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.

Epithelial-mesenchymal transition in cholangiocarcinoma: From clinical evidence to regulatory networks

“…In addition, inhibition of SLUG sensitized CCA cells to cisplatin through upregulation of the proapoptotic protein PUMA [143]. It is noteworthy that the activity of EMTTFs is not restricted to the repression of E-cadherin promoter, but they also trigger cell survival by promoting apoptosis escape of tumor cells through inhibition of PUMA-mediated cell death in CCA [15,144].…”

“…In CCA, EMT has been identified in vitro as a mechanism of resistance against gemcitabine and cisplatin [71,143], both used in combination as the standard of care for patients with CCA [9]. Yamada et al, recently showed that CCA cell lines exhibiting mesenchymal traits were more resistant to gemcitabine than CCA cell lines having a prominent epithelial phenotype [71].…”

Inhibition of active autophagy induces apoptosis and increases chemosensitivity in cholangiocarcinoma

“…23 The QBC939 cells were grown to subconfluency in complete medium. Viable cells (2 Â 10 6 in PBS/100 ml) were injected subcutaneously into the flanks of 4-week-old nude mice.…”

Animal models of cholangiocarcinoma

“…Expressed IL-6 in stable Mz-ChA-1 cell line/5 Â 10 6 (þ) miR-148a /152, (À) DNMT1 [2] Expressed IL-6 in stable Mz-ChA-1 cell line/3 Â 10 6 (À) miR-370 [3] Expressed miR-494 in stable HuCCT1/3.25 Â 10 6 (À) G1-S transition, (À) tumour growth [4,5 & ] Gene knockdown Slug siRNA in stable QBC939 cell line/5 Â 10 6 (À) Tumour growth [6] CypA siRNA in stable M139 cell line /3 Â 10 6 (À) Tumour growth [7] Beclin-1 siRNA in stable QBC939 cell line/2 Â 10 6 (À) Tumour growth [8 & ] Drug treatment during xenograft development Sk-ChA-1 cells/5 Â 10 6 /tamoxifen injections intratumourally (þ) Apoptosis [9] Mz-ChA-1 cells/5 Â 10 6 /resveratrol i.p injection (þ) Sensitivity to a chemotherapeutic agent [10] Mz-ChA-1 cells/5 Â 10 6 /anti-NYP antibody injections intratumourally (þ) Tumour growth [11] Mz-ChA-1 cells/5 Â 10 6 /green tea polyphenol EGCG i.p injection (þ) Chemotherapy-induced apoptosis [12] QBC939 cells/2 Â 10 6 /magnetic nanoparticles injections by caudal vein (þ) Tumour growth [13] catabolic process involving the degradation of a cell's own components through the lysosomal machinery. Autophagy is activated in CCA, and inactivation of autophagy may lead to cell apoptosis and enhance chemotherapy sensitivity.…”

“…Slug, a snail family transcription factor, is a suppressor of p53 upregulated modulator of apoptosis (PUMA), which has been shown to be involved in the control of apoptosis. Slug silencing suppressed the growth of QBC939 xenograft tumours and sensitized the tumour cells to cisplatin, an anticancer chemotherapy drug, through PUMA upregulation and induction of apoptosis [6]. Cyclophilin A (CypA, a cytosolic protein involved in cell growth, immune modulation, tumourigenesis and vascular disease) is upregulated in majority of CCA patients' tissues and confers a significant growth advantage in CCA cells.…”