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Role of ribophorin II in the response to anticancer drugs in gastric cancer cell lines.

Yuan TM, Liang RY, Chueh PJ, Chuang SM - Oncol Lett (2015)

Bottom Line: All the anticancer drugs were found to exert a concentration-dependent decrease on the cell survival rate of each of the cell lines tested, although the RPN2 levels in the various cell lines were not directly correlated with responsiveness to clinical anticancer drugs, based on the calculated IC50 values. siRNA-mediated RPN2 downregulation enhanced cisplatin-induced apoptosis in AGS cells, but did not markedly decrease the cell survival rates of these cells in response to the tested drugs.Furthermore, RPN2 silencing in MKN-45 cells resulted in no additional increase in the cisplatin-induced apoptosis and survival rates.However, the predictive value of RPN2 expression in cancer therapy is questionable in gastric cancer models.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedical Sciences, National Chung Hsing University, Taichung 40227, Taiwan, R.O.C. ; Department of Surgery, Feng-Yuan Hospital, Ministry of Health and Welfare, Taichung 42055, Taiwan, R.O.C.

ABSTRACT

The identification of prognostic markers and establishing their value as therapeutic targets improves therapeutic efficacy against human cancers. Ribophorin II (RPN2) has been demonstrated to be a prognostic marker of human cancer, including breast and pancreatic cancers. The present study aimed to evaluate RPN2 expression in gastric cancer and to examine the possible correlation between RPN2 expression and the response of cells to clinical anticancer drugs, which has received little research attention at present. The gastric cancer AGS, TMC-1, SNU-1, TMK-1, SCM-1, MKN-45 and KATO III cell lines were used as a model to elucidate the role of RPN2 in the response of cells to six common chemotherapeutic agents, comprising oxaliplatin, irinotecan, doxorubicin, docetaxel, cisplatin and 5-fluorouricil. The functional role of RPN2 was assessed by silencing RPN2 using small interfering RNA (siRNA), and the cytotoxicity was determined by an MTS assay and analysis of apoptosis. Molecular events were evaluated by western blotting. All the anticancer drugs were found to exert a concentration-dependent decrease on the cell survival rate of each of the cell lines tested, although the RPN2 levels in the various cell lines were not directly correlated with responsiveness to clinical anticancer drugs, based on the calculated IC50 values. siRNA-mediated RPN2 downregulation enhanced cisplatin-induced apoptosis in AGS cells, but did not markedly decrease the cell survival rates of these cells in response to the tested drugs. Furthermore, RPN2 silencing in MKN-45 cells resulted in no additional increase in the cisplatin-induced apoptosis and survival rates. It was also found that RPN2 depletion increased anticancer drug-mediated cytotoxicity in gastric cancer cell lines. However, the predictive value of RPN2 expression in cancer therapy is questionable in gastric cancer models.

No MeSH data available.


Related in: MedlinePlus

Knockdown of RPN2 enhances apoptotic cell death in the AGS cell line. (A) The MKN-45, TMK-1, AGS and SCM-1 cells were transfected with RPN2 siRNA duplex to specifically silence RPN2 expression. (B) The AGS cells were exposed to 4 μg cispatin for 48 h and the induction of apoptosis was analyzed. The percentage of apoptotic cells was determined by flow cytometry, and the results were expressed as the percentage of total cells in apoptotic populations. (C) Increases in apoptosis were calculated as fold-induction compared to the control, and the values, presented as the mean ± standard deviation, were obtained from at least three independent experiments. (D) The protein levels of activated caspase 3, cleaved PARP, Bcl-2, RPN2 and β-actin in control and RPN2-knockdown cells subsequent to 2μg/ml cisplatin treatment were determined by western blot analysis. **Indicates P<0.01 compared with cisplatin-treated siC. PARP, poly(ADP-ribose) polymerase; Bcl-2, B-cell lymphoma 2; RPN2, ribophorin II; siRNA, small interfering RNA; si-C, control siRNA; si-RPN2, RPN2 siRNA; CDDP, cisplatin.
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f4-ol-09-04-1861: Knockdown of RPN2 enhances apoptotic cell death in the AGS cell line. (A) The MKN-45, TMK-1, AGS and SCM-1 cells were transfected with RPN2 siRNA duplex to specifically silence RPN2 expression. (B) The AGS cells were exposed to 4 μg cispatin for 48 h and the induction of apoptosis was analyzed. The percentage of apoptotic cells was determined by flow cytometry, and the results were expressed as the percentage of total cells in apoptotic populations. (C) Increases in apoptosis were calculated as fold-induction compared to the control, and the values, presented as the mean ± standard deviation, were obtained from at least three independent experiments. (D) The protein levels of activated caspase 3, cleaved PARP, Bcl-2, RPN2 and β-actin in control and RPN2-knockdown cells subsequent to 2μg/ml cisplatin treatment were determined by western blot analysis. **Indicates P<0.01 compared with cisplatin-treated siC. PARP, poly(ADP-ribose) polymerase; Bcl-2, B-cell lymphoma 2; RPN2, ribophorin II; siRNA, small interfering RNA; si-C, control siRNA; si-RPN2, RPN2 siRNA; CDDP, cisplatin.

Mentions: To directly study the importance of the RPN2 protein level in drug responsiveness of gastric cancer cells, a loss of function approach was employed, using siRNA to knock down RPN2 expression in four gastric cancer cell lines. The RPN2 protein level was markedly downregulated by RPN2 siRNA after 72 h in the tested gastric cancer cell lines (Fig. 4A). The subsequent experiments revealed that siRNA-mediated RPN2 knockdown in the AGS cells increased the percentage of apoptotic cells from 5.9% in the siRNA control cells to 8.6% in the RPN2-knockdown cells. Additional induction of apoptosis was observed after treatment with 4 μg/ml cisplatin, which increased the percentage of apoptotic cells between 5.9% in the control siRNA group and 14.4% in the RPN2-knockdown cells (Fig. 4B). Therefore, knockdown of RPN2 significantly enhanced cisplatin-induced apoptosis in the AGS cells compared with the siRNA control group (Fig. 4C). Furthermore, western blot analyses demonstrated that the depletion of RPN2 increased the level of activated caspase 3 and downregulated Bcl-2 expression, which supports the hypothesis of enhanced induction of apoptosis by RPN2 knockdown alone (Fig. 4D).


Role of ribophorin II in the response to anticancer drugs in gastric cancer cell lines.

Yuan TM, Liang RY, Chueh PJ, Chuang SM - Oncol Lett (2015)

Knockdown of RPN2 enhances apoptotic cell death in the AGS cell line. (A) The MKN-45, TMK-1, AGS and SCM-1 cells were transfected with RPN2 siRNA duplex to specifically silence RPN2 expression. (B) The AGS cells were exposed to 4 μg cispatin for 48 h and the induction of apoptosis was analyzed. The percentage of apoptotic cells was determined by flow cytometry, and the results were expressed as the percentage of total cells in apoptotic populations. (C) Increases in apoptosis were calculated as fold-induction compared to the control, and the values, presented as the mean ± standard deviation, were obtained from at least three independent experiments. (D) The protein levels of activated caspase 3, cleaved PARP, Bcl-2, RPN2 and β-actin in control and RPN2-knockdown cells subsequent to 2μg/ml cisplatin treatment were determined by western blot analysis. **Indicates P<0.01 compared with cisplatin-treated siC. PARP, poly(ADP-ribose) polymerase; Bcl-2, B-cell lymphoma 2; RPN2, ribophorin II; siRNA, small interfering RNA; si-C, control siRNA; si-RPN2, RPN2 siRNA; CDDP, cisplatin.
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f4-ol-09-04-1861: Knockdown of RPN2 enhances apoptotic cell death in the AGS cell line. (A) The MKN-45, TMK-1, AGS and SCM-1 cells were transfected with RPN2 siRNA duplex to specifically silence RPN2 expression. (B) The AGS cells were exposed to 4 μg cispatin for 48 h and the induction of apoptosis was analyzed. The percentage of apoptotic cells was determined by flow cytometry, and the results were expressed as the percentage of total cells in apoptotic populations. (C) Increases in apoptosis were calculated as fold-induction compared to the control, and the values, presented as the mean ± standard deviation, were obtained from at least three independent experiments. (D) The protein levels of activated caspase 3, cleaved PARP, Bcl-2, RPN2 and β-actin in control and RPN2-knockdown cells subsequent to 2μg/ml cisplatin treatment were determined by western blot analysis. **Indicates P<0.01 compared with cisplatin-treated siC. PARP, poly(ADP-ribose) polymerase; Bcl-2, B-cell lymphoma 2; RPN2, ribophorin II; siRNA, small interfering RNA; si-C, control siRNA; si-RPN2, RPN2 siRNA; CDDP, cisplatin.
Mentions: To directly study the importance of the RPN2 protein level in drug responsiveness of gastric cancer cells, a loss of function approach was employed, using siRNA to knock down RPN2 expression in four gastric cancer cell lines. The RPN2 protein level was markedly downregulated by RPN2 siRNA after 72 h in the tested gastric cancer cell lines (Fig. 4A). The subsequent experiments revealed that siRNA-mediated RPN2 knockdown in the AGS cells increased the percentage of apoptotic cells from 5.9% in the siRNA control cells to 8.6% in the RPN2-knockdown cells. Additional induction of apoptosis was observed after treatment with 4 μg/ml cisplatin, which increased the percentage of apoptotic cells between 5.9% in the control siRNA group and 14.4% in the RPN2-knockdown cells (Fig. 4B). Therefore, knockdown of RPN2 significantly enhanced cisplatin-induced apoptosis in the AGS cells compared with the siRNA control group (Fig. 4C). Furthermore, western blot analyses demonstrated that the depletion of RPN2 increased the level of activated caspase 3 and downregulated Bcl-2 expression, which supports the hypothesis of enhanced induction of apoptosis by RPN2 knockdown alone (Fig. 4D).

Bottom Line: All the anticancer drugs were found to exert a concentration-dependent decrease on the cell survival rate of each of the cell lines tested, although the RPN2 levels in the various cell lines were not directly correlated with responsiveness to clinical anticancer drugs, based on the calculated IC50 values. siRNA-mediated RPN2 downregulation enhanced cisplatin-induced apoptosis in AGS cells, but did not markedly decrease the cell survival rates of these cells in response to the tested drugs.Furthermore, RPN2 silencing in MKN-45 cells resulted in no additional increase in the cisplatin-induced apoptosis and survival rates.However, the predictive value of RPN2 expression in cancer therapy is questionable in gastric cancer models.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedical Sciences, National Chung Hsing University, Taichung 40227, Taiwan, R.O.C. ; Department of Surgery, Feng-Yuan Hospital, Ministry of Health and Welfare, Taichung 42055, Taiwan, R.O.C.

ABSTRACT

The identification of prognostic markers and establishing their value as therapeutic targets improves therapeutic efficacy against human cancers. Ribophorin II (RPN2) has been demonstrated to be a prognostic marker of human cancer, including breast and pancreatic cancers. The present study aimed to evaluate RPN2 expression in gastric cancer and to examine the possible correlation between RPN2 expression and the response of cells to clinical anticancer drugs, which has received little research attention at present. The gastric cancer AGS, TMC-1, SNU-1, TMK-1, SCM-1, MKN-45 and KATO III cell lines were used as a model to elucidate the role of RPN2 in the response of cells to six common chemotherapeutic agents, comprising oxaliplatin, irinotecan, doxorubicin, docetaxel, cisplatin and 5-fluorouricil. The functional role of RPN2 was assessed by silencing RPN2 using small interfering RNA (siRNA), and the cytotoxicity was determined by an MTS assay and analysis of apoptosis. Molecular events were evaluated by western blotting. All the anticancer drugs were found to exert a concentration-dependent decrease on the cell survival rate of each of the cell lines tested, although the RPN2 levels in the various cell lines were not directly correlated with responsiveness to clinical anticancer drugs, based on the calculated IC50 values. siRNA-mediated RPN2 downregulation enhanced cisplatin-induced apoptosis in AGS cells, but did not markedly decrease the cell survival rates of these cells in response to the tested drugs. Furthermore, RPN2 silencing in MKN-45 cells resulted in no additional increase in the cisplatin-induced apoptosis and survival rates. It was also found that RPN2 depletion increased anticancer drug-mediated cytotoxicity in gastric cancer cell lines. However, the predictive value of RPN2 expression in cancer therapy is questionable in gastric cancer models.

No MeSH data available.


Related in: MedlinePlus