cells. We report that treatment of human melanoma cells with cryptolepine inhibits the growth and viability of melanoma cells in culture and in an in vivo mouse xenograft model and does so by targeting the mechanisms that regulate mitochondrial dynamics and mitochondrial biogenesis. We first determined the short-term effects of cryptolepine on the viability of various human melanoma cell lines (i.e., A375, Hs294t, SK-Mel 28 and SK-Mel 119) using different concentrations of cryptolepine (0, 1.0, 2.5, 5.0 and 7.5 µM) for 24 and 48 h. An MTT assay revealed a dose-dependent decrease in the viability of A375 cells after treatment with cryptolepine for 24 h (13.7 to 40.8%; P < 0.05–P < 0.01) and after 48 h (15.7 to 52.8%; P < 0.05–P < 0.001) (Fig. 1a). Under identical experimental conditions, similar effects of cryptolepine were observed on treatment of Hs294t, SK-Mel28 and SK-Mel119 cells. The sensitivity of normal human epidermal melanocytes (NHEM) to the cytotoxic effects of cryptolepine was significantly lower than the sensitivity of the melanoma cell lines (P < 0.01–P < 0.001) (Fig. 1a). Cryptolepine treatment inhibits the viability and growth of melanoma cells. (a) Treatment of melanoma cells (A375, SK-Mel28, SK-Mel119 and Hs294t) and normal human epidermal melanocytes with cryptolepine (0, 1.0, 2.5, 5.0 and 7.5 µM) for 24 or 48 h inhibits cell viability in a dose- and time-dependent manner. Cell viability was determined using an MTT assay as described in the Materials and Methods. Cells were seeded in 96-well plates in 6 replicates/sample and per treatment group. Cells treated with DMSO alone served as a vehicle control. Statistical significance versus control group, *P < 0.05, ¶P < 0.01, †P < 0.001. (b) Effect of cryptolepine on the colony forming capacity of melanoma cells. Colonies were detected by staining with crystal violet and plates were scanned for photographs. Colonies appear blue-violet. To determine the long-term cytotoxic effect of cryptolepine treatment on melanoma cells, a colony formation assay was employed. For this purpose, melanoma cells (A375, Hs294t, SK-Mel28 and SK-Mel119) were treated with cryptolepine. As shown in Fig. 1b, cryptolepine significantly inhibited colony formation by the melanoma cell lines. The reduction in colony formation in terms of both size and numbers was dependent on the concentration of cryptolepine used to treat the cells prior to the colony formation assay. These data provide further evidence that cryptolepine has a cytotoxic effect on human melanoma cells. To determine whether the cryptolepine-induced reduction in melanoma cell viability or toxicity was associated with disruption of mitochondrial integrity and function, we determined the effect of cryptolepine on mitochondrial membrane potential using the Rhodamine 123 fluorescent probe. As the cytotoxic effect of cryptolepine on the viability of different melanoma cell lines was similar, we selected two melanoma cell lines (A375 and Hs294t) for further experiments. The melanoma cells were treated with various concentrations of cryptolepine (0, 2.5, 5.0 and 7.5 µM) in vitro and the numbers of Rhodamine 123-stained cells quantified using flow cytometry. We found a significant decrease (P < 0.01) in Rhodamine-123-positive cells in the cryptolepine-treated A375 and Hs294t melanoma cells, suggesting that cryptolepine induces a loss of mitochondrial membrane potential in these cells (Fig. 2a). Depending on the dose of cryptolepine, the percentage of A375 cells with a reduced mitochondrial membrane potential was 14.1 to 38.4% (P < 0.05–P < 0.01) as compared to 4.8 percent of vehicle-treated control cells. In Hs294t cells, the percentage of cells with loss of mitochondrial membrane potential was 21.2 to 41.2% (P < 0.05–P < 0.01) as compared to 5.0% of vehicle-treated control cells (Fig. 2b). Treatment with cryptolepine disrupts mitochondrial membrane potential in melanoma cells. (a) Representative melanoma cell lines (A375 and Hs294t) were treated with various concentrations of cryptolepine (0, 2.5, 5.0 and 7.5 µM) for 24 h. Thereafter, cells were harvested, incubated with Rhodamine 123 and then analyzed for loss of mitochondrial disruption using flow cytometry. Data were compared with non-cryptolepine-treated cells. Treatment groups are shown in color. (b) Percentage of cells with mitochondrial disruption in different treatment groups was determined. The resultant data are presented in terms of the percentage of cells with loss of mitochondrial membrane potential and as means ± SD, n = 3. Statistical difference versus control, *P < 0.05 and ¶P < 0.01. As treatment of cryptolepine induces loss of mitochondrial membrane potential in melanoma cells, we investigated whether this effect is due to its effects on mitochondrial fission or fusion proteins. It has been shown that the mitochondria shaping proteins regulate mitochondrial morphology and dynamics by modulating their size and their numbers8, 28. Mitofusins (Mfn1 and Mfn2) and the mitochondrial dynamin-like GTPase, Opa1, control mitochondrial dynamics through regulation of mitochondrial fusion whereas Dynamin-1-like protein (Drp1), which is also a GTPase, regulates mitochondrial fission29, 30. Western blot analysis revealed that treatment of A375 and Hs294t cells with cryptolepine (2.5, 5.0 and 7.5 µM) for 24 h resulted in a concentration-dependent decrease in the levels of Mfn1 and Mfn2 proteins as compared to vehicle-treated control cells (Fig. 3a). In addition, a dose-dependent decrease in Opa1 and Drp1 proteins was observed. It has been shown that overexpression of Drp1 is associated with cancer cell survival and tumor growth and inhibition of Drp1 in tumor cells has been associated with reduced cell viability and inhibition of tumor progression28, 31. Treatment with cryptolepine reduces the protein levels of mitochondrial dynamics and decreases ATP levels. A375 and Hs294t melanoma cells were treated with cryptolepine (0, 2.5, 5.0 and 7.5 µM) for 24 h. (a) Cell lysates were prepared and subjected to analysis of mitochondrial fission and fusion proteins. Equal loading of proteins on the gels was verified by stripping the membrane and reprobing with β-actin. (b) ATP production was determined in cell lysates using an ATP determination kit, as described in Material and Methods. The experiment was performed in triplicate and results are expressed as mean ± SD in terms of percentage of control. Statistical significance versus control, *P < 0.05, ¶P < 0.01 and †P < 0.001. To determine the cause of cryptolepine-induced mitochondrial membrane disruption, we tested the effects of cryptolepine on the levels of ATP in melanoma cells. The levels of ATP in melanoma cells were determined after treatment of the cells with various concentrations of cryptolepine (0, 2.5, 5.0 and 7.5 µM) for 24 h. We found that treatment of cryptolepine significantly decreased the levels of ATP in A375 cells (18 to 71%, P < 0.05 to P < 0.001) and in Hs294t cells (30 to 75%, P < 0.05 to P < 0.001) as compared to the levels of ATP in vehicle-treated control cells (Fig. 3b). We then tested whether the cryptolepine-induced loss of mitochondrial membrane potential and ATP depletion affects the mass of mitochondria in melanoma cells and/or stimulates an increase in the levels of the energy sensing 5′ adenosine monophosphate-activated protein kinase (AMPKα1/2) and its upstream regulator, the liver kinase B1 (LKB1). Cryptolepine (2.5, 5.0 and 7.5 µM)-treated A375 and Hs294t melanoma cells were stained simultaneously with MitoTracker Red CMXRos dye and antibodies specific for p-AMPKα1/2 or LKB1. The MitoTracker Red CMXRos dye accumulates in healthy mitochondria in live cells. The number of red-fluorescent dye containing cells was markedly lower in cryptolepine-treated cells than the non-cryptolepine-treated control cells suggesting that cryptolepine treatment induces mitochondria depletion that contributes to the ATP depletion in A375 and Hs294t melanoma cells (Fig. 4a). Both the phosphorylation of AMPKα1/2 and the levels of LKB1 were increased in the A375 and Hs294t melanoma cells with damaged mitochondria in the cryptolepine-treated groups as compared to vehicle-treated control cells (Fig. 4a,b). Western blot analysis of the same treatment groups revealed that the treatment of melanoma cells with cryptolepine resulted in higher levels of AMPKα1/2 and LKB1 proteins as compared to vehicle-treated control cells (Fig. 4c). We also found that cryptolepine treatment reduced the levels of Drp1 protein as well as the levels of Drp1 phosphorylation with a concomitant increase in AMPKα1/2 phosphorylation in A375 and Hs294t melanoma cells in a concentration-dependent manner (Fig. 5a). These results provide strong evidence that cryptolepine reduces the viability of melanoma cells by targeting mitochondrial dynamics and inducing activation of AMPKα1/2 proteins. Cryptolepine treatment depletes mitochondria and increases the levels of AMPKα1/2 and LKB1 in melanoma cells. (a and b) Approximately 1×105 A375 or Hs294t melanoma cells/well were plated in four-well chambered slides and treated with 0, 2.5, 5.0 and 7.5 µM cryptolepine for 24 h. Cells were stained with MitoTracker Red for 30 min. After fixation, permeabilization and blocking, cells were incubated with p-AMPKα1/2 or LKB1 antibodies overnight at 4 °C. Staining was performed as detailed in Materials and Methods. Images were acquired at 40x magnification using a Keyence Fluorescence Microscope BZ-X710 (Keyence Corporation of America). A representative photomicrograph from each group is shown. (c) After treatment of cells with cryptolepine for 24 h, total cell lysates were prepared and subjected to western blot analysis to determine the levels of AMPKα1/2 and LKB1 proteins. Blots were developed using chemiluminescence-specific ECL detection agents. Equal loading of proteins was verified by stripping the membrane and reprobing with anti-β-actin antibody. Treatment with cryptolepine disrupts mitochondrial dynamics by activating AMPK phosphorylation, reducing Drp1 phosphorylation and inhibiting mTOR signaling in melanoma cells. (a) Immunofluorescence staining of p-AMPKα1/2 and p-Drp1 was performed after the treatment of cells with cryptolepine, as detailed in Materials and Methods. Photomicrographs were obtained using Keyence Fluorescence Microscope BZ-X710 (Keyence Corporation of America). A representative photomicrograph from each group is shown. (b) After treatment of A375 and Hs294t cells with cryptolepine for 24 h, total cell lysates were prepared and subjected to western blot analysis to determine the level of proteins involved in mTOR signaling. Blots were developed using a chemiluminescence-specific ECL system. Equal loading of proteins was verified by stripping the membrane and reprobing with anti-β-actin or vinculin antibodies. Protein synthesis in cells is considered to be one of the most energy consuming processes among those required for cellular growth and survival. The mechanistic target of rapamycin (mTOR) plays a crucial role in multiple cellular processes including protein synthesis, cell growth, cell cycle, cell survival, and autophagy32. mTOR has been shown to be activated in the majority of malignant melanomas33, 34. Cross-talk between AMPK and mTOR signaling is crucial for regulating cellular metabolism, energy homeostasis, cell growth and cell survival and a growing body of evidence indicates that AMPK activation negatively regulates the mTOR signaling pathway35, 36. We therefore determined the effect of cryptolepine on mTOR signaling. Treatment of A375 and Hs294t melanoma cells with cryptolepine greatly reduced the total protein levels of mTOR and its phosphorylation in a concentration-dependent manner (Fig. 5b). Moreover, cryptolepine treatment resulted in reduced phosphorylation of the p70S6K and 4E-BP1 proteins (Fig. 5b), which are considered to be crucial downstream targets in mTOR-regulated protein synthesis. The levels of total p70S6K and 4E-BP1 protein were not significantly affected by cryptolepine. These results suggest that cryptolepine treatment reduces mTOR signaling and suggest that this reduces protein synthesis in the melanoma cells. To evaluate the effect of cryptolepine on mitochondrial biogenesis in melanoma cells, the levels of two mitochondrial proteins succinate dehydrogenase-A (SDH-A), which is a subunit of respiratory Complex II and encoded by nuclear DNA, and cytochrome c oxidase I (COX-I), which is a subunit of respiratory Complex IV and is encoded by mitochondrial DNA, were measured simultaneously using the MitoBiogenesis™ In-Cell ELISA Colorimetric system. A375 and Hs294t cells were treated with 2.5, 5.0 and 7.5 µM cryptolepine for 24 h prior to the assay. Treatment with cryptolepine significantly reduced the levels of nuclear DNA-encoded SDH-A protein in a concentration dependent manner in A375 cells (44 to 78%; P < 0.001) and in Hs294t cells (29 to 78%; P < 0.01 to P < 0.001) as compared to vehicle-treated control cells (Fig. 6a). Similar effects also were observed in terms of the mitochondrial DNA-encoded COX-I protein levels in A375 cells (53 to 80%; P < 0.001) and Hs294t cells (35 to 86%; P < 0.01 to P < 0.001) after cryptolepine treatment as compared to vehicle-treated control cells (Fig. 6a). These results clearly demonstrated that cryptolepine significantly inhibits mitochondrial biogenesis in both of these melanoma cell lines by targeting nuclear and mitochondrial signaling pathways of mitochondrial biogenesis. Treatment with cryptolepine inhibits mitochondrial biogenesis and associated molecular targets in melanoma cells. (a) Mitochondrial biogenesis in cryptolepine-treated and non-treated A375 and Hs294t cells was determined by measuring the levels of SDH-A and COX-I protein expression using the Mitochondrial Biogenesis Kit according to the manufacturer’s protocol. The experiment was performed two times. Protein expression in the vehicle-treated control group was considered as 100%. Statistical significance versus control, ¶P < 0.01 and †P < 0.001. (b) After treatment of cells with cryptolepine for 24 h, total cell lysates from A375 and Hs294t cells were prepared and subjected to western blot analysis to determine the levels of proteins involved in mitochondrial biogenesis. Equal loading of proteins was verified by stripping the membrane and reprobing with β-actin antibody. (c) Immunofluorescence staining for the detection of Opa1-positive and SIRT1-positive cells was performed as detailed in Materials and Methods. Photomicrographs were obtained using Keyence Fluorescence Microscope BZ-X710 (Keyence Corporation of America). Representative photomicrographs are shown. PGC-1α is one of the most important central controllers of cellular bioenergetics and mitochondrial biogenesis1, 37. Western blot analysis revealed that treatment of A375 and Hs294t cells with cryptolepine (2.5, 5.0 and 7.5 µM) greatly reduced the levels of PGC-1α protein in a concentration-dependent manner (Fig. 6b). It has been demonstrated that SIRT1-mediated deacetylation activates PGC-1α under energy deprivation conditions resulting in enhanced mitochondrial biogenesis, cell survival and drug resistance11, 38, 39. We found that cryptolepine treatment reduced the levels of SIRT1 and Opa1 proteins in A375 and Hs294t cells (Fig. 6b,c). It has been shown that c-Myc stimulates expression of approximately 400 nuclear-encoded mitochondrial genes which play a central role in mitochondrial biogenesis2, 9, 40. Relevant to our present study, we found that cryptolepine treatment markedly reduced c-Myc protein levels in A375 and Hs294t cells in a concentration-dependent manner (Fig. 6b). These data demonstrate that cryptolepine targets proteins involved in mitochondrial biogenesis. To assess whether the effects of cryptolepine we observed in the in vitro studies are translatable to an in vivo system, we determined the effects of administration of cryptolepine in a melanoma xenograft model. The A375 cell line was chosen as a representative melanoma cell line as we had found similar effects of cryptolepine on the viability of the different melanoma cell lines (Fig. 1). The A375 melanoma cells were implanted in the flanks of athymic nude mice and cryptolepine was administered intraperitoneally (i.p.) for 24 d. The dose of cryptolepine (10 mg/Kg body weight) was based on the doses used for intraperitoneal administration of some other phytochemicals in experiments to evaluate their anti-carcinogenic effects. Monitoring of the estimated volume of the tumor xenografts indicated that the average tumor growth/size in terms of total tumor volume/mouse was significantly lower (68%, P < 0.001) in the cryptolepine-treated group than the group of mice administered vehicle alone (Fig. 7a). As shown in Fig. 7b, the average wet weight of the tumor at the termination of the experiment in the mice that were treated with cryptolepine was significantly lower than that of tumors from vehicle-treated control mice (61%, P < 0.001). Cryptolepine treatment inhibits the growth of melanoma xenografts in athymic nude mice. The tumor xenograft growth experiment was carried out for a total of 24 d, as detailed in Materials and Methods. (a) Tumor xenograft growth was monitored and is shown in terms of tumor volume/mouse as a mean ± SD (mm3), n = 5/group. (b) At the termination of the experiment, tumors were harvested and the wet weight determined. (c) ATP content was determined in each tumor lysate sample, as described in Materials and Methods. The data are presented in terms of percentage of the non-cryptolepine-treated group and expressed as mean ± SD. *P < 0.05, †P < 0.001. (d) Levels of proteins associated with mitochondrial biogenesis/dynamics were determined in lysates of the excised A375 melanoma tumor xenografts by western blot analysis. Each sample per group was prepared by mixing the tumor tissues from 2–3 tumors from different mice. Equal loading of proteins was verified by stripping the membrane and reprobing with anti-β-actin antibodies. The body weight of the animals was recorded on a weekly basis for the duration of the experiment. No significant differences in the average body weight of the vehicle-treated mice and that of the cryptolepine-treated mice were observed (data not shown). In addition, cryptolepine-treated mice did not exhibit any physical sign of toxicity or abnormal behavior as compared to control mice. These data suggest that cryptolepine treatment at the concentration used in the present study is not associated with any apparent sign of toxicity in mice. We further evaluated the effect of cryptolepine on various biomarkers of regulation of mitochondrial dynamics and biogenesis in the tumor xenograft tissues. The content of ATP was significantly lower (32%, P < 0.05) in tumor tissues of mice treated with cryptolepine compared to the tumor tissues of control mice which were treated with vehicle alone. (Fig. 7c). Cryptolepine treatment also resulted in enhanced phosphorylation of AMPKα1/2 as well as reduced phosphorylation of 4E-BP1 protein, which is a downstream effector of mTOR signaling. These data demonstrate that cryptolepine has the ability to activate the metabolic tumor suppressor and disrupt energy homeostasis signaling under in vivo conditions and suggest that it does so by modulating cross-talk between AMPKα1/2 and mTOR cross-talk. Western blot analysis revealed that administration of cryptolepine to A375 xenograft-bearing mice resulted in a decrease in the levels of phosphorylated form of Drp1 protein that is involved in maintenance of mitochondrial dynamics (Fig. 7d). Further, the levels of c-Myc, SIRT1 and PGC-1α protein were reduced in the tumor samples from mice treated with cryptolepine as compared with the tumor samples from vehicle-treated control mice (Fig. 7d). These results verified our in vitro findings and demonstrated that cryptolepine-induced effects in melanoma cells are translatable to in vivo conditions. The balance between mitochondrial energy production and physiological functions required for cell survival is regulated by mitochondrial dynamics41. Maintenance of mitochondrial mass and the numbers of mitochondria in cells is regulated by the processes of mitochondrial biogenesis, fission, fusion and mitophagy. Uncontrolled mitochondrial function and dysregulated mitochondrial dynamics contribute to the pathogenesis of various diseases42. Thus, the targeting of mitochondrial biogenesis and mitochondrial functions has emerged as a novel preventive and therapeutic strategy for various metabolic diseases including cancer6, 43. Cryptolepine has been shown to possess anti-inflammatory activity and cytotoxic potential that is mediated by direct and indirect interactions with DNA22,23,24,25,26,27, 44, 45. In the current study, we found that cryptolepine treatment induced a highly significant decrease in melanoma cell viability and growth demonstrating that this compound possesses strong anti-melanoma activity. Furthermore, we found that cryptolepine targets mitochondrial dynamics and biogenesis in melanoma cells and that these effects were accompanied by activation of AMPKα1/2-LKB1, inhibition of mTOR signaling, and a reduction in the levels of c-Myc, SIRT1 and PGC-1α protein. AMPKα1/2 is recognized as a central energy-sensing protein that regulates glucose and lipid metabolism and can be activated by various stress-related factors such as ATP depletion, low glucose levels, exercise and fasting13, 46. A growing body of evidence demonstrates that loss of AMPKα1/2 expression is associated with enhanced tumorigenesis whereas induction of AMPKα1/2 expression is related to reduced cancer cell growth13, 14. Activation of AMPKα1/2 has emerged as a novel strategy for prevention and treatment of cancer and several metabolic diseases13, 14, 47. Our data demonstrate that cryptolepine reduces ATP production in melanoma cells and enhances both the levels of AMPKα1/2 protein and its phosphorylation. We also found that expression of LKB1, an upstream regulator of AMPKα1/213, 48, was enhanced in melanoma cells after cryptolepine treatment. It has been demonstrated that in response to energy-deprived conditions, activation of AMPKα1/2 inhibits protein synthesis through inhibition of mTOR signaling35, 36. Our results are in line with these observations in that we found that cryptolepine treatment induced activation of AMPKα1/2 in melanoma cells and caused inhibition of the protein synthesis machinery by reducing the phosphorylation of mTOR, p70S6K and 4E-BP1. Collectively, these results suggest that the ability of cryptolepine to induce activation of AMPKα1/2-LKB1 results in inhibition of mTOR signaling. In addition to ATP depletion, we observed that cryptolepine promotes loss of mitochondrial membrane potential in melanoma cells. These effects of cryptolepine were accompanied by a greater reduction in mitochondrial content in the treated melanoma cells suggesting modulation of mitochondrial dynamics. Cells maintain the numbers of mitochondria by their continuous fission and fusion. Mitochondrial fusion is regulated by a variety of proteins, including Mfn1, Mfn2, and Opa1, whereas mitochondrial fission is associated with Drp1 and mitochondrial fission 1 protein (Fis1)2, 3, 49. Higher expression of Mfn1 and Mfn2 has been linked with cancer cell proliferation, enhanced cell survival and invasion. Conversely, inhibition of Mfn1 and Mfn2 expression inhibits cell growth and induces apoptosis of various cancer cells29, 30, 43, 50. Expression of Opa1 also has been shown to enhance cancer cell proliferation and survival whereas its inhibition leads to apoptosis51, 52. We found that the levels of Mfn1 and Mfn2 and Opa1 protein were considerably reduced in cryptolepine treated melanoma cells. In terms of the fission-related Drp1 protein, it has been shown that activation and enhanced expression of Drp1 enhances cancer cell proliferation, survival and drug resistance to melanoma-targeted therapies3, 28, 53, 54. In the present study, we found that cryptolepine treatment reduced the levels of Drp1 protein as well as its phosphorylation in melanoma cells. Collectively, these results suggest that cryptolepine-induced mitochondrial fission in concert with reduced fusion caused mitochondrial depletion in cryptolepine-treated melanoma cells. Mitochondrial biogenesis is controlled by both nuclear genome-encoded proteins and mitochondrial genome-encoded proteins. Our study demonstrates that treatment of melanoma cells with cryptolepine resulted in reduced levels of SDH-A protein (a nuclear genome-encoded protein which is a component of Complex II enzyme) as well as COX-I protein (a mitochondrial genome-encoded protein, which is a subunit of the COX-IV enzyme complex). PGC-1α, a transcriptional coactivator, is considered to be a master regulator of mitochondrial biogenesis. PGC-1α maintains energy homeostasis and controls bioenergetics through ATP production and mitochondrial biogenesis, thereby promoting cell proliferation and survival. Enhanced expression of PGC-1α has been associated with cancer development and progression17, 55, 56. In our study, we found that cryptolepine greatly reduced protein expression of PGC-1α, which may have resulted in the significant depletion of ATP in cryptolepine-treated melanoma cells. SIRT1 controls mitochondrial biogenesis through deacetylation and activation of PGC-1α11, 39, 57 and overexpression of SIRT1 has been shown to promote melanoma cell proliferation and drug resistance11, 58, 59. We found that cryptolepine treatment markedly reduced the levels of SIRT1 protein in melanoma cells. Activated mTOR kinase regulates mitochondrial biogenesis both at the transcriptional level, through activation of PGC-1α/Yin Yang 1 (YY1) signaling leading to mitochondrial gene expression, and at the translational level, through repression of 4E-BPs that downregulate nuclear-encoded mitochondrial protein translation60. Thus, our data provide ample evidence that cryptolepine-induced inhibition of mTOR signaling and SIRT1 protein levels resulted in reduction of PGC-1α protein levels and mitochondrial biogenesis in cryptolepine-treated melanoma cells. c-Myc, a proto-oncogene that globally regulates key functions in cell growth, cell cycle, cell survival, protein synthesis, cell adhesion, cell metabolism and differentiation is recognized as the most important and central regulator of mitochondrial biogenesis2, 9, 40. We found that cryptolepine treatment greatly reduced the levels of c-Myc protein in melanoma cells. Importantly, the in vitro growth inhibitory potential and effects of cryptolepine on mitochondrial dynamics and biogenesis also were not only observed under in vitro conditions but also in vivo in a melanoma xenograft growth model in nude mice without any apparent sign of toxicity. These in vivo studies further verified that administration of cryptolepine inhibits tumor growth by modulating AMPKα1/2/mTOR, and reducing the c-Myc/SIRT1/PGC-1α signaling cascade involved in mitochondrial dynamics and biogenesis. In summary, the association of dysregulation of mitochondrial dynamics with cancer and other diseases are prompting the development of strategies to prevent or treat these diseases by targeting cellular energetics and mitochondrial biogenesis. Various phytochemicals, including flavonoids, grape polyphenol (resveratrol), green tea polyphenol (EGCG), honokiol, curcumin and berberine, that have demonstrated significant anticancer potential have been shown to target bioenergetics and activation of the metabolic tumor suppressor AMPKα1/2 and other signaling pathways involved in tumor growth and progression14, 61, 62. Interestingly, honokiol, a phytochemical derived from Magnolia grandiflora, also showed activity in vemurafenib-resistant melanoma through induction of respiratory enzyme succinate dehydrogenase in in vivo model63. In other tumor models, honokiol treatment increases AMP-activated protein kinase phosphorylation in breast cancer cells and thus inhibits the migration and invasion of breast cancer cells64. The effect of honokiol on mitochondria was also investigated in cardiac hypertrophy. The anti-hypertrophic effects of honokiol depends on activation of the deacetylase Sirt3. Honokiol treatment increases mitochondrial rate of oxygen consumption and reduces reactive oxygen species synthesis and thus affect the cardiac hypertrophy65. In the present study, by employing in vitro cell culture and an in vivo melanoma xenograft model, we found that an alkaloid isolated from the roots of Cryptolepis sanguinolenta significantly inhibits mitochondrial biogenesis in melanoma cells by inducing mitochondrial fission, reducing enhancers of mitochondrial biogenesis and affecting mTOR signaling, as summarized in Fig. 8. The results of our study suggest more detailed pre-clinical studies as a basis for exploration of cryptolepine as a potential anti-melanoma agent. Schematic diagram showing the effects of cryptolepine on mitochondrial dynamics, biogenesis and the regulatory cascade of events resulting in suppression of melanoma cell growth. The process of melanomagenesis is influenced by dysregulated mitochondrial dynamics and mitochondrial biogenesis associated with dysregulation of regulatory molecules. The dysregulated events in melanoma cells are shown with red arrows, the therapeutic effects of cryptolepine, which result in inhibition of melanoma cell growth, are shown with green arrows. Purified cryptolepine hydrate (≥98% purity by HPLC), MTT dye, 2′,7′-dichlorofluorescin diacetate, and Rhodamine 123 were purchased from Sigma-Aldrich (St. Louis, MO). The MitoBiogenesis In-cell ELISA kit was purchased from Abcam (Cambridge, MA). Antibodies against c-Myc, p-Drp1, LKB1, and AMPKα1/2 were purchased from Cell Signaling Technology (Beverly MA). The Opa1 antibody was obtained from Thermo Scientific (Rockford, IL). Antibodies against PGC-1α, Mitofusin 1, Mitofusin 2, Drp1, p-AMPKα1/2, SIRT1, and β-actin and horseradish peroxidase (HRP)-labeled anti-mouse and anti-rabbit were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The ATP Determination kit, MitoTracker Red CMXRos, AlexaFluore 488- and 594-conjugated secondary antibodies were obtained from Molecular Probes (Eugene, OR). The human melanoma cell lines A375 (human skin amelanotic malignant melanoma cell line) and Hs294t (human skin amelanotic melanoma cells derived from metastatic site lymph node) were obtained from the American Type Culture Collection (Manassas, VA). SK-Mel28 and SK-Mel119 were obtained from Dr. Alan Houghton of the Sloan-Kettering Institute for Cancer Research (New York, NY). Normal human epidermal melanocytes (NHEM) were obtained from Dr. Slominski of the University of Alabama at Birmingham, AL. Cells were cultured and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) or RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 100 mg/ml penicillin-streptomycin solution at 37 oC in a 95% humidified incubator with 5% CO2. NHEM were grown in melanocyte growth medium (MGM-4 Bullet kit; CC-3249) purchased from Lonza Walkersville Inc. (Walkersville, MD) that contains melanocyte basal cell medium (MBM-4; CC-3250) and is supplied with a supplements and growth factors kit (MGM-4 singlequots; CC-4435) including CaCl2, hFGF-B, PMA, rh-insulin, hydrocortisone, BPE, FBS and Gentamycin/Amphotericin-B. The cells were maintained under standard cell culture conditions as described above. All the cell lines were used 3-4 passage in each experiment. When the cells reached approximately 60–70% confluence, the media was replaced with media containing the specified concentrations of cryptolepine or vehicle control. The cryptolepine was dissolved in dimethylsulfoxide (DMSO) prior to addition to the media such that the final concentration of DMSO in the media was not more than 0.1% (v/v). An equivalent amount of DMSO was added to the culture media of control groups. The effects of cryptolepine on melanoma cell viability were determined using the MTT assay as described previously66. The viability of cryptolepine-treated cells was compared to the viability of vehicle-treated control cells, which was arbitrarily defined as 100%. Colony formation assay was performed as described previously66 to evaluate the clonogenic potential of melanoma cells after treatment with cryptolepine. Briefly, 500 cells from each of cryptolepine-treated groups (0, 1.0, 2.5, 5.0 and 7.5 µM for 24 h) were suspended in 3 ml complete growth medium and plated individually in separate wells of a 6-well plate. Cells were allowed to grow for a total 14 days with media being replaced on day 7. On day 14, colonies were washed with chilled PBS buffer, and fixed in chilled methanol for 10 min. Colonies were stained with 0.5% crystal violet (prepared in 25% methanol) for 10 min and excess stain removed by washing with water. The plates were air dried then scanned and microscopically observed. The changes in mitochondrial membrane potential in cryptolepine-treated and non-treated melanoma cells were determined based on uptake of Rhodamine 123 as described previously67. Approximately 2 × 105 cells were treated with 0, 2.5, 5.0 and 7.5 µM cryptolepine for 24 h. The cells were then incubated with Rhodamine 123 for 30 min, harvested, washed with PBS and resuspended in PBS for analysis of mitochondrial membrane potential using BD Accuri C6 flow cytometer (San Jose, CA). To determine the effect of cryptolepine on ATP production by mitochondria, total ATP content was measured in total cell lysates using the ATP Determination Kit purchased from Molecular Probes following the manufacturer’s protocol. Briefly, equal number of A375 or Hs294t cells was treated with 0, 2.5, 5.0 and 7.5 µM cryptolepine for 24 h and total cell lysates prepared. An equal amount of protein (20 µg) from each treatment group was used to determine the ATP content in terms of nMol ATP/mg protein. The data are presented in terms of the percentage of the values from the vehicle-treated control cells. The effect of cryptolepine on mitochondrial biogenesis in melanoma cells was determined using the MitoBiogenesis In-Cell ELISA (Colorimetric) kit (MitoBiogenesis) and as described in the manufacturer’s protocol manual. Using this kit, the levels of two mitochondrial proteins, SDH-A and COX-I, were measured simultaneously in cryptolepine-treated and non-treated cells. Briefly, approximately 2 × 104 cells/well were plated in each well of 96-well culture plates, 24 h later the cells were treated with different concentrations of cryptolepine (0, 2.5, 5.0 and 7.5 µM) for 24 h, and thereafter the manufacturer’s protocol was followed. Melanoma cells were harvested after 24 h of incubation with or without cryptolepine and cell lysates prepared as described previously66. Equal amounts of proteins were resolved electrophoretically on Tris-glycine gels and transferred onto a nitrocellulose membrane. Non-specific sites were blocked by incubating the membrane with blocking buffer for 1 h. The membrane was incubated with specific primary antibodies overnight at 4 °C followed by 2 h incubation with HRP-conjugated secondary antibodies. The bands were visualized by chemiluminescence on X-ray film. Equal loading of proteins was verified by probing the stripped membrane with anti-β-actin or anti-vinculin antibodies. All in vitro immunoblot data are presented from two independent experiments, and samples were run simultaneously on the same gel. In some cases, the membrane was cut into 2 or 3 pieces based on the molecular weights of the proteins and then blotting was performed individually. In these cases, the β-actin loading control remains common to all the proteins identified. Approximately 5 × 104 cells/well were seeded in four-well chambered slides and the next day the cells were treated with 0, 2.5, 5.0 and 7.5 µM of cryptolepine for 24 h. For mitochondrial staining, cells were further incubated with 100 nM MitoTracker Red (Molecular Probes) dye in culture medium. After incubation, the cells were washed twice with chilled PBS, fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.5% Triton-X 100 in PBS for 3 min. Non-specific binding was blocked by incubating the cells with 3% BSA in PBS for 30 min. Cells were incubated with specific primary antibodies over night at 4 °C, washed three times with PBS and then incubated for 1 h with fluorochrome-conjugated secondary antibodies. After washing with PBS, the slides were mounted with Vectashield mounting media containing DAPI and analyzed and imaged using a Keyence BZ-X710 fluorescence microscope (Keyence Corporation of America, Atlanta, GA). Female athymic nude mice of 4–5 weeks of age were purchased from the National Cancer Institute (Bethesda, MD, USA). Mice were housed in the Animal Resource Facility at the University of Alabama at Birmingham in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines. All methods were performed in accordance with the guidelines and regulations of IACUC. The tumor xenograft protocol was approved by the IACUC of the University of Alabama at Birmingham. To determine the in vivo chemotherapeutic efficacy of cryptolepine against human melanoma xenograft growth, exponentially growing A375 melanoma cells (2 × 106 A375 cells in 50 μl PBS + 50 μl matrigel) were injected subcutaneously in each flank of each mouse. 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Fisetin inhibits growth, induces G2/M arrest and apoptosis of human epidermoid carcinoma A431 cells: role of mitochondrial membrane potential disruption and consequent caspases activation. Exp. Dermatol.22, 470–475, doi:10.1111/exd.12181 (2013). ArticleCASPubMedPubMed Central Google Scholar Download references This work was financially supported in part by the funds from Veterans Administration Merit Review Award (1I01BX001410 to S.K.K.). The content of this publication does not necessarily reflect the views or policies of the funding agency. The funding agency had no roles in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The editorial assistance by Dr. Fiona Hunter is gratefully acknowledged. Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL, USA Harish C. Pal, Ram Prasad & Santosh K. Katiyar Environmental Health Sciences, University of Alabama at Birmingham, Birmingham, AL, USA Santosh K. Katiyar Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL, USA Santosh K. Katiyar Birmingham Veterans Affairs Medical Center, Birmingham, AL, USA Ram Prasad & Santosh K. Katiyar Search author on:PubMedGoogle Scholar Search author on:PubMedGoogle Scholar Search author on:PubMedGoogle Scholar H.C.P. and S.K.K. designed experiments, H.C.P. performed all experiments and compiled all the final results and figures, R.P. helped in in vivo experiments and tumor size measurement, S.K.K. and H.C.P. were involved in data analysis and writing of manuscript. All the authors have read and approved the final version of the manuscript for its publication. Correspondence to Santosh K. Katiyar. The authors declare that they have no competing interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Reprints and permissions Pal, H.C., Prasad, R. & Katiyar, S.K. Cryptolepine inhibits melanoma cell growth through coordinated changes in mitochondrial biogenesis, dynamics and metabolic tumor suppressor AMPKα1/2-LKB1. Sci Rep7, 1498 (2017). https://doi.org/10.1038/s41598-017-01659-7 Download citation Received: Accepted: Published: DOI: https://doi.org/10.1038/s41598-017-01659-7 Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article.
