VB6,a New Lignan,Induce Apoptosis And Autophagy Via Activating JNK Pathway

[Background]The incidence of malignant tumors increases dramatically in recent years. Because of the invasive properties, these malignant tumors usually affect several organs simultaneously, and therefore causing huge health burdens. At present the treatments for the malignant tumors include surgery, chemotherapy, radiotherapy and biotherapy. Besides, the emerging treatment, molecular targeted therapy and tumor immunotherapy casing the diversification of clinical treatment play a more and more important role, which result in an improved survival of the patients. Still, most malignant tumor patients have a low survival rate and this is greatly due to the resistance of tumor cells to chemotherapy or radiotherapy and of the toxicity of chemotherapy. What is the key issue for solving this bottleneck is to develop high effective drugs with low toxicity?Fructus Viticis Negundo named Huang-Jing-Zi is the ripe fruits of Vitex negundo L. It is commonly used in clinical practice for treating asthma, malaria, gastrohelcosis, chronic tracheitis and premenstrual syndrome in Traditional Chinese Medicine. Evn-50, ethyl acetate extract from vitex negundo seed, is a new mixture of lignan compounds belonging to a new class of neolignan. In contrast to classical lignans, vitexins play its biological function while they are not metabolized to END and ENL. Our group has systemically studied the effects of Evn-50in treating malignant tumors. We found that Evn-50had the anti-tumor effects at a very wide range. In vitro, it can obviously inhibited the proliferation of several cell lines including MCF7、ZR-75-1、 MDA-MB-435S、MDA-MB-231、LNCaP、PC-3、C0C1; in vivo, Evn-50equally hampered the growth of breast cancer, prostate cancer, liver cancer and cervical cancer in nude mice. We further confirmed that this effect was mainly conducted by modulating the ratio of bax/bcl-2. Evn-50could upregulate the expression of bax and downregulate bcl-2at the same time. The change of the two key transcription factors lead to apoptosis of tumor cells. These results suggested that the small natural molecule Evn-50could induce apoptosis of certain malignant tumor cells.VB6,6-hydroxy-4-(4-hydroxy-3-methoxy-phenyl)3-(hydroxymethyl)-7-methoxy-3,4-dihydro-(3R,4S)-2-aldehyde naphthalene, is a small molecule fractioned from Evn-50. Our group will continue the study on VB6. Our major goal is to explore the anti-tumor effects of VB6in vitro and in vivo; we will further elucidate the molecular pathways underlying this effect and we anticipate this will be the sound experimental foundation for discovering new anti-tumor drugs in future.[Objective]To screen sensitive cell lines and study the anti-cancer mechanism of VB6in vitro. To clear signal transduction pathways that VB6induce apoptosis, autophagy of breast cancer cell lines MCF-7and colon cancer cell lines RKO. To investigate the in vivo therapeutic effect of VB6on nude mice bearing implanted RKO tumor.[Methods]1.Inverted microscope observe the cells morphological changes after VB6treatment of MCF-7and RKO cells;2.The antiproliferative effects of VB6on fourteen human cancer cell lines from different tissues were measured with MTT;3.Transmission electron microscopy observe the subcellular structural changes after VB6treatment of MCF-7and RKO cells;4.The percentage of apoptosis cell and the cell cycle distribution were analyzed by flow cytometry using PI staining;5.The protein expression of JNK、p-JNK、c-Jun、p-c-Jum、p-Bcl-2、Bcl-2、Bax、 Caspase-3、PARP、Beclin-1、LC3B in treated cells were detected by Wersten blot; JNK specific inhibitor SP600125against JNK was used to block activation of JNK;6. To observe the anti-tumor effect of VB6in vivo on Balb/c-nu mice loading RKO cells model.[Results]1. VB6induce MCF-7and RKO morphologic changes with a time-and dose-dependent manner under inverted microscope RKO and MCF-7cells showed the typical morphologic changes of cytopathies after treating with VB6, and became rounded and detached from the plate, with a time-and dose-dependent manner under inverted microscope. Those changes were abrogated by the presence of SP600125.2. Sensitivities of different cell lines to VB6were different IC50of human breast cancer MCF-7cell line, human blood cancer K562cell line, human bladder cancer HTB-95637、RT4cell line, human gastric cancer AGSs、MKN-28cell line, human lung cancer H1975cell line, human liver cancer HepG2cell line, human osteosarcoma MG63cell line, human pancreatic cancer Panc-1cell line, human prostate cancer Pc-3cell line, human colon cancer SW620、SW480、RKO cell line were1.46μM,1.32μM,2.8μM,3.92μM,2.34μM,7.08μM,4.24μM,12.37μM,2.01μM,7.62μM,1.71μM,3.36μM,4.2μM,2.85μM, respectively.3. VB6induce MCF-7and RKO exhibitting the typical morphological features of apoptosis and autophagy Transmission electron microscopy observation after MCF-7and RKO cells was treated with5μM VB6for24h revealed typical morphological features of apoptosis like:cell shrinkage, plasma membrane blebbing, condensation of cytoplasm and chromatin, fragmentation of the nuclear and changes in mitochondrial ultrastructure. We also found cells sharing morphological features of apoptosis and autophagy indicating the possibility of coexistence of both Programmed Cell Death types.These cells exhibited the typical characteristics of autophagy-autophagic double-membraned giant autophagolysosomes containing cytoplasmic fragments and preserved organelles like:RER, mitochondria, dense bodies, lysosomes and ribosomes.4. VB6induce MCF-7and RKO apoptosis with a concentration-dependent manner After MCF-7cells were treated by VB6(1,2μM) for24hours, flow cytometry indicated that the apoptosis percentages is (6.15±.1.07)%and (11.06±0.5)%, respectively, compared with the control group (0.77±0.75)%(p<0.001); In RKO cells after treated with VB6(1,5μM) for12hours, the apoptosis rate is (3.53±1.07)%and (6.7±0.5)%compared with the control group (0.77±0.75)%(p<0.001); The presence of SP600125markedly diminished VB6-induced apoptosis. The proportion of apoptotic cells was29.3%in the cells treated with VB6(5μM) alone but gradually decreased with the increase dose of SP600125pretreatment for2h, and only3.21%in the cells treated with VB6plus SP600125(40μM) in RKO cells.5. VB6restrained the cell cycle progression at G2/M phase in a dose-dependent maner towards MCF-7cells. Cell cycle distributions were measured by flow cytometric assay, treatment of MCF-7with0,1,5μM VB6for24hours resulted in G2/M phase accumulation of cells corresponding to5.06%,17.58%, and27.23%, respectively.VB6treatment induced a significant accumulation of cells in the G2/M phase in a dose-dependent manner.6. VB6induce MCF-7and RKO cells apoptosis via increasing Bax/Bcl-2ratio、 cleavaging Caspase-3、PARP and activating JNK pathway The apoptosis of MCF-7and RKO cells induced by VB6ccompanied in increase the Bax/Bcl-2ratio and the cleavage of Caspase-3and PARP. VB6can inhibit proliferation and induce apoptosis of MCF-7and RKO cells effectively in a time and dose-dependent manner. VB6induced antitumor effect and cytotoxic activity is exerted through proapoptotic process, which is mediated by a decreased Bcl-2/Bax ratio and activation of caspase-3and PARP. SP600125markedly blocked the phosphorylation of JNK in a dose-dependent, with a parallel decrease activation of c-Jun and Bcl-2phosphorylation induced by VB6. SP600125pretreatment also reduced VB6-mediated cleavage of caspase-3and poly(ADP-ribose) polymerase (PARP) in RKO cells. VB6-mediated caspase activation were JNK dependent and that JNK activation had a key role in VB6-mediated apoptosis.7. VB6induce MCF-7and RKO cells autophagy via increasing Beclin-1expression with a parallel of LC3B conversion The levels of Beclin-1expression were gradually increased following the VB6treatment with a parallel of LC3B conversion. Autophagy was induced by VB6in the treated cells by not only the conversion of a fraction of LC3-Ⅰ into LC3-Ⅱ but also caused the accumulation of Beclin-1protein.8. In vivo therapeutic effect of VB6on Balb/c-nu mice loading RKO cells. The results showed that VB6can inhibit the RKO cells implanted tumor growth, compared with the PBS group, the difference was significant (p<0.05), but between the different concentration groups of VB6the RKO cells transplanted tumor volume was no significant difference; At the same time, nude body weight of the each VB6concentration group compared with the PBS group was no significant difference, we can say that VB6almost has no toxicity in nude mice.[Conclusion]1. VB6could effectively inhibit proliferation of human origin cancer and sensitivities of different cell lines to VB6were different;2. VB6can induce MCF-7and RKO cells apoptosis in a dose-and time-dependent manner; VB6restrained the cell cycle progression at G2/M phase in a dose-dependent maner towards MCF-7cells;3. VB6induce Autophagy in MCF-7and RKO cells;4. VB6induced antitumor effect and cytotoxic activity is exerted through proapoptotic process, which is mediated by a decreased Bcl-2/Bax ratio and activation of caspase-3and PARP; 5. VB6induce antitumor effect and cytotoxic activity were JNK dependent and that JNK activation had a key role inVB6-mediated apoptosis;6. VB6can inhibit the RKO cells implanted tumor growth in vivo on Balb/c-nu mice.