Physcion 8-O-β-glucopyranoside induces mitochondria-dependent apoptosis of human oral squamous cell carcinoma cells via suppressing survivin expression
Authors: Fei Tan
Shi-Jiang Xiong
Meng-Dong Liu
Yi Liu
Journal: Acta Pharmacologica Sinica (2016)
Discipline: PHARMACOLOGY & TOXICOLOGY
Keywords: Physcion 8-O-β-glucopyranoside; apoptosis; cell cycle progression; human lung cancer cells; molecular mechanisms
Affiliations: School of Dentistry, Shandong University, Ji-nan 250012, China., School of Stomatology, Qingdao University, Qingdao 266011, China., Shandong Provincial Key Laboratory of Oral Biomedicine, Shandong University, Ji-nan 250012, China.

Abstract:  Aim: A previous study has shown that physcion 8-O-β-glucopyranoside (PG) derived from Rumex japonicusHoutt causes apoptosis and blocks cell cycle progression in human lung cancer cells. In the present study we investigated the molecular mechanisms underlying PG-induced cancer cell apoptosis. Methods: Human OSCC-derived cell line KB was treated PG (10, 20, 50 μg/mL). Cell apoptosis was detected with flow cytometry. Mitochondrial membrane potential (MMP) and release of cytochome C from mitochondria were measured; the expression of relevant signaling proteins was analyzed using Western blotting or qRT-PCR. For evaluation of in vivo anticancer action, nude mice grafted with KB cells were treated with PG (10, 20, 40 mg·kg(-1)·d(-1), ip) for 24 days. Results: PG dose-dependently suppressed cell proliferation and induced apoptosis in KB cells. PG-induced apoptosis was mediated via the intrinsic mitochondrial pathway, as evidenced by the decreased Bcl-2, increased Bax and Bax/Bcl-2 ratio, as well as the loss of MMP, caspase-9 activation, and increased cytosolic cytochrome c. Furthermore, PG suppressed the expression of survivin, whereas overexpression of survivin markedly attenuated PG-induced apoptosis. Meanwhile PG increased the expression of tumor suppressor PTEN, and decreased p-Akt, p-GSK3β and miR-21 levels. Pharmacological activation of Akt/GSK3β signaling or transfection with miR-21 mimic abolished PG-induced survivin reduction and cell apoptosis. Similar results were observed in PG-treated nude mice grafted with KB cells. Conclusion: Physcion 8-O-β-glucopyranoside induces mitochondria-dependent apoptosis of human OSCC cells by suppressing survivin expression via miR-21/PTEN/Akt/GSK3β signaling pathway. 

Retracted: No

PubPeer comments:
 This publication from DiNardo appears to be a review of a subject that has only 1 previous publication. That paper (Huo, et al) reports on a case-control study and in vitro cell-line study. However, there were several limitations in their methodologies, assumptions, and final relevance of the conclusions (should be reviewed elsewhere on Pubpeer). Because the Huo paper is inconclusive and fails to demonstrate a significant link, it is unusual that a scientific journal would publish a short review that does not add to the body of scientific evidence. Despite the wide body of evidence supporting the safety of oxybenzone, the authors make many unsupported claims without reference and attempt to weave together several unrelated studies to support the weak claims of the Huo paper. It is clear they are using the scientific publishing platform to push their own agenda related to mineral sunscreen (Downs runs a company that certifies oxybenzone-free sunscreen). The paper reads like an amateurish book report. They describe various aspects of the Huo study incorrectly u2013 for example they report that Huo matched subjects and controls within the population by age, weight, education and behaviors (smoking, drinking). But Huo only mentioned matching using motheru2019s age and adjusting for BMI. The authors attempt a risk assessment by making assumptions and calculations that demonstrate a lack of basic understanding of human absorption, distribution, metabolism, and excretion (ADME) mechanisms. BP-3 does not accumulate in the body the same way they estimate in their computed estimates. In fact, detection in the urine demonstrates that it is excreted and will not continue to accumulate indefinitely. There are several assumptions that are based on selective literature that does not represent the weight of evidence. Statement that BP-3 is an endocrine disruptor (EDC) is based on 3 studies only which does not represent the whole body of evidence and published literature on the topic. The weight of evidence overwhelmingly supports the safe use of oxybenzone. In general, the DiNardo paper presents nothing new, and only adds more assumptions on top of inconclusive results from Hupo, et al. , 
 #1 First, this short communication was meant to do nothing more than 1) show that all 101 of the neonates requiring surgery for Hirschsprungu2019s Disease (HD), reported by Huo et al, had oxybenzone in their body which matched moderate to high levels of oxybenzone in their motheru2019s blood and umbilical chord; (2) that oxybenzone levels have been reported in the urine of 96.8% of the US population according to the Center for Disease Control as well as at significantly higher levels than expected in the blood by other scientists including the Food and Drug Administration (FDA); (3) that the dose reported to damage the neurological cells involved in HD can be easily achieved using a sunscreen product containing 6% oxybenzone under normal u201cconditions of useu201d (not 35 - 110 years as previously reported by others) as recommended by FDA, dermatologists, pediatricians and skin cancer organizations. Second, oxybenzone has been reported to bioaccumulate and biomagnify in the body of many species and in humans has been shown to continue to be released into the urine even 5 days after whole body applications have stopped. Additionally the recent FDA study noted, accumulation of oxybenzone in the body with continued use with a mean u201chalf-lifeu201d between 24 u2013 31 hours u2026 presence in the urine does not mean that that the chemical has been removed from the body u2013 continued use of products containing oxybenzone means continued buildup of the chemical in the body and continued exposure to a fetus. With respect to u201cStatement that BP-3 is an endocrine disruptor (EDC) is based on 3 studies only which does not represent the whole body of evidence and published literature on the topic.u201d The following link https: www.regulations.gov docketBrowser?rpp=25 so=DESC sb=commentDueDate po=0 s=Joe%2BDiNardo dct=PS D=FDA-1978-N-0018 will yet take you to another amateurish book report I wrote to the FDA outlining roughly a 178 scientific publications demonstrating some level of toxicological concern. There are literally dozens of papers from scientist from all over the world clearly demonstrating endocrine disrupting effects in numerous species. There is no doubt that oxybenzone as well as many of the other organic sunscreen actives are endocrine disruptors u2026 even according the European Union and World Health Organization regulatory bodies. Based on the PUBLISHED data, you decide: One hundred and one (101) newborns needing surgery for HD, all with oxybenzone in their blood along with moderate to high levels of oxybenzone found in their motheru2019s blood u2026 findings that were not observed in the surgical and or non-surgical HD control groups reported in the study. In vitro testing with oxybenzone demonstrating toxicity of neuro crest cells associated with the HD u201cat levels commonly achieved with daily use of a 6% oxybenzone sunscreenu201d. One hundred and seventy-eight (178) scientific publications on oxybenzone demonstrating that it is chemically aggressive, forming free radicals stimulating caspases (involved in melanoma production) as well as reacts with chlorine, bromine and ozone to form toxic byproducts in pools, spas and tap water. Environmentally, the chemical bioaccumulates and biomagnifies and should be considered a water and food contaminant since it can be measured in virtually all bodies of water and in many edible fish. It demonstrates general toxicity (acute and sub-chronic) in addition to mutagenic, cytotoxic, genotoxic, neurotoxic, numerous endocrine disruption activities, proliferates human breast lung prostate cancer cells, produces phototoxic, contact and photo-contact allergic reactions as well as contact urticaria and, to a lesser degree contact mediated anaphylaxis, can be found in urine, blood, breast milk, semen and sperm in numerous aquatic and terrestrial species including humans. AND here is the real kicker u2013 In February 2019, FDA delisted oxybenzone (and 13 other organic sunscreen actives from the u201csafe and effectiveu201d sunscreen monograph category) until carcinogenicity and REPRODUCTIVE toxicology studies can be conducted reviewed demonstrating that it is safe and effective for its intended use! The only people that I can think of that WOULD NOT be concern about using oxybenzone products are people who donu2019t like themselves, their loved ones and or their children OR perhaps someone from industry trying to hold onto a $10 BILLION a year sunscreen business? According to the Food, Drug and Cosmetic Act, the LAW clearly states that INDUSTRY is responsible to PROVE their products are safe and effective u201cprioru201d to being sold (not our federal tax dollars) u2026 industry and those supported by them have not only grossly misrepresented the safety of sunscreen products, but have made a $100+ BILLION doing so over the years u2013 it is time for industry to put their money were their mouth is and either prove these actives products are safe by actually testing them (to at least FDA standards) or stop making such erroneous statements based on personal opinion. The testing conducted on these sunscreen actives products is over 40 years old and was submitted to FDA by industry u2026 this information is no longer valid or accurate!!!!!! , 
 While I cannot ascribe motives to either of the previous commenters, I would suggest that the potential conflict of co-author Downs is the type of COI disclosure that would benefit readers of the paper. Currently, the conflicts section of the paper reads: **Joseph C. DiNardo**: no conflict(s) of interest. **Craig A. Downs**: Haereticus Environmental Laboratory received a donation $1,100 from the W.S. Badger Company to cover the costs of 105 T-shirts for a u201cSave The Reefu201d Rally at 5:00 pm EST on January 15, 2019 in Key West, Florida. This donation had no influence on this on the data, arguments or conclusions stated in this manuscript. The authors have no other financial relationships relevant to this article to disclose However, #1 is apparently correct that Dr. Downs is Executive Director of [Haereticus Environmental Laboratory] (HEL) which issued a [press release](http: haereticus-lab.org can-oxybenzone-cause-hirschsprungs-disease ) in conjunction with the publication of this paper, including an infographic which appears to be a revised version of Figure 1 (see below), HEL appears to be an advocacy organization which solicits donations and will certify products for a fee. As always, such potential COI disclosures are not taken as an indication of the bias of the authors, but *failure* to disclose an apparent association could be interpreted as misleading. I believe Dr. Downs leadership of an environmental advocacy organization (HEL) which is using this paper to support their fundraising and certification business is clearly a  financial relationship that is relevant to disclose, and would recommend a correction to the existing disclosures in the paper. Below: Figure 1 and infographic from HEL site. *** ![file] *** ![file] , 
 DiNardo states that paper was  meant to do nothing more than show points 1-3 above, and therein lies some of the important problems. Please re-read the Huo paper. (1) They did not test blood levels in the mothers, cord blood, or the infants as you assert above. They did a spot urine analysis an unspecified length of time after the birth of the child. There is no reason to believe that level is related to early trimester pregnancy. (2) that s well known, and if that indeed cause Hirschprung s Disease, then every child in the US would be born with it. (3) Cell culture test may be mechanistically informative, but considering the relevance of dosing cell line studies as compared to whole organism physiological conditions, the choice to treat cells at 0.1 u2013 100 u00b5M BP-3 is notably high. (As noted, the Huo paper deserves it s own Pubpeer assessment). DiNardo is also incorrect in asserting that the EU and WHO have designated oxybenzone an endocrine disruptor. Neither have done such a thing, because oxybenzone does not meet the definitions https: www.who.int ceh risks cehemerging2 en . DiNardo s  real kicker is a false flag. The US FDA is currently revising the sunscreen monograph, and in the process requesting data based on new criteria. However, they have continuously asserted that consumers should continue to use sunscreens. u201cOTC sunscreen products will continue to be available on the market for consumer use. FDA communications have been confusing, but they want people to keep using the sunscreens currently on the market. The last paragraph in DiNardo s rant above clearly shows that his viewpoint is misinformed and biased, and not based on science. , 
 I apologize for misspeaking my facts u2026 in the paper, I reference who has identified oxybenzone in the blood and in umbilical chord blood u2026 Huo indeed only identified oxybenzone in the mothers urine and in the fetus. Again, I misspoke about EU and WHO designations - I was thinking of octinoxate that both the EU and WHO classified as endocrine disruptors, both references are below. However, the second reference also identifies benzophenone-3 (oxybenzone) as an endocrine disruptor. Additionally, of the many references listed in the FDA comments there are many scientists who identify oxybenzone as an endocrine disruptor. 1) World Health Organization (2012): State of the science of endocrine disrupting chemicals u2013 2012. An assessment of the state of the science of endocrine disruptors prepared by a group of experts for the United Nations Environment Programme (UNEP) and WHO. Available at http: www.who.int ceh publications endocrine en 2) Evaluation of 22 SIN List 2.0 substances according to the Danish proposal on criteria for endocrine disrupters. Available at http: ec.europa.eu health scientific_committees consumer_safety docs scc_o_9.pdf Lastly, I am neither misinformed nor biased, but rather frustrated and overwhelmed by the amount of data that everyone wants to ignore or make believe is fake science. I do not want anyone to follow my viewpoint, I want people to follow the science u2026 take the time to read the research that is published and if you can come up with a better conclusion good for you. As for my 43+ years of toxicology in the personal care industry, sunscreens are inefficient at best when it comes to preventing skin cancer, they are clearly toxic as evidenced by the number of scientific papers published in multiple species and are the primarily reason why there is a global skin cancer epidemic currently at hand. , 
 DiNardo: You are still wrong on facts. Huo did not check oxybenzone levels in the fetuses. Only a single spot urine sample of the mothers an unspecified amount of time after birth. There s no definitive correlation with the fetus. This simple, willful misunderstanding (or misrepresentation) of the facts is exactly why you should retract your paper. Conflating oxybenzone and octinoxate is another example of uncareful analyses. The WHO report you link does not identify octinoxate (Octyl-methoxycinnamate) as an EDC, but makes uncited reference to examples of potential endocrine active compounds. (Look up risk vs hazard. ) Nevertheless, it has nothing to do with the paper in question. The SIN list is not an EU authority, but an activist NGO. Despite DiNardo throwing various red herrings out, he fails to explain why this paper deserves to be in print. It does not add to the scientific discourse. , 
  clearly the reason , again you are not speaking correctly. to say that sunscreen causes cancer is not true and you have no evidence stick to what you know: cosmetics. I would venture you know nothing about the etiology of melanoma or really what melanoma even is. you could not identify a melanoma from normal tissue. a pubmed search or two and now you are a molecular oncologist? , 
 #6 #6 The Hirschsprungu2019s paper has been peer reviewed in an acceptable scientific journal and identifies all of the referenced material accurately in the paper. Comments to date have been very positive and I hope will stimulate more research in this area that will prove or disprove the reproductive toxicity of oxybenzone u2013 FDA has requested industry to provide such data u2026 perhaps you can conduct a study of your own and add something to the scientific discourse? You started with nasty comments and I unfortunately responded in kind u2026 I was under the impression u201cpubpeeru201d had something to do with open discussion of information and apparently it is nothing more than an open invitation for people to insult you without any regards to the issue at hand. Read all of the papers published on the topic, come to your own conclusion and then do something about it u2026 best of luck! , 
 #7 Another very interesting and insightful comment from someone who must be an oncologist dedicated to helping the 3 to 5 million people each year who get skin cancer just in the US. Perhaps you could provide FDA with an explanation as to why the following groups outlined in the table below have a problem with sunscreens and the prevention of skin cancer u2026 surely, you know the answer. With respect to sunscreens causing cancer, we will let industry answer that question since FDA is asking that exact question of them u2026 or perhaps you can do your own research and add something to the scientific discourse? The issues at hand are simple u2026 oxybenzone and octocrylene (and most likely other benzophenone based chemicals) breakdown and release a known carcinogen called benzophenone (FDA banned it a few months ago in foods, International Agency for Research on Cancer also identified it as a carcinogen and California placed it on Prop 65). Oxybenzone, octinoxate and octocrylene (and probably most sunscreens) have been reported to allow enough UV into the skin after ~1 hour of sun exposure that free radicals develop u2013 this cannot only produce apoptotic cells, but also release caspases which my pubmed searches appear to tell me are involved in the melanoma pathway. And of course there is the issue of sunscreen abuse u2013 u201cuse of sunscreens extend sun exposure ... which increases the risk of melanomau201d. There are several other concerns as well, but please donu2019t believe me just read the research u2026 Best of luck! Agency Reference Findings: 1) American Cancer Society: Cancer Facts 2018: Siegel RL, et al. Cancer statistics, 2018. CA Cancer J Clin., 68(1):7-30.tMelanoma has increased from 8 100,000 to 32 100,000 for men and from 7 100,000 to 21 100,000 for women between 1975 to 2014. 2) FDA Labeling Guidelines: https: www.fda.gov downloads drugs guidancecomplianceregulatoryinformation guidances ucm330696.pdf. Allowable Claims u2013 u201chelps prevent sunburnu201d and u201cif used as directed with other sun protective measures (meaning sun avoidance and protective clothing), decreases the risk of skin cancer and early skin aging caused by the sun.u201d US Health and Human Resources: Guy G.P. Jr., et al. (2015). Prevalence and costs of skin cancer treatment in the U.S., 2002-2006 and 2007-2011. Am J Prev Med., 48(2):183-187.tu201cSkin cancer cases increased from 3.4 million in 2002u20132006 to 4.9 million in 2007u2013 2011.u201d International Agency for Research on Cancer: Vainio H., et al. (2000). An international evaluation of the cancer-preventive potential of sunscreens. 3) Int. J. Cancer., 88, 838u2013842. Autier P. (2009). Sunscreen abuse for intentional sun exposure. Br J Dermatol., 161 Suppl 3:40-45. tu201cNo conclusion can be drawn about the cancer-preventive activity of sunscreens against basal cell carcinoma and melanoma. Use of sunscreens extend sun exposure ... which increases the risk of melanomau201d 4) Division of Dermatology, David Geffen School of Medicine - UCLA: Chesnut C., Kim J. (2012). Is there truly no benefit with sunscreen use and Basal cell carcinoma? J Skin Cancer., 480985tu201cThere has only been one randomized and controlled study to examine sunscreenu2019s role in the prevention of Basal Cell Carcinoma, and no significant protective benefit was found.u201d 5) University of Granada Spain - School of Medicine: Martu00edn Garcu00eda E., et al (2017). Changes in the Incidence of Skin and Lip Cancer Between 1978 and 2007. Actas Dermosifiliogr., 108(4):335-345. tu201cSkin cancer incidence via World Health Organization data from the Cancer Incidence in Five Continents - observed increases in global skin and lip cancers for the period 1978 to 2007.u201d 6) University of Melbourne Australia - Department of Medicine: Fransen M., et al. (2012). Non-melanoma skin cancer in Australia. Med J Aust., 197(10):565-568.tu201cNon-melanoma skin cancers increased from 412,493 in 1997 to 767,347 in 2010 and estimated 938,991 in 2015.u201d 7) Cancer Council of Western Australia: Slevin T. (2013). Sunscreen, skin cancer and the Australian summer. The Conversation: (Retrieved from) http: theconversation.com sunscreen-skin-cancer-and-the-australian-summer-11633. u201cItu2019s important to understand that sunscreen is a useful adjunct to other sun protection measures (sun avoidance and protective clothing). Rather than being our first line of defense, it should be the last.u201d , 
 #9 To anyone on this site that has been reading this slanderous attack, I apologize! Is this what science has come to, the trading of insults? I apologize for not being able to resist being somewhat sarcastic to these two individuals u2026 it is sad to see that some have substituted science with personal opinions and apparently forgotten how to do research. Perhaps they have even been bought into the myopic views of a $10 BILLION industry selling us the Emperoru2019s New Clothes. Industry and many medical professionals have known for over 15 years that the rate of skin cancer has been significantly rising globally (sunscreens are a failed technology). It appears that many have turned a blind eye thinking that what industry claims - u201csunscreens save livesu201d - is true u2026 unfortunately, the data strongly disagrees u2026 please read the research that literally hundreds of scientist from all over the globe have written. I will not have any additional communications on this site, you can look at the paper I wrote and communicate with me directly if you have any other questions. Again, sorry for the inappropriate discussions u2013 perhaps pubpeer could ban such individuals from using this site for their own political purposes, Regards! , 
 #10 I would still appreciate a response to my COI disclosure concern mentioned in #3. , 
 The sarcasm, especially since the real victims are the patients, not me, is sad. That said, you are  cherry-picking the literature and thus confounding cause and effect. Maybe more people are spending time in the sun tanning beds? It certainly is more plausible that UV from the sun, known to damage DNA, is the cause of increased skin cancer rather than sunscreen. Think for a second: people putting on sunscreen are *going out in the sun*. How can you therefore say that the sunscreen causes cancer? Isn t more likely that it s the sun exposure that s really causing it? Another way of explaining this to you: A = B B = C therefore A=C Dogs are animals, cats are animals, therefore dogs are cats. People who spend time in the sun frequently wear sunscreen. People who spend time in the sun are more likely to get skin cancer. Therefore sunscreen increases risk of cancer. Really really try to think critically of all literature. Feynman put it best when he said, and I paraphrase :  Do *everything* you can to disprove your model. As a human with an ego, it s much much easier to ignore confounding evidence. ,