Valproic Acid Enhances Sensitivity Of Triple-Negative Breast Cancer Cells To Paclitaxel

Part one:Valproic Acid induces the apoptosis of Triple-Negative Breast Cancer cells and its underlying mechanismObjective:Valproic acid(VPA),a broad-spectrum anti-seizure drugs,also defined as one of the histone deacetylase inhibitors,was showed to have anti-cancer ability by many studies in recent years but the underlying mechanism of which has not been fully elucidated yet.Currently,the Triple-negative breast cancer(TNBC)remains incurable due to lacking of efficient therapy.In our present study,we focused on exploring the potential therapeutic effect of VPA against TNBC as well as its underlying molecular mechanism.Methods:1?Different doses of VPA(0?0.125?0.25?0.5?1?2 mmol/L)were used to treat the human TNBC cell lines MDA-MB-231 and BT20 for 72 hours.MTS assay was carried out to analysze the survival rates of the treated cells.The cell survival curves were plotted for comparative analysis by using untreated cells' survivals rates as control.2?Different doses of VPA(0?0.125?0.25?0.5?1?2 mmol/L)were used to treat the human TNBC cell lines MDA-MB-231 and BT20.Clonogenic assay was carried out to analyze the effects of VPA on clone formation of TNBC cells.3?The same dose of VPA(0.5 mmol/L)was used to treat the human TNBC cell lines MDA-MB-231 and BT20 for 24h or 48h.Then western blotting analysis was used to detect the induced cleavage of PARP.The density of the corresponding bands was measured quantitatively using image analysis software and corrected by reference to the value of p-actin.4?The same dose of VPA(0.5 mmol/L)was used to terat the human TNBC cell lines MDA-MB-231 and BT20 for 24h or 48h.Then western blotting analysis was used to detect the protein expression levels of key proteins in related cell signaling pathways,as well as the phosphorylation levels of key components.The density of the corresponding bands was measured quantitatively using image analysis software and corrected by reference to the value of ?-actin.5?Different dosesof VPA(0?0.25?0.5?1mmol/L)were used to terat the human TNBC cell lines MDA-MB-231 and BT20 for 24 hours.Then western blotting analysis was used to detect the protein expression levels of key proteins in related cell signaling pathways,as well as the phosphorylation levels of key components.The density of the corresponding bands was measured quantitatively using image analysis software and corrected by reference to the value of ?-actin.6?Both regular RT-PCR and quantitative RT-PCR were carried out to analyze the mRNA levels of EGFR and Survivin in TNBC cells untreated or treated with VPA(0.5 mmol/L)for 24h or 48h.The relative expression levels of mRNAs were plotted for comparative analysis.Results:1?The MTS assay showed that as compared with the untreated cells,the survival rates of both human TNBC cell lines MDA-MB-231 and BT20 were gradually decreased with the increase of dose of VPA.There was significant difference between groups(P<0.05).The IC50(half maximal inhibitory concentration)of VPA to MDA-MB-231 and BT20 was 0.4137 mmol/L and 0.8262 mmol/L,respectively.2?Our clonogenic assay showed that the cell growth/proliferation of both human TNBC cell lines MDA-MB-231 and BT20 were significantly inhibited with the increase of dose of VPA.There was significant difference between groups(P<0.05).3?Our western blotting results showed that treatment of cells with VPA(0.5 mmol/L)could induce significant apoptosis in both MDA-MB-231 and BT20,as indicated by increased degradation of PARP(C-PARP)as compared with untreated control.As for MDA-MB-231,the induced apoptosis was significantly enhanced with the prolongation of VPA's treatment.4?Our western blotting results showed that treatment with VPA significantly induced down-regulation of expression of EGFR in both MDA-MB-231 and BT20.The downstream PI3K/Akt signaling pathway was also significantly inhibited,as indicated by decreased p-Akt levels in VPA-treated cells comparing with that of untreated cells.Both the effects of down-regulation of EGFR and inhibition of PI3K/Akt signaling pathway by VPA were showed to be both in a time-and dose-dependent manners.Besides,the gradually enhanced inhibition of ERK was also showed in BT20 with the increased dose of VPA or prolonged treatment of VPA.5?Interestingly,downregulated expression of Survivin in both MDA-MB-231 and BT20 by VPA in a time-and dose-dependent manner,a member of IAPs(inhibitor of apoptosis proteins),was also showed by our western blotting analysis.6?Both regular RT-PCR and quantitative RT-PCR results showed that the mRNA levels of both EGFR and Survivin in MDA-MB-231 and BT20 treated with VPA remais much the same as in untreated control.These results indicated that downregulation of EGFR and Survivin by VPA may be via a transcription-independent mechanism.Conclusion:VPA can inhibit the growth/proliferation as well as induce apoptosis of human TNBC cells in vitro in a time-and dose-dependent manner.The possible molecular mechanism may be that VPA could down-regulate expression of both EGFR and Survivin via a transcription-independent mechanism in TNBC cells.Downregulation of EGFR leads to inhibition of downstream PI3K/Akt and/or ERK signaling pathways,while decreased expression of Survivin directly sensitizes TNBC cells to induced apoptosis.Part two:Valproic acid enhances the sensitivity of TNBC cells to paclitaxel induced apoptosis and its underlying mechanismObjective:Paclitaxel(or taxol)is the first-line anti-cancer drug for TNBC treatmemt.However,primary or acquired resistance to paclitaxel occurs frequently in patients with TNBC,which being one of the major causes of treatment failure.Therefore,it is of great significance to develop new drug that can enhance the sensitivity of cancer cell to paclitaxel.Based on the new findings in our previous study in part one,we focus on exploring the possible synergistic effect as well as the underlying molecular mechanism of VPA and paclitaxel on the treatment of TNBC in vitro.Methods:1?Different doses of paclitaxel(0?2?4?8?16?32 nmol/L)alone or different doses of paclitaxel in combination with VPA(0.25 mmol/L)were used to treat the human TNBC cell MDA-MB-231 for 72 hours.And then MTS assay was carried out to analysze the survival rates of the cells.The cell survival curves were plotted for comparative analysis by using untreated cells' survivals rates as control.2?MDA-MB-231 were treated with VPA alone,Paclitaxel alone,or VPA in combination with Paclitaxel.The western blotting analysis was used to detect the induced cleavage of PARP.The density of the corresponding bands was measured quantitatively using image analysis software and corrected by reference to the value of ?-actin.3?Technology of molecular cloning was employed to construct the lentiviral expression vector of EGFR shRNA After identification,the successful recombinant lentiviral vector pLKO.1-EGFR shRNA and the gene knockdown control vector pLKO.1-SHCOO2 were transfected into the HEK293T cells with the packing plasmids psPAX2 and pMD2.G to produce the lentiviruses.The supernatant with lentiviruses was harvested stored at-80? for further use.4?Lentivirus infection and puromycin screening were used to knockdown expression of EGFR in human TNBC cell lines MDA-MB-231 and BT20.The knockdown effect was detect by western blotting analysis.5?Different doses of paclitaxel(0?2?4?8?16?32 nmol/L)were used to treat the human TNBC cell lines MDA-MB-231 and BT20 with or without EGFR-knockdown.And then MTS assay was carried out to analysze the survival rates of the cells.The cell survival curves were plotted for comparative analysis by using untreated cells' survivals rates as control.6?Paclitaxel(2.5 nmol/L)was used to treat the human TNBC cell lines MDA-MB-231 and BT20 with or without EGFR-knockdown.Clonogenic assay was carried out to analyze the effects of paclitaxel on clone formation of TNBC cells with or without EGFR-knockdown.7?MDA-MB-231 with or without EGFR-knockdown were treated with VPA alone or paclitaxel alone and western blotting analysis was used to detect the induced cleavage of PARP.The density of the corresponding bands was measured quantitatively using image analysis software and corrected by reference to the value of ?-actin.Results:1?The MTS assay showed that as compared with the cells with paclitaxel treatment alone,paclitaxel in combination with VPA treatment resulted in significantly decreased survival rates of the human TNBC cell line MDA-MB-231(P<0.05).Meanwhile,western blotting results also demonstrated that as compared with untreatment control,VPA treatment alone,or paclitaxel treatment alone,paclitaxel in combination with VPA treatment significantly enhanced apoptosis of MDA-MB-231,as indicated by increased degradation of PARP.Taken together,these results suggested that VPA synergistically enhance the anti-cancer activity of paclitaxel against TNBC in vitro.2?Restriction analysis of the recombinatant plasmid showed that EGFR knockdown lentiviral vector pLKO.1-EGFR shRNA was successfully constructed.The EGFR knockdown effect of our self-prepared lentiviral system in TNBC cells was verified by the result of our western blotting analysis.3?Both the results of MTS assay and the clonogenic assay showed that knockdown of EGFR could sensitize the TNBC cell lines MDA-MB-231 and BT20 to paclitaxel.Our western blotting results demonstrated that knockdown of EGFR could also enhanced the paclitaxel-induced apoptosis in TNBC cell MDA-MB-231,as indicated by increased degradation of PARP(C-PARP)as compared with the EGFR-unknockdown control.Collectively,our current results suggested that VPA may synergistically enhance the sensitivity of TNBC cells to paclitaxel via specific down-regulation of EGFR expression.Conclusion:VPA can synergistically enhance the sensitivity of TNBC cells to paclitaxel in vitro.The possible molecular mechanism may be that VPA could specifically down-regulate the expression of EGFR in TNBC cells,which in turn results in inhibition of downstream PI3K/Akt and/or ERK signaling pathways and sensitizes TNBC cells to paclitaxel-induced apoptosis.Taken together,our current study offers a theoretic basis as well as experimental data for VPA-based of new therapy for TNBC.