Honokiol Suppresses Renal Cancer Cells’ Metastasis via Dual-Blocking Epithelial-Mesenchymal Transition and Cancer Stem Cell Properties through Modulating miR-141/ZEB2 Signaling
Authors: Chang An
Qian Wang
Qiaozhen Su
Lei Ma
Dandan Ma
Weidong Li
Hongfeng Liang
Journal: Molecules and Cells (2014)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Renal cell carcinoma; Honokiol; Metastasis; Epithelial-mesenchymal transition; Cancer stem cells
Affiliations: Department of Medical Oncology, Affiliated Cancer Hospital of Guangzhou Medical University, Cancer Center of Guangzhou Medical University (CCGMU)., The Medical Faculty of Jinan University., Department of Neurology, the Second Affiliated Hospital of Guangzhou University of Chinese Medicine., Internal Medicine of Traditional Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou, People's Republic of China.

Abstract:  Renal cell carcinoma (RCC) is associated with a high frequency of metastasis and only few therapies substantially prolong survival. Honokiol, isolated from Magnolia spp. bark, has been shown to exhibit pleiotropic anticancer effects in many cancer types. However, whether honokiol could suppress RCC metastasis has not been fully elucidated. In the present study, we found that honokiol suppressed renal cancer cells' metastasis via dual-blocking epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties. In addition, honokiol inhibited tumor growth in vivo. It was found that honokiol could upregulate miR-141, which targeted ZEB2 and modulated ZEB2 expression. Honokiol reversed EMT and suppressed CSC properties partly through the miR-141/ZEB2 axis. Our study suggested that honokiol may be a suitable therapeutic strategy for RCC treatment. 

Retracted: No

PubPeer comments:
 The authors report (p. 4 of the PDF) that data were collected from 2020-03-01 through 2020-05-31. Several things make me wonder if these dates have been accurately reported. 1. On 2020-02-26 (a Friday), the Spanish Health Ministry reported that 32 cases of COVID-19 had been recorded in Spain, of which 3 were in Catalonia (the autonomous community of which Barcelona is the principal city). Yet by this time the authors had apparently already obtained ethical approval to start a prospective clinical trial of two different treatments (with and without calcifediol) for this new disease. 2. The treatment offered to all patientsu2014described on p. 5 of the PDF as  standard therapy u2014included hydroxychloroquine and azithromycin. I can find no trace of these being discussed in the literature before the Gautret et al. preprint , published on 2020-03-20. 3. The article mentions (p. 8 of the PDF file)  a *prior* [emphasis added] pilot trial in Cordoba in which calcifediol treatment lead to a reduction of more than 50% of ICU admission in hospitalized COVID-19 patients . The accompanying reference is to this article https: www.ncbi.nlm.nih.gov pmc articles PMC7456194 , which was published on 2020-08-29. Even if that article (which does not mention the dates of data collection) took a long time to prepare, it does not seem that it would have been possible to recruit enough patients to that trial, in a much smaller city than Barcelona, in such a way that it could be described as  prior to one that started in 2020-03-01. , 
 In Table 1 the authors present  ethnicity data for their patients. Can they provide some details of how these data were obtained, and what the possible categories were? Does  Asian include people of Turkish, Pakistani, and Japanese origin? Does  African American mean  Black , or were all 10 Black participants actually from (North) America? I have discussed this with a Spanish hospital doctor (from a different region to Catalonia) who informed me that in their hospital they do not collect  ethnicity data, whether via self-report or some kind of observation by staff. I am more familiar with the situation in France and Germany, where I believe that to ask patients or any other research participants for any  ethnicity information, apart perhaps from their citizenship (passport nationality), would be considered rather unethical. ![file] , 
 https: twitter.com fperrywilson status 1360944814271979523 , 
 It seems that the design was, at best, a cluster randomized trial. So the analyses should have taken into account the effect of the ward, but it seems they did not. We don t have the data to run such a model, but this thread shows that it could have quite an impact. https: twitter.com lycraolaoghaire status 1360765704849481731?s=20 , 
 https: twitter.com sTeamTraen status 1361032138116710402 , 
 https: twitter.com GidMK status 1361063430745022467 , 
 There are also some comments in the preprint commenting system: https: papers.ssrn.com sol3 papers.cfm?abstract_id=3771318 , 
 https: twitter.com maureviv status 1361273275385577475 , 
 https: twitter.com sTeamTraen status 1361299968494043141 , 
 There does not appear to be any record of the required registration of this  randomized controlled trial (see extract from p. 5 of the preprint)in the [Spanish national clinical trials database] . If this was an RCT then it should have been registered. If it was not then the claim about it being an RCT should be withdrawn. ![file] https: twitter.com maureviv status 1361273275385577475?s=20 , 
 #4 Dear collegues, Here some comments about the study: Our research group, which besides basic scientists includes internists, endocrinologists, and infectious disease experts, has a large experience in the Vitamin D field associated with both classic bone-health related issues as well as to extraskeletal effects (see pubmed Nogues and Vitamin D in addition to Gu00fcerri-Fernu00e1ndez and vitamin D). Vitamin D has known immunomodulatory effects, and since the beginning of the pandemic we were intrigued by the potential effects of vitamin D baseline levels and supplementation on infection-related outcomes. By March 1st, we had more than 3 COVID-19 patients admitted in our wards (by March 14th the hospital was already saturated and the country entered lock down); perhaps these late-February early March cases were still not officially registered. Formal ethical approval was obtained shortly after the study started although verbal approval from the ethics committee was given at the time it was started while we completed all the bureaucratic process. This is irregular but you have to understand that this happened in the setting of an exceptional epidemiological moment. We were also sure that our intervention would be safe for patients given calcifediolu2019s pharmacodynamics effects and doses employed. In addition, we never say in the article that it is a randomized control trial (RCT) but we consider an open randomized trial, and an observational study. Hospital del Mar has one the most important and well-known Infectious disease departments in Spain. Our experts were in continuous communication with other international experts, and given the situationu00b4s exceptionality, decided to quickly include hydroxychloroquine and azithromycin as part of the standard-of-care protocol for all COVID-19 patients admitted in the hospital during the early phase of the pandemic. These decisions were taken with the available knowledge at that time, albeit they had not been formally studied or published. A previous study, The Cordoba study, was conducted by one of our study co-authors (Dr. Quesada-Gomez) so both studies were started at very similar times. However, given that the Cu00f3rdoba study only included 76 patients, and had a simpler design, it was analyzed and published earlier. In summary, it is not a RCT as we explain in methods, although patients were randomly assigned to treated or non-treated group. About that: This study was performed in the worst moment of the first wave of coronavirus pandemic and patients were allocated in the empty beds available at the admission time regardless patient conditions. All the hospital staff followed the same protocol designed to treat COVID-19 but Calcifediol was randomly assigned to 5 wards and the other 3 wards remained only with the standard protocol without Calcifediol. Both groups, treated and non-treated, are Vitamin D deficient and differences between groups are not clinically relevant in terms of median. Moreover, analysis assessing risk ratio are adjusted by these baseline vitamin D levels and other Covid-19 associated risk factors. The study is totally transparent as we show all data; raw data and adjusted results and all patient characteristics. All comments about RCT are not applicable here since the study does not have a placebo group that it is clearly specified in limitations. It is an observational study as is clearly specified in introduction and methods. Thanks so much for all your interest in this work which is a preprint and I appreciate your constructive comments. , 
 #11 we never say in the article that it is a randomized control trial (RCT) Please look at the snippet that I pasted in #10. It literally says that this was a  randomized controlled trial . What is the reader meant to conclude from this, other than that you are indeed claiming that it was an RCT? If this is not the case, I think you should consider withdrawing the preprint and replacing it with one that is much clearer about the method (and, indeed, the provisional nature of the ethical approval on day 1). , 
 #11 I hope that after making this spectacular discovery you didn t stop administering vitamin D to COVID patients. After all as you note it is quite a harmless treatment. So is COVID-19 mortality in your hospital now still 60% lower than other hospitals in the area? And didn t any of your colleagues noticed this and asked how you were doing it? It just does not make sense to me that you conducted a study with such amazing results and then waited over half a year to tell the world about it. , 
 #11 Dear authors, Thank you for your explanation. This explanation appears to be in direct contradiction with numerous claims made in the manuscript itself. In addition, the statistical analysis and study design do not at all support the claims made. I urge you to quickly retract the pre-print and any publication submissions made, and strongly consider all of these issues. It is crucial that the public receives well-vetted, rigorous, and ethical scientific information, and at this time the pre-print does not serve these purposes. Best regards, Noah Haber , 
 https: twitter.com StuartJRitchie status 1361386414026461196 , 
 https: twitter.com NoahHaber status 1360949846643982337 , 
 Now that the authors have confirmed that randomisation was done by ward, can they explain why their statistical analyses apparently did not include a design effect to take this into account? And in view of their claim that  The study is totally transparent , will the authors make a suitably anonymised version of the full dataset available? , 
 Can the authors please confirm that every patient in the trial, starting on March 1 2020 and ending on May 31 2020, received hydroxychloroquine and azithromycin? Can they also tell us whether that is still the standard treatment protocol for COVID-19 at Hospital de Mar? And is vitamin D used in addition to this for every patient? If the standard treatment has changed, how are we to interpret the results of adding vitamin D to a treatment protocol that is no longer used? Or is the claim that although hydroxychloroquine and azithromycin were useless, vitamin D saved the day? , 
 https: twitter.com sTeamTraen status 1362145352804159494 , 
 It seems that the preprint has been removed from the SSRN site: https: retractionwatch.com 2021 02 19 widely-shared-vitamin-d-covid-19-preprint-removed-from-lancet-server , 
 Subsequent to the Castillo et al 2020 and Nogues et al 2021 trials, the province of Andalucia incorporated calcifediol treatment: into their care homes, their general practitioners and possibly their hospital system for treatment of COVID-19 in November 2020. It is notable that within a matter of a few weeks, from the beginning of December, hospitalisations and deaths fell sharply, reversing the usual annual trend, so that by the beginning of January daily deaths had dropped from around 60 to approximately 10. It appears to have risen since before falling back down. It is unclear exactly how much calcifediol was dispensed, how long this was sustained for and to what extent the recommendations were followed, but something certainly appears to have triggered this dramatic downturn. It could not have been vaccination as the vaccination programme in Andalusia started on 27 December 2020. This intervention and response are perhaps the reasons why the COVID-19 death counts and profile in Spain have been so much better in the 2nd wave (Sept 01 to December 31, 2020) than in the 1st (Feb 01 to June 01). Certainly better than its contiguous neighbours for the second wave For the 2nd wave: Spain experienced a 20% decrease, Portugal a 258% increase, France a 17 % increase and Italy a 16% increase according to Worldometer, the WHO and the Spanish Ministry of Health. ![file] , 
 #21 You do realise that this isn t how any of this works, right? You can t just make a claim that one region may or may have not introduced a treatment under some unclear circumstances and this must be why the country of which that region represents less just under 18% of the population is responsible for an apparent drop in the national figures? For what it s worth, here are the per-region Covid-19 statistics for cases, hospitalisations, ICU admissions, and deaths for the period March-May 2021. As you can see, Andalucia comes in solidly below the midpoint on all measures. Spreadsheet here https: docs.google.com spreadsheets d 1XAKJjXkGcJpYke6dj3rISVFhe0oxdaUUjBGPNTFLALU edit?usp=sharing, data sources https: www.mscbs.gob.es profesionales saludPublica ccayes alertasActual nCov documentos Actualizacion_387_COVID-19.pdf and https: cnecovid.isciii.es covid19 #documentaci%C3%B3n-y-datos ![file] , 
 #20 The study is now published and peer reviewed. Seems pretty strong. https: academic.oup.com jcem advance-article doi 10.1210 clinem dgab405 6294179 , 
 #23 The peer-reviewed article has its own thread on here. https: pubpeer.com publications 226B408B633BA2281793EC6F5A18C9 ,