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, 11 (10), 4926-4933
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Inhibition of WNT7A-β-catenin Signaling Pathway Sensitizes Oral Squamous Cell Carcinoma to Cisplatin

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Inhibition of WNT7A-β-catenin Signaling Pathway Sensitizes Oral Squamous Cell Carcinoma to Cisplatin

Jiangang Tian et al. Int J Clin Exp Pathol.

Abstract

Oral squamous cell carcinoma (OSCC) is the most common type and most threatening head and neck cancer worldwide. Here, we aim to study the relationship between the WNT7A-β-Catenin signaling pathway and the chemotherapy resistance of OSCC patients. We analyzed 42 OSCC patients and 19 adjacent non-tumor tissues, evaluated the expression levels of WNT7A mRNA, and subsequently studied WNT7A dependent cisplatin resistance in OSCC cell line KB cells. Moreover, we also utilized an in vivo mouse model to validate our findings. We first found a significant upregulation of WNT7A mRNA in OSCC patients. Our results showed that the knockdown of WNT7A sensitized KB cells to cisplatin. Moreover, our results revealed that nuclear β-catenin was dramatically reduced and cleaved caspase-3 and cleaved PARP were dramatically induced when WNT7A was knocked down in cisplatin treated KB cells. Besides, we found that the knockdown of WNT7A significantly reduced the weight and volumes of xenograft tumors. Moreover, we examined apoptotic cells and found that the combination of WNT7A knockdown and cisplatin treatment resulted in many more apoptotic cells than cisplatin treatment alone, suggesting that the knockdown of WNT7A sensitized KB cells to cisplatin treatment in vivo. Our results revealed that inhibition of WNT7A-β-catenin signaling sensitizes OSCC to cisplatin, which has provided insights into the molecular diagnosis and treatment of OSCC.

Keywords: OSCC; WNT7A; cisplatin; drug resistance; β-Catenin.

Conflict of interest statement

None.

Figures

Figure 1
Figure 1
WNT7a is highly expressed in oral squamous cell carcinoma (OSCC). (A) qPCR analysis showed WNT7a was upregulated in 42 OSCC tissues compared to 19 normal tissues. (B) Immunohistostaining of WNT7a in OSCC patients. Non-tumor, adjacent normal tissues; tumor, cancerous tissues from OSCC patients. (C) Western blot analysis of WNT7a in OSCC patients. NT, non-tumor; T, tumor. qPCR (D) and Western blot (E) analysis of WNT7a in human oral cancer cell line KB and primary mucosal epithelial cell (CON). GAPDH was used as normalization control. The experiments were repeated at least three times. *, P < 0.05 compared to control. Values represent mean ± SD.
Figure 2
Figure 2
WNT7a knockdown sensitizes KB cells to cisplatin. Forty-eight hours after transfection with WNT7a siRNA or scrambled siRNA, RNA interference efficiency was evaluated by qPCR (A and D) and western blot (B and E) in KB cells and CAL-27 cells repectively. GAPDH was used as a normalization control. (C) WNT7a downregulated KB cells were treated with cisplatin with various concentrations: 0.1, 1, and 10 μM for 48 hours. Cell survival rates were determined by MTT assay. (F) WNT7a downregulated CAL-27 cells were treated with cisplatin with various concentrations: 0.1, 1, and 10 μM for 48 hours. Cell survival rates were determined by MTT assay. *, P < 0.05 compared to normal KB cells transfected with scrambled siRNA. Data were collected from three independent experiments. Values represent mean ± SD.
Figure 3
Figure 3
WNT7a knockdown deactivates β-catenin pathway. A. Forty-eight hours after RNA interference, nuclear proteins in KB cells were extracted and analyzed by Western blot, showing that active β-catenin was decreased in WNT7a-downregulated KB cells. Histone H3 was used as a loading control. B. WNT7a knockdown further activates caspase-3/PARP in cisplatin-treated KB cells. KB cells trasfected with WNT7a siRNA or scrambled siRNA were treated with 10 μM cisplatin and total proteins were extracted for Western blot analysis. GAPDH was used as a loading control.
Figure 4
Figure 4
Downregulation of WNT7a sensitizes OSCC to cisplatin in vivo. Athymic nude mice were injected subcutaneously with 3 × 106 WNT7a-downregulated (siWNT7a+Vehicle and siWNT7a+Cisplatin) or control cells (Scramble+Vehicle and Scramble+Cisplatin). Chemotherapeutic groups were treated with cisplatin (2 mg/kg) by intraperitoneal injection every 3 days. (A) Representative overview of KB-xenografted tumors. Scale bar, 1 cm. Tumor weight (B) and volume (C) were decreased in WNT7a-downregulated mice. Statistical analysis was performed using Student’s t test; significant differences were shown in groups sharing the same letter. (D) Intratumoral apoptosis was analyzed by TUNEL assay. Scale bars, 50 μm.

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