RNA interference targeting slug increases cholangiocarcinoma cell sensitivity to cisplatin via upregulating PUMA

Abstract

Slug is an E-cadherin repressor and a suppressor of PUMA (p53 upregulated modulator of apoptosis) and it has recently been demonstrated that Slug plays an important role in controlling apoptosis. In this study, we examined whether Slug's ability to silence expression suppresses the growth of cholangiocarcinoma cells and/or sensitizes cholangiocarcinoma cells to chemotherapeutic agents through induction of apoptosis. We targeted the Slug gene using siRNA (Slug siRNA) via full Slug cDNA plasmid (Slug cDNA) transfection of cholangiocarcinoma cells. Slug siRNA, cisplatin, or Slug siRNA in combination with cisplatin, were used to treat cholangiocarcinoma cells in vitro. Western blot was used to detect the expression of Slug, PUMA, and E-cadherin protein. TUNEL, Annexin V Staining, and cell cycle analysis were used to detect apoptosis. A nude mice subcutaneous xenograft model of QBC939 cells was used to assess the effect of Slug silencing and/or cisplatin on tumor growth. Immunohistochemical staining was used to analyze the expression of Slug and PUMA. TUNEL was used to detect apoptosis in vivo. The results showed that PUMA and E-cadherin expression in cholangiocarcinoma cells is Slug dependent. We demonstrated that Slug silencing and cisplatin both promote apoptosis by upregulation of PUMA, not by upregulation of E-cadherin. Slug silencing significantly sensitized cholangiocarcinoma cells to cisplatin through upregulation of PUMA. Finally, we showed that Slug silencing suppressed the growth of QBC939 xenograft tumors and sensitized the tumor cells to cisplatin through PUMA upregulation and induction of apoptosis. Our findings indicate that Slug is an important modulator of the therapeutic response of cholangiocarcinoma cells and is potentially useful as a sensitizer in cholangiocarcinoma therapy. One of the mechanisms is the regulation of PUMA by Slug.

Keywords: E-cadherin; PUMA; chemotherapy; cisplatin; slug.

Figure 1

Slug regulation-induced processing of Slug and PUMA. (A) The protein fractions of Slug in QBC939, RBE, ICC-9810, and FRH 0201 cells were subjected to Western blot analysis. FRH 0201 exhibited the lowest expression level of Slug and QBC939 exhibited the highest expression level of Slug. (B) The protein fractions of Slug, E-cadherin, and PUMA in QBC939 cells after transfection with Slug siRNA for 48 h were subjected to Western blot analysis. Slug expression was barely detectable compared with parental cells (**P < 0.01), and a remarkble upregulation of E-cadherin and PUMA protein was shown compared with parental cells (*P < 0.05). (C) The protein fractions of Slug, E-cadherin, and PUMA in the FRH 0201 cell line after transfection with Slug cDNA for 48 h were subjected to Western blot analysis. Remarkable upregulation of Slug protein was shown compared with parental cells (**P < 0.01), and remarkable downregulation of PUMA and E-cadherin proteins was shown compared with parental cells (*P < 0.05).

Figure 2

TUNEL staining was performed for QBC939 cells transiently transfected with Slug siRNA or mock transfected for 48hs. Green nuclear staining indicates apoptotic cells. The percentage of TUNEL-positive cells was quantified. Columns and bars represent the mean and standard deviation of three independent determinations, respectively. Significant differences between the controls (mock) and the Slug siRNA groups are indicated by *P < 0.05.

Figure 3

Cisplatin-induced apoptosis in QBC939 and FRH 0201 cells. A, C, QBC939 and FRH0201 cells (1 × 10⁶/mL) were exposed to the designated concentrations of cisplatin for the indicated times, after which time the percentage of apoptotic cells was determined by flow cytometric analysis, as described in “Materials and Methods”. The data are expressed as a mean value of the percentage of apoptotic cells from three independent experiments performed in duplicate (*P < 0.05, **P < 0.01, ***P < 0.001). B, D, At the indicated time points after treatment with 20 μg/mL cisplatin, the cells were harvested and fixed in 70% ethanol. After staining with PI, the apoptotic DNA content was analyzed by flow cytometry. The number of apoptotic cells in the sub-G1 fraction is expressed as a percentage of the total number of cells (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 4

Induction of apoptosis by cisplatin is independent of PUMA mechanism. (A, B) The QBC939 and FRH 0201 cells were exposed to various cisplatin concentrations for 72 h. The PUMA and E-cadherin protein expression levels in the cell lysate were examined by Western blot analysis using anti-PUMA (E-cadherin) antibody, and the antibodies against ß-actin which served as an internal control. (C, D) The QBC939 and FRH 0201 cells were exposed to 20 μg/mL cisplatin for the indicated times. The PUMA and E-cadherin protein expression level was measured by Western blot. (E, F) QBC939 and FRH0201 cells (1 × 10⁶/mL) were exposed to 20 μg/mL cisplatin combined with PUMA siRNA for 72 h, PUMA protein expression level was measured by Western blot. (G, H) QBC939 and FRH0201 cells (1 × 10⁶/mL) were exposed to 20 μg/mL cisplatin combined with PUMA siRNA for 72 h, after which time the percentage of apoptotic cells was determined by flow cytometric analysis, as described in “Materials and Methods”. Cisplatin combined with PUMA siRNA did not induce obvious apoptosis in QBC939 and FRH 0201 cells (^*P < 0.01).

Figure 5

TUNEL staining was performed to assess the potential cooperation of Slug silencing and cisplatin treatment in inducing apoptosis in QBC939 cells. Green nuclear staining indicates apoptotic cells. The percentage of TUNEL-positive cells was quantified. Columns and bars represent mean and standard deviation of three independent determinations, respectively. Significant differences between the controls (mock) and the Slug siRNA groups are indicated by *P < 0.05, **P < 0.01.

Figure 6

Slug silencing sensitizes cholangiocarcinoma xenograft tumors to cisplatin. A, growth bar of QBC939 tumors (n = 6 per group) subjected to Slug siRNA, mock, and PBS and/or combined with cisplatin treatment for 28 days. B, Immunohistochemistry analysis for Slug and PUMA in different groups. C, TUNEL-positive cells in three groups were detected in the control, cisplatin, or Slug siRNA plus cisplatin treatments. Columns and bars represent the mean of three independent determinations and S.D., respectively. *P < 0.05, **P < 0.01, ***P < 0.01.