Combinatorial Effect Of Proteasome Inhibitor Bortezomib And Cisplatin On Human Cervical Cancer Cells

The most important risk factor for cervical cancer is infection by HPV(human papillomavirus).Its carcinogenic mechanism is still under investigation while we understand more and more on it.Association and subsequently degradation of the cellular tumor suppressor proteins p53 and Rb by HPV E6 and E7 proteins through the ubiquitin pathway have been considered the major mechanism by which HPV induces cervical cancer.Degradation of the tumor suppressors leads to deregulation of the cell cycle,resulting in accumulation of somatic mutations of oncogenes and tumor suppressors that will ultimately lead to cancer.1.Combinatorial effect of proteasome inhibitor bortezomib and cisplatin on human cervical cancer cellsClinical application of bortezomib in combination with other conventional chemotherapeutic agents in the treatment of multiple myeloma patients has achieved good results.On the other hand,cisplatin is a commonly used non-specific potent anti-cancer drug in the clinics.The goals of our study are:examing the combinatorial effect of bortezomib and cisplatin on the proliferation of cervical cancer HeLa cells,alteration of tumor suppressor protein p53 and Rb levels in HeLa cells,and whetherthese changes can be used to provide a rationale for the treatment of cervical cancer.Objective:To investigate the effects of proteasome inhibitor bortezomib,cisplatin and their combinatorial effect on cervical cancer cells and the underlying mechanisms.Methods:Cell Counting Kit-8(CCK-8)was used to examine HeLa cell proliferation after treatment with different drugs:cisplatin,bortezomib and the combined treatment group.Western blotting was used to investigate the steady-state levels of p53 and Rb in the treated cells.Flow cytometry was used to study DNA fragmentation after drug treatment.Results and Conclusions:Compared with control group,less adherent and a few floating cells were observed in cisplatin or bortezomib treated cells.The inhibitory effect on the proliferation of HeLa cells in the combined treatment group was more significant compared with the other groups.WB analysis showed that,compared with the control group,the level of p53 was higher in the cisplatin group and the combined treatment group,but no obvious difference in the bortezomib group;Compared with other three groups,the level of Rb was higher in bortezomib group,and no obvious change was observed among these three groups.Flow cytometry analysis demonstrated a synergistic effect of bortezomib and cisplatin on HeLa cell apoptosis.Cisplatin may inhibit the proliferation of HeLa cells via regulating the level of p53;Bortezomib may inhibit the proliferation of HeLa cells via regulating the level of Rb;Bortezomib could enhance the inhibitory effects of cisplatin on HeLa cells.2.Screening for proteasome inhibitors on cervical cancer cellsPI-1840 is a l,2,4-oxadiazole compound,which has a high IC50 value for the proteasome ?5 subunit,and our collaborators have used it as a precursor to synthesize a series of derivatives,the potential proteasome inhibitors.Objective:We screened these compounds for identifying ones with IC50s close to oreven lower than that of PI-1840 on cervical cancer cells,providing a rationale for the next round screening.Methods:First,the IC50 of PI-1840 in HeLa cells was determined,and then the concentration was used as the reference to screen other compounds.We used CCK-8 kit for the screen.Results and Conclusions:The results showed that the activity of 003 series compounds was low,while in the 007 and 013 series,some compounds had high HeLa cell inhibitory activity.Therefore,we tentatively concluded that the structure of the 003 series of compounds was not the ideal protein medium inhibitor structure we expect,and the compounds with higher activity in the 007 and 013 series met our expectations and can be further experiments.