The Mechanism Of Marchantin M Induce Drug-resistant Prostate Cell To Premature Senescence And Regulate Senescent Secretion

Prostate cancer is the most common malignancy in men.In recent years,the incidence rate in China has been increasing rapidly.Most of the patients diagnosed with advanced and progressive patients.Its mortality rate is the second highest among Asian countries,after Vietnam.PCa is initially androgen-dependent cancer.Therefore,androgen deprivation therapy has become the standard first-line treatment for advanced patients.Unfortunately,the development of metastatic castration resistant prostate cancer(mCRPC)has been observed in two years after androgen deprivation therapy,which is distant metastasis,high mortality.At present,chemotherapy,radiotherapy and immunotherapy are the main treatments.Docetaxel-based chemotherapy in the only one which has been verified that can be appropriately extended life time of mCRPC patients.However,50%of mCRPC patients have no response to docetaxel and produce strong toxic effects.Patients who sensitive to Docetaxel easy to produce multi-drug resistance.At present,to Docetaxel resistant prostate the lack of effective treatments in clinical.Based on high expression of" Drug pump" protein(which will pump out drugs,completely different in structures)P-gp,which is one of the mechanism of Docetaxel resistance.Cabazitaxel is a second line drug,Low affinity to P-gp.There,however,is not treatment to other drug resistance mechanism.Due to tumor cells is infinite proliferation,senescent cells can't proliferate,inducing tumor cells to senescent(Prosenescence therapy)is recognized as a tumor suppressor mechanism in recent years.Cell senescence Cellular senescence is a process in which cells cease dividing and undergo distinctive alterations,such as an enlarged,flattened morphology,metabolism activation,cycle arrest and senescence-associated ?-galactosidase(SA?-gal)activity,is a irreversible life process.The advantage of prosenescence therapy:lower drug will induce premature senescence to inhibit cell proliferating,control tumor proliferation,potentially limit treatment related side effects,extend the survival rate,etc.However,senescent cells along with sene scence-associated secretory phenotype(SASP).The SASP entails the secretion of numerous inflammatory cytokines,growth factors and proteases,has an paracrine effect in neighboring cells and micro environment.In one way,SASP will attract and active immune cells,which can remove nearby senescent cells,suppress cancer.In an another way,the secretion of inflammatory cytokines and matrix metalloproteinases will lead to nearby tissue cell proliferation,angiogenesis,invasion and metastasis,has a role to promote the development of tumor.So,taking advantage of inducing cancer cells senescent and reducing its effect on promoting cancer by regulating senescent secretion play an important role in presenescent therapy.Marchantin M is new naturally bisbibenzyl and small molecule compound isolated from liverwort plants.We have previously identified Marchantin M is a research compound exert antitumor,anti-inflammatory and low toxicity effects.Whether Marchantin M can regulate senescence-associated secretory phenotype and exert antitumor effects by its anti-inflammatory.In our study,we induce tumor cell senescent in 1?M Marchantin M and analyzes its effect on senescence-associated secretory phenotype.The results indentified that 1?M Marchantin M significantly induced cancer cell senescent and reduce the secretion of SASP and has a synergistic effect with Doxorubicin,to low it toxicity.Part I Marchantin M induce drug-resistant prostate cell to premature senescence1.1?M Marchantin M induced many drugs-resistant cells senesce1.1 Drug-resistant cells are sensitive to Marchantin M:Preliminary screening result indicated that Marchantin M inhibit many cancer cells proliferating,the IC50 is 10?M.Drug-resisitant cells are more sensitive to Marchantin M,significantly inhibiting drug-resistant cells proliferating,than sensitive ones.1.2 Low dose Marchantin M induce drug-resisitant cells senescent:After treated with 1?M or 2?M Mar M 3 days,multi-resistant prostate cancer cells(PC3/DOC)undergo senescent phenotype,enlarging and flatting.After 5 days,cells maintained an enlarged,flattened morphology and ceased dividing,80%of cells are positive for SA-?-gal activity.PI staining results indicated that senescent cells were mainly blocked during S period owing to overexpression of p21 cycle inhibitor.Meantime,2 ?M induce many drugs-resistant cells senesce,including 460/RT,EC109/CDDP,RM1/DOC.EDU incorporation results indicated that Mar M inhibited proliferating of PC3/DOC,has no effect on,however,the proliferation of normal prostate cells(RWPE1)and normal human fibroblast(NHF).2.Mar M mediated inhibition of proteasome activity induces induce docetaxel-resistant prostate cancer cells to premature senescence.Western blotting results indicated that DNA-damaged observed in PC3/DOC,treated with Mar M 5 days.Proteasome activity analysis display Mar M inhibited proteasome activity.A combined application of Mar M and an decreasing in the activity level of proteasome achieved suppressing cell senesce,suggesting that Mar M mediated inhibition of proteasome activity induces induce multi-resistant prostate cancer cells to premature senescence.Part II Marchantin M regulate senescence-associated secretory phenotype1.Marchantin M suppress senescent cells expressing SASP1.1The analysis of quantitative protein chipWe collect of supernatant of senescent cells to analyze of quantitative protein chip.The results indicated that Mar M suppressed expression of IL1?,IL1?,IL6 secreted by senesce cells,lower than doxorubicin,in a time-dependent manner.1.2The analysis of RT-PCR:In order to further explore Marchantin M regulate SASP,we take RT-PCR to Marchantin M treatment with PC3/DOC.The results indicated that Marchantin M suppressed expression of IL1?,IL1?,IL62.Marchantin M reduced the effect on promoting proliferation of SASP senescent cells:we take supernatant of senescent cells,according to the different proportion of added to RWPE1 cells and PC3 cells.After 24 hours,analysis of cell proliferation.Determined by MTT results suggesting that the supernatant of Marchantin M induction of cell aging cells will not promote cancer cell proliferation.The supernatant of doxorubicin can obviously promote cell proliferation.3.Marchantin M decrease TFEB?TFE3 and p-p65 detectable in the nuclear fraction.3.1 As a result of NF-kB,TFEB,TFE3 are important transcription factors regulating SASP,so we first analyze whether Marchantin M through these transcription factors regulating the expression of SASP.Western blotting and Immunofluorescence results indicated that Mar M exerts inhibitory effect on TFEB.TFE3 and p65 in a time-dependent manner.Mar M markedly reduced thapsigargin or starvation-induced TFEB.TFE3 nuclear localization in PC3/DOC.3.2Mar M inhibited NLRP3 inflammasome activationELISA results indicated that Mar M not only down-regulates the expression of IL1? but also inhibits the expression of p65 overexpress-stimulated IL1?.Western blotting results indicated that Mar M inhibited caspase-1 cleavage and subsequent IL1? secretion.So we concluded that Mar M inhibited NLRP3 inflammasome activation.Part? There was a synergistic effect between Marchantin M and Doxorubicin.1.Through the analysis of the drug combination software,it was found that there was a synergistic effect between MarM and Doxorubicin.2.Low doses of Marchantin M can reduce the side effects of Doxorubicin.RT-PCR results show that low doses of Marchantin M could reverse in Doxorubicin induce a variety of inflammatory cytokines secretion.PART IV Conclusions and Innovation1.Conclusions1.1.Drug-resistant prostate cells are resistant to Docetaxel and Doxorubicin and sensitive to Bisbizenzyls compound Marchantin M.Which indicates.that Marchantin M reverse drug resistance.1.2.Low dose of Marchantin M induced drug-resistant prostate cells senescent and other drug-resistant cells including paclitaxel-resistant lung cancer cells.cisplatin-resistant esophageal cancer cells.and Docetaxel-resistant prostate cells from murine1.3.Low doses of Marchantin M inhibits prostate cancer resistant cells in S phase,induction of DNA damage,and p21 signals play an important role in induction of the senescent process,and the role of p16 and p27 was not significant.1.4.Low dose of Marchantin M inhibited NLRP3 inflammasome activation,further to induce DNA lesions,promote cells senescent.1.5.Compared with senescence induced by Doxorubicin,Marchantin M significantly reduced the secretion of proinflammatory,can significantly inhibit the inflammatory corpuscle NLRP3 activity,inhibiting secretion of SASP.at the same time reduce the SASP effect on normal cells,and other tumor cell proliferation.1.6.Marchantin M decrease TFEB,TFE3 and p-p65 detectable in the nuclear fraction to reduce secreting proinflammatory.In particular by inhibiting NLRP3 activity,significantly reduce the mature and secretion of IL1??1.7.There was a synergistic effect between MarM and Doxorubicin.Marchantin M can reduce the side effects of Doxorubicin and contributes to the sensitization of prostate cancer cells to Doxorubicin.2.Innovation and defects2.1 We firstly reported multi-resistant prostate cancer cells are sensitive to Marchantin M.Low dose of Marchantin M induced drug-resistant cells to cell cycle block,DNA lesion,cell senescence.However,the mechanism need to be further studied.2.2 We firstly reported that anti-inflammatory of Marchantin M is relevant with inhibition of transcription factors TFEB.TFE3 and activation of NF-?B.Marchantin M inhibited proteasome activity,But the role of accurate target is needed by synthesis with the tag to Marchantin M to Omics analysis and make sure the association of proteasome inhibition with transcription factors inhibiton.