Re-expression of ARHI (DIRAS3) induces autophagy in breast cancer cells and enhances the inhibitory effect of paclitaxel

Abstract

Background: ARHI is a Ras-related imprinted gene that inhibits cancer cell growth and motility. ARHI is downregulated in the majority of breast cancers, and loss of its expression is associated with its progression from ductal carcinoma in situ (DCIS) to invasive disease. In ovarian cancer, re-expression of ARHI induces autophagy and leads to autophagic death in cell culture; however, ARHI re-expression enables ovarian cancer cells to remain dormant when they are grown in mice as xenografts. The purpose of this study is to examine whether ARHI induces autophagy in breast cancer cells and to evaluate the effects of ARHI gene re-expression in combination with paclitaxel.

Methods: Re-expression of ARHI was achieved by transfection, by treatment with trichostatin A (TSA) or by a combination of TSA and 5-aza-2'-deoxycytidine (DAC) in breast cancer cell cultures and by liposomal delivery of ARHI in breast tumor xenografts.

Results: ARHI re-expression induces autophagy in breast cancer cells, and ARHI is essential for the induction of autophagy. When ARHI was re-expressed in breast cancer cells treated with paclitaxel, the growth inhibitory effect of paclitaxel was enhanced in both the cell culture and the xenografts. Although paclitaxel alone did not induce autophagy in breast cancer cells, it enhanced ARHI-induced autophagy. Conversely, ARHI re-expression promoted paclitaxel-induced apoptosis and G2/M cell cycle arrest.

Conclusions: ARHI re-expression induces autophagic cell death in breast cancer cells and enhances the inhibitory effects of paclitaxel by promoting autophagy, apoptosis, and G2/M cell cycle arrest.

Figures

Figure 1

ARHI re-expression in breast cancer cells. (A) SKBr3 cells were transfected with pcDNA3-ARHI or pcDNA3 vector constructs. Lipofectamine alone was used as a negative control. The ARHI expression was detected by western blot. (B) MDA-MB-231 cells and (C) SKBr3 cells were treated with DAC, TSA or a combination of DAC and TSA. The untreated cells and cells treated with DMSO diluent were used as negative controls. The ARHI mRNA was detected by real-time quantitative RT-PCR and is presented as the fold change compared to the negative control. *P < 0.05 or ***P < 0.001 compared to the DMSO diluent controls. The data were obtained from three independent experiments. (D) ARHI expression in MDA-MB-231 xenografts was detected by real-time quantitative RT-PCR. It is presented as the fold change compared to the PBS control. Each group included xenografts from two mice, and the data were replicated in three independent experiments. **P < 0.01 compared to pcDNA3 vector/liposomes control.

Figure 2

ARHI induces autophagy in breast cancer cells. (A) The detection of LC3 punctae. SKBr3 cells were co-transfected with GFP-LC3 and ARHI or pcDNA3 vector. The cells transfected with GFP-LC3 alone and treated with rapamycin served as a positive control. The cells were observed by fluorescence microscopy. The white arrows indicate LC3 punctae. (B) The autophagosomes were detected by TEM in xenografts after treatment with ARHI-liposomes (N, nucleus; L, lysosome). The white arrows indicate autophagosomes. (C) ARHI re-expression increased autophagy induction. SKBr3 cells were transfected with ARHI expression vector (left), MDA-MB-231 cells were transfected with ARHI expression vector (middle) or SKBr3 cells were treated with TSA (right), and flow cytometry-AVO analysis was performed to examine autophagy induction. The data were obtained from three independent experiments. (D) LC3 conversion and signaling. (Left) SKBr3 cells were stably transfected with GFP-LC3 and subsequently transfected with ARHI or pcDNA3 vector constructs. LC3-I and LC3-II were detected by western blot. (Right) SKBr3 cells were transfected with ARHI expression vector. The pcDNA3 vector was used as the negative control. The pAKT, AKT, pmTOR, mTOR and ARHI proteins were detected by western blot.

Figure 3

ARHI siRNA blocks TSA-mediated autophagy induction. (A) ARHI expression was knocking-down by siRNA. SKBr3 cells were treated with TSA and siRNA-ARHI (siARHI). siRNA-control (siControl) was used as the negative control. ARHI expression was detected by real-time quantitative RT-PCR. (B) TSA-mediated autophagy was blocked by ARHI siRNA. SKBr3 cells were treated with siControl or siARHI only, TSA+siControl or TSA+siARHI. Autophagy was detected by Flow cytometry-AVO analysis. The experiments were repeated three times, *p < 0.05, comparing with TSA/siControl. (C) One representive case of Flow cytometry-AVO analysis from (B). (D) Confocal fluorescent imaging of SKBr3 cells. The cells were co-transfected with a GFP-LC3 construct, and treated with siControl or siARHI only, TSA+siControl or TSA+siARHI.

Figure 4

ARHI re-expression enhances the inhibitory effects of paclitaxel in vitro and in vivo. (A) SKBr3 cells were treated with paclitaxel (P), TSA (4-15 nM), or a combination of both drugs. The inhibitory rate was detected by SRB assays after three days of culture. **P < 0.01, ***P < 0.001 compared to paclitaxel alone. (B) MDA-MB-231 xenografts were treated with liposomes, pcDNA3 vector/liposomes or ARHI/liposomes. PBS was used as a control. *P < 0.05 compared to control group. (C) MDA-MB-231 xenografts were treated with paclitaxel, ARHI-liposomes or a combination of paclitaxel and ARHI-liposomes. PBS was used as a control. *P < 0.05 compared to control group.

Figure 5

Autophagy and apoptosis are enhanced by the combination of ARHI re-expression and paclitaxel. ARHI-induced autophagy is increased by the addition of paclitaxel in SKBr3 (A) and MDA-MB-231 (B) cells. The cells were transfected with pcDNA3-ARHI or pcDNA3 vector constructs. Cells treated with lipofectamine only or DMSO diluent served as negative controls. After 24 hours, paclitaxel was added. Flow cytometry-AVO analysis was performed after 48 hours. *P < 0.05 compared to control. The data were obtained from three independent experiments. (C) Paclitaxel-induced apoptosis is increased by ARHI re-expression. MDA-MB-231 cells or SKBr3 cells were treated with paclitaxel, DAC, TSA, or a combination. The rate of apoptosis was analyzed by annexin-flow cytometry after 48 hours. **P < 0.01 compared to control. The data were obtained from three independent experiments. (D) Both apoptotic cells (left) and autophagic cells (right) were detected by TEM in xenograft tumors that received paclitaxel/ARHI combination treatment.

Figure 6

G2/M cell cycle arrest rate is increased in cells treated with a combination of TSA/DAC and paclitaxel. (A) MDA-MB-231 cells were treated with paclitaxel, DAC, TSA, or a combination. The cell cycle status was detected by PI staining and flow cytometry analysis 48 hours after treatment. The data were obtained from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control. (B) Flow cytometry data from one representative experiment.