CACNA2D3 Enhances the Chemosensitivity of Esophageal Squamous Cell Carcinoma to Cisplatin via Inducing Ca2+-Mediated Apoptosis and Suppressing PI3K/Akt Pathways

Abstract

Resistance to platinum-based combination chemotherapy is the main cause of poor prognosis in patients with advanced esophageal squamous cell carcinoma (ESCC). Previously, we showed that CACNA2D3 (voltage-dependent subunit alpha 2 delta 3 of a calcium channel complex) was significantly downregulated and functioned as a tumor suppressor in ESCC, but its role in the chemosensitivity of ESCC to cisplatin remained unknown. Here, we found that the expression of CACNA2D3 was significantly associated with poor platinum response in ESCC patients from the Gene Expression Omnibus database. Overexpression of CACNA2D3 increased sensitivity to cisplatin in ESCC in vitro, whereas knockdown of CACNA2D3 increased cisplatin resistance. CACNA2D3 promoted cisplatin-induced apoptosis by modulating intracellular Ca²⁺ stores. In vivo experiments further showed that overexpression of CACNA2D3 enhanced cisplatin anti-tumor effects in a xenograft mouse model. CACNA2D3 overexpression also resulted in the attenuation of PI3K and Akt phosphorylation. Treatment with the PI3K/Akt inhibitor LY294002 restored the chemosensitivity of CACAN2D3-knockdown cells to cisplatin. In conclusion, the results of the current study indicate that CACAN2D3 enhances the chemosensitivity of ESCC to cisplatin via inducing Ca²⁺-mediated apoptosis and suppressing PI3K/Akt pathways. Therefore, regulating the expression of CACNA2D3 is a potential new strategy to increase the efficacy of cisplatin in ESCC patients.

Keywords: CACNA2D3; ESCC; LY294002; chemosensitivity; voltage-gated calcium channel.

Figure 1

Downregulation of CACNA2D3 is associated with poor chemotherapy response in ESCC. (A) Box and whisker plot of CACNA2D3 mRNA levels in neoadjuvant chemotherapy responder and non-responder groups (GSE45670). ^*p < 0.05. (B) IC50 values for cisplatin in six ESCC cell lines. Cells were treated with a range of concentrations of cisplatin for 72 h, and IC50 was calculated. Data are represented as the mean ± SD of three independent experiments. (C) Correlation of CACNA2D3 mRNA levels and IC50 values for cisplatin in six ESCC cell lines. r, Pearson correlation coefficient.

Figure 2

CACNA2D3 promotes chemosensitivity to cisplatin in vitro. (A,E) Stable expression of CACNA2D3 in KYSE30 cells generated by pCDNA3.1-CACNA2D3 transfection and silencing of CACNA2D3 in KYSE180 cells by siRNA were examined by western blotting and qRT-PCR. GAPDH and β-Actin were employed as a loading control. (B,F) CACNA2D3-overexpressing KYSE30 cells and CACNA2D3-knockdown cells were treated with cisplatin at the indicated concentrations for 72 h. The number of viable cells was measured by CCK-8 assay. (C,G) IC50 values were calculated using linear or logarithmic regression (R² > 0.9). Values are presented as the mean ± SD of three wells. (D,H) Colony forming assays were used to determine colony forming ability after cisplatin treatment. Data are presented as the mean ± SD of three wells. ^**p < 0.01, ^***p < 0.001.

Figure 3

CACNA2D3 enhances cisplatin-induced apoptosis through mitochondria-dependent pathway. (A) Intracellular Ca²⁺ levels were detected by fluorescence-activated cell sorting (FACS) with Fluo-3 AM. (B,C) Annexin V/PI staining in CACNA2D3 overexpression and knockdown cell lines treated with cisplatin for 48 h; apoptosis was analyzed by FACS. The percentages of apoptotic cells are presented as the mean ± SD of three independent experiments. (D,E) The mitochondrial membrane potential was measured using fluorescent dye JC-1 after treatment with cisplatin for 48 h. Percentages of green fluorescence from JC-1 in cells are presented as the mean ± SD of three independent experiments. (F) Levels of cleaved caspase-3, total caspase-3, cleaved caspase-9, and total caspase-9 proteins were analyzed by western blotting in CACNA2D3-overexpressing and knockdown cell lines treated with or without cisplatin. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Figure 4

CACNA2D3 increases chemosensitivity of ESCC cells to cisplatin in vivo. (A) Representative images of xenografted tumors derived from Balb/c-nu mice at day 33. (B) The mice in the treatment groups were intraperitoneally injected with 2 mg/kg every 3 days. Tumor volumes were measured at 4 days intervals. Data are presented as mean ± SD. (C) Tumor weights were measured after mice were sacrificed. (D) Detection of CACNA2D3 from tumor sections by immunohistochemical staining. Quantification was performed by calculating the percentage of the staining intensities using ImageJ. (E) TUNEL staining of tumor sections from each group; the number of TUNEL-positive cells was quantified by counting. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Figure 5

CACANA2D3 regulated the sensitivity of ESCC to cisplatin through inhibiting the PI3K/Akt pathways. (A) DEG heatmap and hierarchical clustering results for CACNA2D3-overexpressing KYSE30 ESCC cells. Red and green indicate high and low gene expression, respectively. (B) GO enrichment analysis of the DEGs. The genes were divided into three categories: cellular component, biological process, and molecular function genes. (C) KEGG pathway enrichment analysis of differentially expressed pathways upon CACNA2D3 overexpression. The ordinates represent the enriched KEGG pathway. p < 0.05 was considered statistically significant. (D) GSEA analysis of differentially expressed pathways upon CACNA2D3 overexpression. NES, normalized enrichment score. (E) Levels of P-PI3K, PI3K, P-Akt, and Akt proteins were analyzed by western blotting in CACNA2D3-overexpressing and knockdown cell lines treated with or without cisplatin.

Figure 6

LY294002 restores the sensitivity of ESCC to cisplatin in CACNA2D3-knockdown cells. (A) CACNA2D3-knockdown KYSE180 cells and control cells were treated with cisplatin at the indicated concentrations with or without LY294002 for 72 h. Levels of P-Akt and Akt proteins were analyzed by western blotting. (B) The number of viable cells was measured by CCK-8 assay. (C) IC50 values were calculated using linear or logarithmic regression (R2 > 0.9). Data are presented as the mean ± SD from triplicate wells. (D) Colony forming assays were used to determine the colony forming ability after cisplatin with or without LY294002 treatment. Data are presented as the mean ± SD from three wells. ^**p < 0.01.