Scutellarein inhibits cancer cell metastasis in vitro and attenuates the development of fibrosarcoma in vivo
Authors: Chen Zhang
Yan Zhang
Xiaoqing Liu
Xiujuan Shi
Yu Qiu
Shuzhang Yang
Guangfeng Chen
Xuexun Fang
Xiaoqiang Liu
Journal: International Journal of Molecular Medicine (2015)
Discipline: CLINICAL MEDICINE
Keywords: Fibrosarcoma; Scutellarein; Anticancer potential; Cell proliferation; Apoptosis
Affiliations: Tenth People's Hospital, Tongji University School of Medicine, Shanghai 200072, P.R. China., Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, Changchun, Jilin 130023, P.R. China.

Abstract:  Fibrosarcoma is an aggressive and highly metastatic cancer of the connective tissue, for which effective therapeutic methods are limited. Recently, there has been a renewed interest in small molecular compounds from natural products in the treatment of cancer. In the present study, we investigated the compound, scutellarein, extracted from the perennial herb Scutellaria lateriflora, and it was found to possess anticancer potential. Cell proliferation assay and cell cycle analysis revealed that the proliferation rate of HT1080 human fibrosarcoma cells was significantly suppressed by treatment with scutellarein through the induction of apoptosis. Moreover, an in vivo experiment using Balb/c nude mice revealed that the volume and weight of the tumors were markedly reduced following treatment with scutellarein. We also analyzed the effects of scutellarein on the markers of metastasis, using the HT1080 cells. The results indicated that scutellarein potently inhibited cell migration, invasion and the expression and activity of matrix metalloproteinase (MMP)-2, -9 and -14. Furthermore, MMP activation and cell survival were suppressed due to the scutellarein-mediated downregulation of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) activation. In conclusion, our data suggest that scutellarein has the ability to attenuate the development of fibrosarcoma and inhibit cancer cell metastasis. 

Retracted: No

PubPeer comments:
 Cordycepin is a broad spectrum and cytotoxic inhibitor of many enzymes: https: www.mdpi.com 1420-3049 24 12 2231 htm Due to its structural similarity to adenosine, it was established as an inhibitor of RNA synthesis more than 50 years: https: pubmed.ncbi.nlm.nih.gov 5783871 https: pubmed.ncbi.nlm.nih.gov 5275385 https: www.ncbi.nlm.nih.gov pmc articles PMC3504674 https: www.sciencedirect.com science article abs pii 0306362380900804 Its action on circadian rhythms is has been known for more than 20 years: https: journals.physiology.org doi pdf 10.1152 ajpcell.1996.271.5.C1646 https: www.ncbi.nlm.nih.gov pmc articles PMC3040569 http: www.plantphysiol.org content 113 4 1167 It is rather astonishing that neither the authors nor the reviewers were aware of this and did not check the prior literature. Any effect on circadian rhythms or clock protein complexes is very likely a stress response to transcriptional inhibition. The former was not adequately controlled for, as these only occurred at one circadian phase and did not use drugs that inhibit transcription in a similar manner to cordycepin, the latter was not controlled for at all. Of note, systemic insults reset circadian such as UV treatment of cells have similar effects on circadian rhythms e.g. https: www.ncbi.nlm.nih.gov pubmed 19465696 It is undoubtedly the case that RUVBL-2 proteins interact with CoTP, since they also bind ATP. This does not mean RUVBL-2 proteins are the mechanism by which cordycepin affects circadian rhythms. , 
 We obtained cordycepin as a hit of drastic clock phase-shifter in an unbiased screen, using a chemical library which contains purified natural product compounds (Materials Methods of this paper). Since the antiphasic phenotype of this hit was so striking, and has never been seen in the senior author s previous genome-wide siRNA screen (Zhang, Cell 2009), or reported by other colleagues chemical screens (Hirota, PNAS 2008, PLoS Biol 2010, Science 2012; Isojima, PNAS 2009; Chen, PNAS 2012; He, Cell Metab 2016 etc), we deemed that this phenotype was novel and of highly interest. The questioner is right that cordycepin has been considered as an inhibitor of RNA synthesis. We thus immediately conducted compound tests to evaluate the impact of inhibitors of general transcription to the cellular clock oscillation. As shown in Fig S1C, neither three well-known transcription inhibitors (u03b1-amanatin, Triplotide, Actinomycin D) could generate a drastic phase-shift, except killing the oscillation as a whole in high doses, in this culture condition. Furthermore, if the inhibition of RNA synthesis in general was the mechanism, one would expected to see similar antiphasic effects by the administration of other nucleoside analogs. We demonstrated in the main Fig 1B that, however, neither 3 -Deoxy-guanosine cytidine urdine thymidine nor 2 ,3 -Deoxy-adenosine have a clock-phase phenotype in the regular or 20X higher doses. In fact, we synthesized hundreds of nucleoside analogs in the current (main Fig1C) and follow-up studies, and found only a few adenosine (but not G C U T)-like analogs gave rise to the phase-shift phenotype, and some are clearly not involved in transcriptional inhibitions. That is, adenosine-like structure of the compound is the key for phase-shifting. Regarding the effect of cordycepin in non-mammalian clock systems, we also observed a phase-shift phenotype in plants. Indeed, we included one FigS in our initial submission, reporting that the cca1::luc reporter was phase-shifted in Arabidopsis upon the cordycepin admin. However, one reviewer believed that the above mentioned O Neill et al paper was irrelevant to ours that was focused on the mammalian clockwork, and recommended us to drop the panel. Of note, RUVBL2 is conserved among species and even kingdoms. The questioner is welcome to join us to explore how cordycepin RUVB-like ATPase (At5g67630 in Arabidopsis) regulates the clock phase in plants in the follow-up studies. Every treatment may cause some degree of stress. The question is specificity. Our mass-action kinetics modeling (main Fig 5A), originally developed by Dr. Daniel Forger s group, does not support that a general transcriptional inhibition, nor a time-point-specific inhibition, could generate the drastic phase-shifting phenotype. The model predicted, and subsequently proven by our vigorous experiments in vivo and in vitro (main Fig 5B,C,D), that the time-specific *activation* of E-box genes led to the antiphasic phenotype. This activation, not inhibition, can only be explained by a  de-repression mechanism (Fig S20), in which the circadian megadalton repressive complex is disassembled by the conformation changes from CoTP RUVBL2 interaction. When we scientists talk about experimental results, one should always bear in mind the  quantification . The UV-treatment paper is interesting, but the phenotype was much milder with maximal 6 h phase-shift than ours, which was up to 12 h antiphase. Even for this phenotype, one may also use our  de-repression model to, at least partially, interpret the mechanism. For example, the UV-treatment may introduce chromatin-remodeling which causes the disassembly of circadian repressive complex. Therefore, we encourage the questioner to read carefully about our paper, and practice our model to explain some previous old data, which may give him her some joyful surprises. In searching for the functional target of cordycepin in regulating the clock phase, we conducted a chemical-genetic screen with nearly 2000 genes, coupled with IP-MS for the physical interaction (Fig S7). We confirmed that reduction of RUVBL2 expression would substantially attenuate the phase effect of cordycepin treatment (Fig 3A). With the help of CoTP RUVBL2 co-crystal structure, we were able to identify two variants, D299Q and A85T, of RUVBL2 protein that respond in different degrees to the cordycepin treatment (Fig 4F). Given that RUVBL2 is a component of circadian repressive supercomplex (Fig 6A,B), and the treatment of cordycepin releases it from the bound chromatin of E-box loci along with repressive CRY proteins (Fig 6D,E,F), we believe that we have established our claim that RUVBL2 *IS* the mechanism for cordycepin s clock-phase effect. Last but not the least, cordycepin and adenosine-like analogs have been considered as a wide range of applications in multiple preclinical investigations and clinic trials in treating sleep disorders, depression, diabetes, inflammatory diseases, and cancer (Reviewed in PMID: 28324458; PMID: 28560504). Regarding its potential usage in the clock shifting, we argued in this paper that  the relatively low potency of cordycepin requires high dosing to the animals to obtain the behavioral effect. It is thus desirable to develop analogs for better pharmacokinetic and pharmacodynamic properties, as well as testing in human cells, before going to clinical examinations. ,