ANGPTL4 suppresses progression and improves cisplatin sensitivity in cervical cancer

Figures

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  Fig. 1. Expression of ANGPTL4 and its effects on CC cell proliferation. ANGPTL4 mRNA levels in (a) GSE63514 and (b) CC cell lines. Levels of ANGPTL4 protein were assessed using western blot in SiHa and CaSki cells transfected with (c) ANGPTL4 plasmid (OE_ANG) or empty vector (OE_C), and (d) ANGPTL4 siRNA (KD_ANG) or control siRNA (KD_C). Original blots are available in Supplementary Fig. S1. Relative ANGPTL4 level was normalized to GAPDH. Relative cell proliferation was evaluated using MTS assays in (e) ANGPTL4-overexpressing cells and (f) ANGPTL4-knockdown cells. *P < 0.05, **P < 0.01, and ***P < 0.001.

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  Fig. 2. Effects of ANGPTL4 on CC cell migration and invasion. (a) Representative images and (b) bar graphs illustrate the decreased migration and invasion capabilities of ANGPTL4-overexpressing cells compared to their control counterparts (OE_ANG vs. OE_C), as analyzed using transwell migration and Matrigel invasion assays. (c) Representative images and (d) bar graphs show the increased migration and invasion of ANGPTL4-knockdown cells in comparison to their respective controls (KD_ANG vs. KD_C). Expression levels of E-cadherin (e) mRNA and (f) protein were examined using real-time PCR and western blot, respectively in ANGPTL4-overexpressing cells. Levels of E-cadherin (g) mRNA and (h) protein in ANGPTL4-knockdown cells. (Supplementary Fig. S1). *P < 0.05, **P < 0.01, and ***P < 0.001.

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  Fig. 3. ANGPTL4 enhances sensitivity of CC cells to cisplatin treatment. The viability of ANGPTL4-overexpressing cells was determined using MTS assay after cisplatin treatment for 24 and 48 h in (a) SiHa and (b) CaSki cells (OE_ANG vs. OE_C). The viability of ANGPTL4-knockdown cells was assessed following cisplatin exposure for 24 and 48 h in (c) SiHa and (d) CaSki cells (KD_ANG vs. KD_C). Cell viability was expressed as a percentage relative to the untreated controls. *P < 0.05 and **P < 0.01.

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  Fig. 4. ANGPTL4 influences CC cells apoptosis. (a) Representative FACS density plots and (b) relative apoptosis of SiHa and CaSki cells transfected with ANGPTL4 plasmid (OE_ANG) or empty vector (OE_C) with or without 10 µM cisplatin treatment, as determined by Annexin V/PI staining. (c) Representative FACS density plots and (d) relative apoptosis of SiHa and CaSki cells transfected with ANGPTL4 siRNA (KD_ANG) or control siRNA (KD_C) in the absence or presence of 10 µM cisplatin. *P < 0.05, ** P < 0.01, and ***P < 0.001.

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  Fig. 5. ANGPTL4 increases cleaved caspase-3 and cleaved-PARP. Caspase-3, PARP, and their cleaved forms were analyzed using western blot in ANGPTL4-overexpressing cells in (a) the absence or (b) presence of cisplatin. These proteins were determined in ANGPTL4-knockdown cells (c) without or (d) with cisplatin treatment (Supplementary Fig.S1) Relative levels of cleaved PARP were normalized to GAPDH. (e) The illustration summarizes the effects of ANGPTL4 on caspase-3 activation and proposes that this activation, evidenced by increased levels of cleaved caspase-3 and cleaved PARP, enhances the sensitivity of cervical cancer cells to cisplatin, leading to reduced viability and increased apoptosis in cisplatin-treated cells. (Created with BioRender.com / Mahidol University). *P < 0.05, **P < 0.01, and ***P < 0.001.