Pannexin1 inhibits autophagy of cisplatin-resistant testicular cancer cells by mediating ATP release.

Pannexin1 (Panx-1) is a gap junction channel protein that mediates the release of intracellular ATP during autophagy, and thus plays an important role in tumor cell apoptosis and chemo-resistance. However, the role of Panx-1 in cisplatin-resistance of testicular cancer cells remains unclear. We found that cisplatin-resistant I-10 testicular cancer cell lines (I-10/CDDP) autophagy-associated proteins (p62, p-mTOR, mTOR and LC3) exhibited high levels of autophagy in their expression, while LC3-II expression was more significantly in the presence of lysosomal degradation blocked by chloroquine (CQ). Xenograft models using I-10/CDDP cells with knockdown ATG5 and ATG7 were established in mouse models and showed blockade of autophagic flux and inhibition of tumor growth. In addition, inhibition of Panx-1 by carbenoxolone (CBX) and probenecid (PBN), as well as shRNA-mediated knockdown promoted autophagy in the I-10/CDDP cells, which was accompanied by a decrease in the levels of extracellular ATP. In contrast, overexpression of Panx-1 decreased autophagy of I-10/CDDP cells and increased extracellular ATP levels. To further determine the effect of panx-1-mediated ATP release on the autophagy of I-10/CDDP cells, apyrase was used to hydrolyze the extracellular ATP. Apyrase promoted autophagy in I-10/CDDP cells city by decreasing extracellular ATP, regardless of Panx-1 expression. This study demonstrated for the first time that Panx-1-mediated ATP release inhibits autophagy of I-10/CDDP cells, which provides a potential therapeutic strategy for cisplatin-resistant testicular cancer.

LYAR Promotes Colorectal Cancer Progression by Upregulating FSCN1 Expression and Fatty Acid Metabolism.

Colorectal cancer (CRC) is a highly malignant tumor associated with poor prognosis, yet the molecular mechanisms are not fully understood. In this study, we showed that LYAR, a nucleolar protein, is expressed at a higher level in CRC tissue than in adjacent normal tissue and that LYAR expression is closely associated with distant CRC metastasis. LYAR not only significantly promotes the migration and invasion of CRC cells in vitro, but knockdown (KD) of LYAR in CRC cells also inhibits xenograft tumor metastasis in vivo. Microarray analysis of LYAR KD cells combined with a chromatin immunoprecipitation (ChIP) assay, gene reporter assay, and rescue experiment indicated that FSCN1 (encoding fascin actin-bundling protein 1 (Fascin-1)) serves as a novel key regulator of LYAR-promoted migration and invasion of CRC cells. Knockdown of FSCN1 significantly inhibits subcutaneous tumorigenesis of CRC cells and leads to the downregulation of FASN and SCD, genes encoding key enzymes in fatty acid synthesis. In summary, this study reveals a novel mechanism by which LYAR promotes tumor cell migration and invasion by upregulating FSCN1 expression and affecting fatty acid metabolism in CRC.

[Down-regulation of pannexin 2 channel enhances cisplatin-induced apoptosis in testicular cancer I-10 cells].

OBJECTIVE: To investigate the effect of down-regulation of pannexin 2 (Panx-2) channels on cisplatin-induced apoptosis in I-10 cells. METHODS: The expression of Panx-2 protein in testicular cancer cells was detected with Western blotting. The testicular cancer cell line I-10 was transfected with two short hairpin RNA (shRNA1 and shRNA2) via Lipofectamine2000, the empty vector (NC group) or Lipofectamine2000 (blank control group), and the changes in the expression of Panx-2 was detected with Western blotting. The effects of transfection with a Panx-2 inhibitor on surviving fraction of the cells treated with cisplatin (16 µmol/L) for 24 h, 48 h and 72 h was assessed with MTT assay, and the clonogenic capacity of the cells was evaluated with colony-forming assay. At 8 h after incubation with 16 µmol/L cisplatin, AnnexinV/PI double staining was used to detect the early apoptosis of the cells. After 24 h of treatment with 16 µmol/L cisplatin, the cells were examined for expressions of caspase-3, Bcl-2 and Bax using Western blotting. RESULTS: The expression of Panx-2 was significantly increased in cisplatin-resistant I-10/DDP (P < 0.001) cells and Tcam-2/DDP (P < 0.01) cells as compared with I-10 cells and Tcam-2 cells. Transfection of I-10 cells with shRNA1 and shRNA2 resulted in significantly decreased Panx-2 expression (P < 0.05) and significantly reduced cell surviving fraction (P < 0.001). In the presence of cisplatin, the cells in NC group showed a higher clonogenic efficiency than those in shRNA1 and shRNA2 groups (P < 0.001). The early-stage apoptosis rate of the cells in shRNA1 and shRNA2 groups were significantly higher than that in NC group (P < 0.01). Panx-2 knockdown in I-10 cells significantly increased caspase-3 and Bax expressions (P < 0.05) and significantly decreased the expression of Bcl-2 (P < 0.01). CONCLUSIONS: Down-regulation of Panx-2 channel enhances cisplatin-induced apoptosis in cultured testicular cancer cells.

[Inhibitory effect of connexin43 protein on autophagy in cisplatin-resistant testicular cancer I-10 cells].

OBJECTIVE: To investigate the effect of connexin43 (Cx43) protein on autophagy in cisplatin (DDP)-resistant testicular cancer I-10 cells. METHODS: The expression of Cx43 proteins in testicular cancer I-10 cells and I-10/DDP cells were detected with Western blotting. I-10/DDP cells were transfected with a full- length mouse Cx43 vector (mCx43) via Lipofectamine2000, the empty vector or Lipofectamine2000 (blank control group), and the changes in the expressions of LC3 and p62 proteins were determined with Western blotting. mCherry-GFP-LC3B transfection and transmission electron microscopy were used to analyze the changes in autophagy of the cells with Cx43 overexpression. RESULTS: Cx43 was significantly decreased in I-10/DDP cells compared with I-10 cells (P < 0.01). Transfection of the I-10/DDP cells with mCx43 vector resulted in significantly increased Cx43 expression in the cells (P < 0.01) and caused significantly decreased expression of LC3-Ⅱ (P < 0.01) and increased expression of p62 (P < 0.05) as compared with the negative control cells. Both transmission electron microscopy and mCherry-GFP-LC3B transfection showed that the number of autophagosomes was obviously reduced in mCx43-transfected cells as compared with the negative control cells. CONCLUSIONS: Cx43 inhibits autophagy in cisplatin-resistant testicular cancer I-10 /DDP cells.

In vitro effect of Pannexin 1 channel on the invasion and migration of I-10 testicular cancer cells via ERK1/2 signaling pathway.

Pannexin (Panx) plays a crucial role in several cellular processes such as immune cell death, cell proliferation, invasion, and migration, apoptosis, and autophagy. However, the role of Panx in regulating cell migration and invasion in testicular cancer remains to be elucidated. In the present study, we determined the correlation between Panx-1 channel function and migration and invasion in I-10 testicular cancer cells. Transwell and wound healing assays showed that inhibition of Panx-1 by carbenoxolone (CBX) and probenecid (PBN) attenuated the migration and invasion of testicular cancer cells in vitro. Moreover, knockdown of Panx-1 with short hairpin RNA (shRNA) remarkably decreased the migration and invasion ability of I-10 cells. In shRNA-transfected cells, extracellular ATP (released through Panx channel) was also found to be decreased. Similarly, overexpression of Panx-1 with mPanx-1 increased the migration and invasion ability of I-10 cells. Moreover, we found that in mPanx-1-transfected cells treated with U0126 (inhibitor of p-ERK1/2), the migration and invasion of I-10 cells were remarkably attenuated. Overall, increased Panx-1 promotes migration and invasion in testicular cancer cells, and the effect is probably be related with ERK1/2 kinase activity. Thus, Panx-1 can serve as a potential therapeutic target for the treatment of testicular cancer.

[Effect of SRC Kinase on Adriamycin Resistance and Invasion and Metastasis in Human Breast Cancer Cells].

OBJECTIVE: To investigate the role of SRC kinase inhibitor PP2 in drug resistance to adriamycin (ADM) in breast cancer cells and invasion, metastasis of cells. METHODS: MTT assay was used to detect the inhibitory effect of ADM on MCF-7 and MCF-7/ADM cells. The 50% inhibitory concentration (IC50) and resistance index (RI) of cells were calculated. The expression of MDR1, connexin 43 (Cx43) and SRC proteins in breast cancer cells were detected by Western blot assay. Transwell experiment and cell scratch test were used to determine the invasion and migration of cells respectively [MCF-7, MCF-7/ADM, PP2 (1, 2, 4 µmol/L)]. Standard colony formation assay was used to detect the cytotoxicity effect of 4 µmol/L PP2 pretreatment on ADM. RESULTS: ADM inhibited the proliferation of MCF-7 more than MCF-7/ADM cells (P<0.01). The IC50 of MCF-7/ADM cells was 24.55 µmol/L, the IC50 of MCF-7/ADM cells was 770.57 µmol/L, the RI was 31. Compared with MCF-7 cells, expressions of the multidrug resistance proteins MDR1 and SRC were significantly increased (P<0.01). The invasion and migration ability of the MCF-7/ADM cells was stronger than that of the sensitive cells (P<0.01). When MCF-7/ADM was exposed to SRC inhibitor PP2, the invasion and metastasis ability of cells were inhibited (P<0.01) and the rate of colony formation was decreased, that is, more sensitivity to ADM (P<0.01). CONCLUSION: The resistance of MCF-7 to ADM is accompanied by increased expression of SRC. SRC inhibitor PP2 can reduce the cell resistance, ability of invasion and metastasis.