Mechanism Of Dicerandrol B Inducing Apoptosis Of Human Cervical Carcinoma Cells By Endoplasmic Reticulum Stress-Mitochondrial Damage

OBJECTIVE:The natural compound Dicerandrol B with antitumor activity was screened from six compounds derived from the endophytic fungus Phomopsis,and the human cervical carcinoma HeLa cells were used as the research object.The anti-tumor mechanism of the compound Dicerandrol B was explored from the perspective of apoptosis,which could be induced by the endoplasmic reticulum stress--mitochondrial injury pathway.This experiment provides a theoretical basis for the development of new natural anti-tumor drugs in the future.METHODS:1.MTT method was used to detect the six kinds of compounds’ ability of inhibiting proliferation of human cervical cancer HeLa cells and human gastric cancer MGC803 cells at different times and at different doses.The compounds were HX03,HX05,HX06,TPB3,HTT46 and TPBR1 respectively,which were extracted from endophytic fungus Phomopsis.The Human mammary epithelial cells MCF10A were selected as benign control cells.The best antitumor activity compound HX06,Dicerandrol B,was screened.2.The cell cycle distribution of HeLa cells incubated with Dicerandrol B was detected by flow cytometry.The levels of the cell cycle-associated genes MKi-67 and ubiquitin-binding enzyme E2S(UBE2S/E2-EPF)were detected by real-time fluorescent quantitative PCR to determine the effect of Dicerandrol B on HeLa cell cycle.3.Annexin V-FITC/PI staining flow cytometry was used to detect the percentage of cells that have undergone apoptosis in HeLa cells incubated with Dicerandrol B.The expression level of cleaved-PARP related to apoptosis was detected by Western blot to determine whether Dicerandrol B caused HeLa cells apoptosis.4.Western Blot was used to detect the expression of endoplasmic reticulum stress-related protein Grp78 and ubiquitinated protein in HeLa cells incubated with Dicerandrol B to determine whether Dicerandrol B induced apoptosis in HeLa cells via the ERS pathway.5.The intracellular ROS levels were detected by reactive oxygen sensitive fluorescent probe DCFH-DA combined with anti-oxidant N-acetylcysteine(NAC).At the same time,MTT was used to detect cell survival rate to determine whether Dicerandrol B plus NAC could up-regulate cell survival rate.6.Western Blot and real-time quantitative PCR were used to detect the changes of p62 protein,Keapl protein,nuclear Nrf2 protein,p62 gene,Nrf2 gene and GPX1 gene in HeLa cells incubated with Dicerandrol B for 6 hours,and the expression of nucleus Nrf2 protein and GPX1 gene in HeLa cells incubated with Dicerandrol B for 24 hours.The effect of Dicerandrol B on the p62-Keapl-Nrf2 signaling pathway in HeLa cells was analyzed.7.Western Blot was used to detect the expression levels of mitochondria-related proteins Bcl-2,Bax and CytC,and the change of Bax/Bcl-2 ratio was observed to determine whether mitochondria was damaged and the apoptosis of HeLa cells was induced by this pathway.RESULTS:1.Compared with the other five compounds(codes HX03,HX05,TPB3,HTT46 and TPBR1,respectively),Dicerandrol B(HX06)had strong anti-tumor activity and was weak in inhibiting the proliferation of human benign breast epithelial cells.HeLa cells were incubated with Dicerandrol B for 24h,48h and 96h,and their IC50 values were 7.13μg/mL,3.00μg/mL and 1.84μg/mL,respectively.Dicerandrol B inhibited the proliferation of HeLa cells in a dose-and time-dependent manner.2.The HeLa cells were incubated with Dicerandrol B for 24h.Flow cytometry results showed that the number of G2/M phase HeLa cells increased by 67.24%and S phase HeLa cells decreased by 17.91%in the 3μg/mL group compared with the control group.The number of G2/M phase HeLa cells increased by 71.27%and S phase HeLa cells decreased by 29.08%in the 5μg/mL group.The results of real-time PCR showed that the expression of the cell cycle-related gene MKi-67 was up-regulated and the UBE2S gene was decreased in HeLa cells incubated with Dicerandrol B.These data indicated that HeLa cells underwent a cell cycle G2/M phase block under the action of Dicerandrol B.3.Flow results showed that the percentage of apoptotic cells was 30.9%in the 3μg/mL group,and 45%in the 5μg/mL group in the HeLa cells incubated with Dicerandrol B for 24h.The difference was significant statistically(p<0.01)compared to the control group.At the same time,Dicerandrol B caused the up-regulation of protein cleaved-PARP in HeLa cells,indicating that HeLa cells underwent apoptosis under the action of Dicerandrol B.4.Western Blot results showed that the levels of endoplasmic reticulum stress-related proteins Grp78 and Ub increased in HeLa cells incubated with Dicerandrol B for 12 h,indicating that HeLa cells underwent endoplasmic reticulum stress under the action of Dicerandrol B.5.The HeLa cells were incubated with Dicerandrol B for 24h.Compared with the control group,ROS levels increased significantly in HeLa cells.The ROS levels decreased after combined application of NAC in HeLa cells.MTT results showed that the cell survival rate was higher after Dicerandrol B combined with NAC than that without NAC,indicating that Dicerandrol B caused ROS production in HeLa cells.6.Western Blot and real-time PCR showed that the levels of p62 and Keapl protein were decreased,the level of Nrf2 protein of the nucleus increased,and the expression of Nrf2 and GPX1 genes increased in HeLa cells incubated with Dicerandrol B for 6h,indicating that the p62-Keapl-Nrf2 signaling pathway was activated in HeLa cells,so the expression of the antioxidant enzyme glutathione peroxidase GPX1 gene downstream was increased in order to counteract oxidative stress.The level of Nrf2 protein of the nucleus decreased and the expression of GPX1 gene also decreased in HeLa cells incubated with Dicerandrol B,indicating that the activation of p62-Keapl-Nrf2 signaling pathway was weakened with prolonging the action time of Dicerandrol B.7.Western Blot showed that the ratio of Bax/Bcl-2 increased significantly in HeLa cells incubated with Dicerandrol B for 24h,and the expression of cytochrome C of the cytoplasm was up-regulated.This indicated that HeLa cells underwent mitochondrial damage and cytochrome C was released into the cytosol under the action of Dicerandrol B.Dicerandrol B induced apoptosis by the mitochondrial pathway.CONCLUSIONS AND INNOVATIONS:1.Dicerandrol B with strong ability to inhibit the proliferation of HeLa cells is screened from endophytic fungus Phomopsis metabolites.2.The mechanisms by which Dicerandrol B inhibites HeLa cell proliferation were identified.On the one hand,it causes the cell cycle arrest of HeLa cells in the G2/M phase;on the other hand,it induces endoplasmic reticulum stress-mitochondrial damage-mediated apoptosis in HeLa cells.3.The regulation of p62-Keapl-Nrf2 signaling pathway under the action of Dicerandrol B is clarified.The innovation of this study is that the mechanism of Dicerandrol B inhibiting the proliferation of human cervical cancer HeLa cells is identified.