Abstract
Bax and Bak are regarded as key mediators for cytochrome c (Cyt c) release and apoptosis. Loss of Bax or Bak is often reported in human cancers and renders resistance of these cancerous cells to chemotherapy. Here, we investigated that phenylarsine oxide (PAO) could induce Bax/Bak-independent apoptosis. Annexin V/propidium iodide (PI) staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, and caspase activation assays were conducted to detect apoptosis in Bax/Bak-deficient mouse embryonic fibroblasts (MEF) and HCT116 bax(-/-) colorectal cancer cells. Cyt c release and Bim expression were assessed by Western blotting and immunostaining. Bim was stably knocked down by short hairpin RNA. Immunoprecipitation was applied to detect the interaction between Bim and Bcl-2. Both subcutaneous and colorectal orthotopic tumor implantation models were used in nude mice to investigate the effect of PAO in vivo. PAO triggered Cyt c release and apoptosis in a Bax/Bak-independent manner. Bim and Bcl-2 were both involved in this process. PAO augmented the expression of Bim and strengthened the interaction between Bim and Bcl-2. Furthermore, PAO attenuated the growth of Bax-deficient cancer cells in vivo. Our results showed that PAO induced apoptosis in chemotherapy-resistant cancer cells, which suggests that PAO has the potential to serve as a chemotherapeutic agent for Bax- and Bak-deficient cancers.
Highlights
Most anticancer drugs exert their effects through the induction of intrinsic apoptosis via mitochondrial pathway characterized by the release of cytochrome c (Cyt c) and caspase activation [1]
phenylarsine oxide (PAO) attenuated the growth of Bax-deficient cancer cells in vivo
Our results showed that PAO induced apoptosis in chemotherapy-resistant cancer cells, which suggests that PAO has the potential to serve as a chemotherapeutic agent for Bax- and Bak-deficient cancers
Summary
We screened a number of compounds for their capacity to induce cell death in SV40 T antigen–immortalized DKO MEFs by MTT assay and found that PAO induced a significant reduction of viability in these cells (data not shown). Annexin V/PI double staining assay showed that PAO-induced apoptotic cells were decreased significantly in the absence of Bim, regardless of the presence of Bax/Bak (Fig. 4C) All these data revealed that Bim was involved in and initiated PAO-induced apoptosis in DKO MEFs. It has been reported that Bim could suppress Bcl-2 which is a primary inhibitor of apoptosis and constitutively localizes on outer mitochondrial membrane [2]. Fluorescence microscopy images showed an increase of apoptotic cells in tumors from the group treated with PAO, compared with those from control group (Fig. 5E) Both subcutaneous and orthotopic xenograft tumor implantation models were used here to test the effects of PAO on the growth of Bax-deficient cancer cells. PAO-induced apoptotic cell death was confirmed by TUNEL staining (Fig. 6I), and PAO-induced apoptosis was not detected with TUNEL assay in normal colorectal tissues (Supplementary Fig. S3)
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