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Suppression of REV7 enhances cisplatin sensitivity in ovarian clear cell carcinoma cells.

Niimi K, Murakumo Y, Watanabe N, Kato T, Mii S, Enomoto A, Asai M, Asai N, Yamamoto E, Kajiyama H, Shibata K, Kikkawa F, Takahashi M - Cancer Sci. (2014)

Bottom Line: Enhanced immunoreactivity to REV7 was associated with poor prognosis represented by reduced progression-free survival in advanced stage (stage II-IV) EOC as assessed using Kaplan-Meier curves and log-rank tests.Knockdown of REV7 in CCC cells decreased cell proliferation without affecting cell cycle distribution.In a nude mouse tumor xenograft model, inoculated REV7-knockdown tumors showed significantly reduced tumor volumes after cisplatin treatment compared with those of the control group.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Nagoya University Graduate School of Medicine, Nagoya, Japan; Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Nagoya, Japan.

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REV7 knockdown enhances sensitivity to cisplatin (CDDP) in ovarian clear cell carcinoma (CCC) cells. (a) Cell viability analysis of CDDP-treated CCC cells. The IC50 of each cell line is indicated. Experiments were carried out in triplicate and the means ± SDs of relative absorbance is shown. *P < 0.05; **P < 0.01. (b) Western blot analysis for cleaved poly(ADP-ribose) polymerase (PARP) in cells after DNA-damaging treatment. The REV7-knockdown (shREV7) and control (shCont) CCC cells were treated with CDDP for 48 h and used for Western blotting (upper panels). The shREV7 and shCont CCC cells were also treated with UV at the indicated doses and used for Western blotting 12 h after UV irradiation (lower panels). The blots of β-actin are indicated as internal controls. (c) Assessment of apoptosis in the shREV7 and shCont CCC cells after CDDP treatment. Immunoreactivity for TUNEL and cleaved caspase-3 in cells treated with CDDP was fluorescently analyzed. The means ± SDs of the percentage of TUNEL-positive (upper panels) or cleaved caspase-3-positive (lower panels) cells are shown. *P < 0.05; **P < 0.01.
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fig03: REV7 knockdown enhances sensitivity to cisplatin (CDDP) in ovarian clear cell carcinoma (CCC) cells. (a) Cell viability analysis of CDDP-treated CCC cells. The IC50 of each cell line is indicated. Experiments were carried out in triplicate and the means ± SDs of relative absorbance is shown. *P < 0.05; **P < 0.01. (b) Western blot analysis for cleaved poly(ADP-ribose) polymerase (PARP) in cells after DNA-damaging treatment. The REV7-knockdown (shREV7) and control (shCont) CCC cells were treated with CDDP for 48 h and used for Western blotting (upper panels). The shREV7 and shCont CCC cells were also treated with UV at the indicated doses and used for Western blotting 12 h after UV irradiation (lower panels). The blots of β-actin are indicated as internal controls. (c) Assessment of apoptosis in the shREV7 and shCont CCC cells after CDDP treatment. Immunoreactivity for TUNEL and cleaved caspase-3 in cells treated with CDDP was fluorescently analyzed. The means ± SDs of the percentage of TUNEL-positive (upper panels) or cleaved caspase-3-positive (lower panels) cells are shown. *P < 0.05; **P < 0.01.

Mentions: Next, we investigated the effect of REV7 expression on chemosensitivity to DNA damaging agents in CCC cells. REV7-knockdown and control cells were treated with CDDP at various concentrations for 48 h, and cell viability was assessed. REV7 knockdown rendered cells more sensitive to CDDP, and the IC50 values were decreased by REV7 depletion compared with those in the shCont cells (1.61-fold, 1.98-fold and 1.93-fold decrease in IC50 in ES-2, KOC-7C, and TOV-21G cells, respectively) (Fig.3a). The chemosensitivity of REV7-knockdown cells was confirmed by colony formation assay, in which the number of colonies formed after CDDP treatment was decreased by REV7 depletion (Fig. S3, Data S1). In addition, chemosensitivity was also examined in shCont and shREV7 ES-2 cells with ectopic REV7 expression. Ectopic REV7 expression rescued enhanced chemosensitivity in shREV7 cells, however, its expression did not significantly affect chemosensitivity in shCont cells, although the IC50 of REV7-expressing shCont cells was elevated a little (Fig. S4), suggesting that endogenous REV7 expression is high enough for chemoresistance in ES-2 cells.


Suppression of REV7 enhances cisplatin sensitivity in ovarian clear cell carcinoma cells.

Niimi K, Murakumo Y, Watanabe N, Kato T, Mii S, Enomoto A, Asai M, Asai N, Yamamoto E, Kajiyama H, Shibata K, Kikkawa F, Takahashi M - Cancer Sci. (2014)

REV7 knockdown enhances sensitivity to cisplatin (CDDP) in ovarian clear cell carcinoma (CCC) cells. (a) Cell viability analysis of CDDP-treated CCC cells. The IC50 of each cell line is indicated. Experiments were carried out in triplicate and the means ± SDs of relative absorbance is shown. *P < 0.05; **P < 0.01. (b) Western blot analysis for cleaved poly(ADP-ribose) polymerase (PARP) in cells after DNA-damaging treatment. The REV7-knockdown (shREV7) and control (shCont) CCC cells were treated with CDDP for 48 h and used for Western blotting (upper panels). The shREV7 and shCont CCC cells were also treated with UV at the indicated doses and used for Western blotting 12 h after UV irradiation (lower panels). The blots of β-actin are indicated as internal controls. (c) Assessment of apoptosis in the shREV7 and shCont CCC cells after CDDP treatment. Immunoreactivity for TUNEL and cleaved caspase-3 in cells treated with CDDP was fluorescently analyzed. The means ± SDs of the percentage of TUNEL-positive (upper panels) or cleaved caspase-3-positive (lower panels) cells are shown. *P < 0.05; **P < 0.01.
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fig03: REV7 knockdown enhances sensitivity to cisplatin (CDDP) in ovarian clear cell carcinoma (CCC) cells. (a) Cell viability analysis of CDDP-treated CCC cells. The IC50 of each cell line is indicated. Experiments were carried out in triplicate and the means ± SDs of relative absorbance is shown. *P < 0.05; **P < 0.01. (b) Western blot analysis for cleaved poly(ADP-ribose) polymerase (PARP) in cells after DNA-damaging treatment. The REV7-knockdown (shREV7) and control (shCont) CCC cells were treated with CDDP for 48 h and used for Western blotting (upper panels). The shREV7 and shCont CCC cells were also treated with UV at the indicated doses and used for Western blotting 12 h after UV irradiation (lower panels). The blots of β-actin are indicated as internal controls. (c) Assessment of apoptosis in the shREV7 and shCont CCC cells after CDDP treatment. Immunoreactivity for TUNEL and cleaved caspase-3 in cells treated with CDDP was fluorescently analyzed. The means ± SDs of the percentage of TUNEL-positive (upper panels) or cleaved caspase-3-positive (lower panels) cells are shown. *P < 0.05; **P < 0.01.
Mentions: Next, we investigated the effect of REV7 expression on chemosensitivity to DNA damaging agents in CCC cells. REV7-knockdown and control cells were treated with CDDP at various concentrations for 48 h, and cell viability was assessed. REV7 knockdown rendered cells more sensitive to CDDP, and the IC50 values were decreased by REV7 depletion compared with those in the shCont cells (1.61-fold, 1.98-fold and 1.93-fold decrease in IC50 in ES-2, KOC-7C, and TOV-21G cells, respectively) (Fig.3a). The chemosensitivity of REV7-knockdown cells was confirmed by colony formation assay, in which the number of colonies formed after CDDP treatment was decreased by REV7 depletion (Fig. S3, Data S1). In addition, chemosensitivity was also examined in shCont and shREV7 ES-2 cells with ectopic REV7 expression. Ectopic REV7 expression rescued enhanced chemosensitivity in shREV7 cells, however, its expression did not significantly affect chemosensitivity in shCont cells, although the IC50 of REV7-expressing shCont cells was elevated a little (Fig. S4), suggesting that endogenous REV7 expression is high enough for chemoresistance in ES-2 cells.

Bottom Line: Enhanced immunoreactivity to REV7 was associated with poor prognosis represented by reduced progression-free survival in advanced stage (stage II-IV) EOC as assessed using Kaplan-Meier curves and log-rank tests.Knockdown of REV7 in CCC cells decreased cell proliferation without affecting cell cycle distribution.In a nude mouse tumor xenograft model, inoculated REV7-knockdown tumors showed significantly reduced tumor volumes after cisplatin treatment compared with those of the control group.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Nagoya University Graduate School of Medicine, Nagoya, Japan; Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Show MeSH
Related in: MedlinePlus