Inhibition of cytosolic phospholipase A2 alpha increases chemosensitivity in cervical carcinoma through suppressing β-catenin signaling

Abstract

Cytosolic phospholipase A2alpha (cPLA2α) is a key mediator of tumorigenesis. In this study, by using a combination of pharmacological and genetic approaches in cell models and patient samples, we identify cPLA2α as a selective target to increase chemosensitivity in cervical cancer. We found that transcript and protein levels of cPLA2α but not other forms of cPLA2 (e.g., cPLA2β and cPLA2αδ) were consistently increased in all tested malignant cervical cancer cells and tissues compared to normal counterparts, suggesting that cPLA2α upregulation is a common feature in cervical cancer. We further found that promoting growth and survival rather than invasion were the predominant roles of cPLA2α on cervical cancer. In addition, chemotherapeutic agents achieved ~100% inhibition efficacy in cPLA2α-depleted cervical cancer cells, demonstrating the important role of cPLA2α in chemoresistance. Importantly, we identify that β-catenin is critically involved in the molecular mechanism of cPLA2α's action in cervical cancer. In summary, our work demonstrates the multiple essential roles of cPLA2α in cervical cancer, particularly in chemoresistance, via a β-catenin-dependent manner. Our work also suggests that targeting cPLA2α has a therapeutic value in overcoming chemoresistance in cervical cancer or other cPLA2α-regulated cancers.

Keywords: Akt; Cpla2α; cervical cancer; chemoresistance; β-catenin.

Figure 1.

cPLA2α is specifically upregulated in cervical cancer tissues. (a) Representative immunohistochemistry (IHC) analysis shows higher level of cPLA2α but not cPLA2β or cPLA2δ in cervical cancer tissues from patients compared to normal cervical tissues from healthy donors. Scale bar represents 50 µm. (b) Quantification analysis using Image J software shows a significant increase in cPLA2α but not cPLA2β or cPLA2δ level in cervical cancer tissues from patients (n = 20) compared to normal cervical tissues from healthy donors (n = 12). The result is shown as relative IHC staining intensity per high power field (HPF) to normal. *p < 0.05 compared to normal. (a) Representative immunohistochemistry analysis shows higher level of cPLA2α but not cPLA2β or cPLA2δ in cervical cancer tissues from patients compared to normal cervical tissues from healthy donors. Scale bar represents 50 µm. (b) Quantification analysis using Image J software shows a significant increase in cPLA2α but not cPLA2β or cPLA2δ level in cervical cancer tissues from patients (n = 20) compared to normal cervical tissues from healthy donors (n = 12). The result is shown as relative IHC staining intensity per HPF to normal. *p < 0.05 compared to normal.

Figure 2.

cPLA2α is upregulated in cervical cancer cells. RT-PCR analysis and representative Western blot analysis show the increased mRNA (a) and protein (b) levels of cPLA2α but not cPLA2β or cPLA2δ in multiple cervical epithelial cancer cell lines: HeLa, CaLo, CasKi, SiHa, C33A, and ViBo cells than normal cervical epithelial cells: Ect1/E6E7 and HCvEpC. The result is shown as relative mRNA and protein level to HCvEpC. (c) Quantification of cPLA2α but not cPLA2β or cPLA2δ protein levels in normal and cancer cervical epithelial cells. *p < 0.05 compared to HCvEpC.

Figure 3.

Inhibition of cPLA2α inhibits the growth of cervical cancer cells. cPLA2α inhibitors pyrrophenone (a) and RSC-3388 (b) significantly inhibit cPLA2α enzyme activity. cPLA2α inhibitors pyrrophenone (c) and RSC-3388 (d) significantly inhibit proliferation of cervical cancer cells. *p < 0.05 compared to dimethyl sulfoxide (DMSO) control.

Figure 4.

Inhibition of cPLA2α inhibits survival without affecting migration of cervical cancer cells. Pyrrophenone (a) and RSC-3388 (b) at 100 nM significantly induce apoptosis in cervical cancer cells. Pyrrophenone (c) and RSC-3388 (d) up to 100 nM do not affect cervical cancer migration. *p < 0.05 compared to DMSO control.

Figure 5.

cPLA2α inhibition using a pharmacological approach enhances chemosensitivity in cervical cancer cells. cPLA2α inhibitors pyrrophenone and RSC-3388 significantly enhance chemotherapeutic agents’ (paclitaxel and cisplatin) effects in inhibiting proliferation (a) and inducing apoptosis (b) in CaLo, SiHa, and CaSki cells. RSC-3388 and pyrrophenone at 100 nM and paclitaxel at 10 nM and cisplatin at 100 nM were used in the combination studies. *p < 0.05 compared to cPLA2α inhibitor or chemotherapeutic agent alone.

Figure 6.

cPLA2α inhibition using a genetic approach enhances chemosensitivity in cervical cancer cells. Specific cPLA2α siRNA knockdown inhibits proliferation (a) and induces apoptosis (b) and significantly augments the inhibitory effects of paclitaxel and cisplatin. Two independent siRNAs (siRNA1 and siRNA2) were used. Paclitaxel at 10 nM and cisplatin at 100 nM were added to the cell medium at 48 h post-transfection. Cells were harvested for growth and apoptosis analysis after 3 d of treatment. *p < 0.05 compared to cPLA2α siRNA or chemotherapeutic agent alone.

Figure 7.

cPLA2α inhibition suppresses Akt/eIF4E/β-catenin signaling in cervical cells. cPLA2α inhibition by inhibitors (a) or siRNA (b) significantly decreases cPLA2α enzyme activity in cervical cancer cells. cPLA2α inhibition by inhibitors (c) or siRNA (d) decreases βAkt (Ser473), p-eIF4E (Ser209), and β-catenin in CaLo cells. (e) Pyrrophenone and RSC-3388 at 100 nM significantly inhibit TOPflash activation. CaLo cells transfected with TOPflash plasmid were treated as indicated. The graph represents mean ± s.e.m. of TOPflash signal relative to control. cPLA2α inhibitors (f) and siRNA (g) significantly decrease the transcript level of C-MYC, BCL9, and CYCLIN D genes in CaLo cells. *p < 0.05 compared to control or siRNA con (scrambled siRNA).

Figure 8.

cPLA2α inhibition acts on cervical cancer cells in a β-catenin-dependent manner. No significant change on β-catenin levels by pyrrophenone and RSC-3388 in CaLo cells treated with LiCl (a) or transfected with β-catenin overexpression plasmid (b). cPLA2α inhibitors (c) and siRNA (d) are ineffective in decreasing C-MYC transcript level in the presence of LiCl or β-catenin-overexpressing CaLo cells. The inhibitory effects of cPLA2α inhibition on proliferation or survival were abolished by LiCl (e and g) and β-catenin overexpression (f and h). Cells are transfected with P-Vector or P-β-catenin plasmid. Cells were harvested for Western blot, RT-PCR, and growth and apoptosis assays at 24 h post-transfection. *p < 0.05 compared to -LiCl or P-Vector.