Periplogenin Induced Apoptosis Of Colon Cancer Cells And The Underlying Molecular Mechanisms

Colorectal cancer(CRC)is the third leading cause of cancer-associated mortality worldwide.In China,CRC has become the fourth leading cause of death among all malignant tumors.By far,the major therapeutic strategy for CRC is the combination of surgery and chemotherapy.But drug-resistance and the side effects of chemotherapeutic drugs not only reduces the cure rate of cancer,but also brings serious adverse prognosis to patients.Thus,developing new safe and effective anticancer drugs has become an inevitable trend.Natural compounds are the main source of new anti-cancer drugs.Periplogenin(PPG)is a natural component extracted from traditional Chinese herb Cortex Periplocae.At present,there is no report about the anti-tumor mechanism of PPG.In the present study,MTT assay was used to examine the effects of PPG on tumor cells.Then,HCT116 colon cancer cells were chosen to perform the following experiment,and the apoptotic effect of PPG on HCT116 cells and the underlying molecular mechanisms was revealed by TUNEL assay,Flow cytometry,Western blot and RNAi Transfection.The achieved results mainly include the following aspects:(1)The natural compound PPG inhibits a variety of tumor cells viability and triggers colon cancer cell apoptosis.MTT assay was used to examine the effects of PPG on several tumor cell lines including HCT116,Hep G2,Hela,MCF7 and the normal cells line IEC6.The results showed that PPG inhibited the proliferation of HCT116,Hep G2,Hela and MCF7 cells in a time-and dose-dependent manner,and colon cancer HCT116 cells displayed the highest sensitivity to PPG,the IC50 is 10.56 ± 0.28?M.However,the inhibitory effect of PPG on IEC6 cell activity was not obvious,indicating that PPG has low toxicity to normal cells.Flow cytometry and TUNEL assay were used to detect the effects of PPG on HCT116 cell apoptosis.The results indicated that PPG promotes tumorcell apoptosis in a dose-dependent manner.To further determine the apoptosis induced by PPG,Western blot was used to detect the effect of PPG on the expression of apoptosis-related proteins.The results showed that PPG promoted the expression of pro-apoptotic factor Bax,decreased the expression of anti-apoptotic factor Bcl2,and increased the cleavage of caspase-3 as well as PARP.(2)ROS signal was involved in regulation of PPG-evoked apoptosis in the HCT116 cells.To determine whether ROS was involved in PPG-induced cell apoptosis in HCT116 cells,DCFH-DA assay was applied to measure the ROS generation.The results showed that PPG promoted ROS levels in a concentration-dependent manner.After antioxidant N-acetyl-L-cysteine(NAC)treatment,both PPG-induced ROS generation and cell apoptosis was attenuated.Moreover,the cell viability was increased by NAC.We further examined the effect of NAC on cleaved caspase-3 protein expression.The results showed that NAC significantly inhibited the PPG-induced cleavage of caspase-3.Therefore,ROS is involved in the regulation of apoptosis induced by PPG on HCT116 cells.(3)PPG activates ER stress pathway to trigger colon cancer apoptosis.Western blot was used to detect the effect of PPG on the expression of ER stress-related proteins.The results showed that PPG increased the protein levels of Bip,p-e IF2?(Ser51)and CHOP.Simultaneously,PPG also increased the protein levels of IRE1? and p-JNK(T183/Y185),but decreased the protein levels of p-ASK1(Ser966).Importantly,CHOP or JNK si RNA inhibited the PPG-increased cleavage of PARP.Meantime,JNK inhibitor SP600125 also inhibited the PPG-increased cleavage of PARP.The results showed that PPG induced colon cancer cell apoptosis via Bip-e IF2?-CHOP and IRE1?-ASK1-JNK signaling route in the ER stress pathway.(4)(1)ROS is upstream of ER stress in PPG-induced apoptosis.Western blot was used to detect the effect of NAC on the expression ofER stress-related proteins.The results showed that NAC attenuated PPG-increased protein levels of Bip,p-e IF2?(Ser51)and CHOP,inhibited the PPG-increased protein levels of IRE1? and p-JNK(T183/Y185)but increased the PPG-decreased p-ASK1(Ser966).Therefore,PPG activates the ROS-ER stress pathway to evoke colon cancer cell apoptosis.In conclusion,the results suggest that PPG inhibited a variety of tumor cells viability and displayed weak toxicity to normal cells.Moreover,PPG activates ROS-ER stress pathway to induce colon cancer cell apoptosis through the Bip-e IF2?-CHOP and Bip-ASK1-JNK signaling routes.Therefore,hopefully PPG can be developed as a promising candidate of chemotherapeutic drug for the treatment of cancers,at least for colon cancer.