Re-expression Of ARHI(DIRAS3) Induces Autophagy In Breast Cancer Cells And Enhances The Inhibitory Effects Of Paclitaxel On Growth

Breast cancer remains a leading cause of morbidity and mortality in women. ARHI is a Ras-related imprinted gene that inhibits cancer cell growth and motility despite 54-59% homology to Ras protooncogenes. ARHI is downregulated in the majority of breast and ovarian cancers and its loss is associated with progression from DCIS to invasive disease. In ovarian cancer, re-expression of ARHI induces autophagy and leads to autophagic cell death in cell culture, but enables ovarian cancer cells to remain dormant when they were grown in mice as xenografts. Paclitaxel, a naturally occurring anti-microtubule agent that causes mitotic arrest, has significant activity against a variety of tumor types by inducing apoptosis. This research tries to explore the mechanism that combination of autophagy induced by ARHI reexpression and apoptosis induced by paclitaxel functions inhibitory effects on breast cancer in vitro and in vivo. The research will provide theoretical and experimental proof for a new strategy in clinical treatment of breast cancer.Objectives:To examine if ARHI reexpression can induce autophagy in breast cancer cells and evaluate the effects of ARHI re-expression in combination with paclitaxel, and also explore the mechanism in combination of ARHI reexpression with paclitaxel.Materials and methods:(1) Re-expression of ARHI was induced in cell culture by the demethylating agent 5-aza-2'-deoxycytidine (DAC) and the histone deacetylase inhibitor Trichostatin A (TSA). ARHI expressing construct was transfected into cells by lipofectamine in vitro. SYBR-Green realtime RT-PCR and Western blot assay were adopted respectively to detect ARHI mRNA and protein expression. (2) Autophagy was quantitatively determined by acidic vacuole organelle (AVO)-FACS analysis and GFP-LC3 accumulation in vacuoles by fluorescent microscopy. Autophagic marker protein microtubule-associated proteinl light chain 3 (MAP-LC3) cleavage LC3-Ⅱwas detected by Western blot assay. (3) Sulforhodamine B (SRB) assay was used to detect the effect of TSA and paclitaxel on the proliferation of breast cancer cells in vitro. (4) The impact of ARHI re-expression on tumor growth in vivo was evaluated using xenografts of the human breast cancer cell MDA-MB-231 in immunosuppressed nu/nu mice. MDA-MB-231 cells were injected into mammary pad of nude mice to establish xenografts model. When tumor reached 3-5mm in diameter, mice were randomly distributed into several groups and administration began. An ARHI expressing construct encapsulated in liposomes was delivered into xenograft by intra-tumoral injection. Paclitaxel (10mg/kg/day) was injected intravenously. Combination group was treated with both ARHI/liposome and paclitaxel. Control group, vector group and liposome group were also set as the controls. Tumors were measured twice a week and volumes were calculated. At the termination, mice were sacrificed and tumors were taken and stored for transmission electronic mircroscopy (TEM) and mRNA detection respectively. (5) Autophagy, apoptosis and cell cycle analysis determinded with fluorescence activated cell sortor (FACS) after acridine orange staining, Annexin-PI staining and PI staining respectively. TEM was used to observe autophagy and apoptosis in tumor tissues. (6) Data was expressed as mean±standard deviation. Homogeneity of variances test was performed with Bartlett's test and p values<0.1 was considered variances were not equal between groups. Student's t-test and Wilcoxon ranksum test was performed to compare the effect of groups. For more than two groups, ANOVA and LSD method were used for multiple comparisions.Results:(1) Re-expression of ARHI can be achieved by transfection and drugs (TSA or TSA/DAC combination) in vitro and by liposome delivery in vivo. (2) ARHI re-expression induces excessive autophagy in breast cancer cells. (3) Re-expression of ARHI enhances the inhibitory effects of paclitaxel in cell culture and xenografts. (4) Although paclitaxel could not induce autophagy in breast cancer cells, it enhanced ARHI-induced autophagy. Conversely, ARHI re-expression promoted paclitaxel-indcuced apoptosis and cell cycle G2/M arrest. Conversely, ARHI re-expression promoted paclitaxel-indcuced apoptosis and cell cycle G2/M arrest. Both autophagic and apoptosic cells were often seen in combination group by TEM.Conclusions:ARHI can be reactivated by epigenetic modulation in vitro and re-expressed by liposomal delivery in xenografts. ARHI re-expression induces autophagic cell death in breast cancer cells and enhances the inhibitory effects of paclitaxel by promoting autophgy, apoptosis and cell cycle G2/M arrest. In conclusion, the combination of autophagy and apoptosis pathways can enhance cell-killing effects on breast cancer. ARHI could be a promising strategy for breast cancer treatment, which is worthy of more detailed investigation.