1α,25(OH)2D3(VD3) promotes Raddeanin A-induced anti-proliferative effects on HeLa cell apoptosis and autophagy through negative regulation of HPV18E6-E7/PD-L1/VDR axis

Abstract

Raddeanin A (RA) has indicated suppressive effects on various human tumor cells, and insufficient vitamin D was associated with human papillomavirus (HPV) persistence and gynecological tumors. However, combined effects of RA and vitamin D on HPV-positive cells remain elusive. Herein, we aimed to investigate the combined effects of RA and 1ɑ,25(OH)₂D₃ (VD3) on cellular viability and modulation of HPV18E6/E7, programmed cell death 1 ligand (PD-L1) and vitamin D receptor (VDR) expression in HeLa cells in vitro. HeLa cells were treated with RA alone or VD3 combined with RA. Cell viability was measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), and apoptosis was detected by flow cytometry. Real-time PCR (qRT-PCR) and Western blot were used to determine the gene/protein expression levels. The autophagosomes were observed by Transmission electron microscopy (TEM). The result showed that cell viability was inhibited by RA, and apoptosis in HeLa cells treated with RA was elevated accordingly. The expression of Bax, Cleaved-caspase-3, Cleaved-caspase-9 and Cleaved-PARP increased, and Bcl-2 decreased. The autophagy was induced by RA, as evidenced by elevated autophagosomes and the increased LC3-II/I ratio and Beclin-1. The expression of HPV18E6/E7, PD-L1 and VDR was reduced by RA. Moreover, RA combined with VD3 had a stronger effect on HeLa cells than RA alone. In conclusion, RA inhibits HeLa proliferation and induces apoptosis and autophagy via suppressing HPV18E6/E7, PD-L1 and VDR, and VD3 showed reinforced effects of RA on HeLa cells. Therefore, combined usage of VD3 with RA might be a potential novel immunotherapy strategy for HPV-related diseases.

Keywords: HeLa cells; PD-L1; Raddeanin A (RA); Vitamin D; apoptosis; autophagy.

Graphical abstract

Figure 1.

Cell viability of the HeLa cells. (a) Following treatment with RA (2.5, 5, 10, 20 and 40 μM/L) and RA combined with 100 nM/L VD3 for 48 h, the viability of HeLa cells was determined using MTT assay. Data was presented as mean ± SD of three independent experiments. P < 0.01 versus the control groups (0 μM/L RA and 0 μM/L VD3). (b) HeLa cells under microscope. HeLa cells were cultured for 48 h with different concentrations of RA (2, 4 and 8 μM/L) and RA combined with 100 nM/L VD3. The morphological changes were investigated under microscope. (×100). RA: Raddeanin A; RA+VD3: Raddeanin A combined with 100 nM/L VD3.

Figure 2.

The effect of RA and RA combined with 100 nM/L VD3 on apoptosis in HeLa cells. HeLa cells were treated with various doses of RA (2, 4 and 8 μM/L) and RA with the addition of 100 nM/L VD3 for 48 h, stained with Annexin V-FITC/PI for flow cytometry analysis (n = 3 per group). (a) The Figure was a representative of three independent trials. Apoptosis rate was defined as the percentage of dead and apoptotic cells (quadrants 2 and 3). (b) RA and RA coupled with 100 nM/L VD3 induced HeLa cells apoptosis, and the data was presented as mean ± SD from 3 independent experiments. * P < 0.05 and ** P < 0.01 versus control groups (0 μM/L RA and 0 μM/L VD3). The apoptosis ratio increased in HeLa cells treated with the combinational compounds compared with RA alone (P < 0.05). (c) Western blotting showed the alterations of Bax, Cleaved-caspase-3, Cleaved-caspase-9, Cleaved-PARP and Bcl-2 with the cell lysates. The protein contents were collected at 48 h after treatment. (d) TUNEL staining showed the changes in the number of TNUEL-positive HeLa cells treated with RA (8 μM/L) and RA combined with 100 nM/L VD3. RA: Raddeanin A; RA+VD3: Raddeanin A combined with 100 nM/L VD3.

Figure 3.

Effect of RA and RA with the addition of VD3 on autophagy in HeLa cells. (a) TEM showed the alterations of autophagosomes in HeLa cells treated with RA (8 μM/L) and RA combined with 100 nM/L VD3 (red arrows), and no autophagosome was observed in the control. (b) Immunofluorescence staining showed the changes in the number of autophagic puncta in HeLa cells treated with RA (8 μM/L) and RA combined with 100 nM/L VD3. (c, d) The relative mRNA expression of LC3 and Beclin-1 increased in HeLa cells treated with RA and RA plus 100 nM/L VD3 in a dose-dependent manner. The data is presented as mean ± SD from 3 independent experiments. * P < 0.05 and ** P < 0.01 versus control groups. And VD3 reinforced the effect of RA on autophagy in HeLa cells (P < 0.01). (e) The Western blotting showed that LC3 and Beclin-1 protein expression increased in HeLa cells treated with RA alone or RA combined with 100 nM/L VD3 in a dose-dependent manner. RA: Raddeanin A; RA+VD3: the combination of Raddeanin A and 100 nM/L 1,25(OH)₂D_3.

Figure 4.

The effect of RA and RA with the addition of 100 nM/L VD3 on HPV18E6 and HPV18E7 expression in HeLa cells. (a, b) Expression of HPV18E6 and HPV18E7 in each sample was normalized to that of GAPDH. Data was presented as mean ± SD of three independent experiments. ** P < 0.01 versus the control groups (0 μM/L RA and 0 μM/L VD3). (c) The protein alterations of HPV18E6 and HPV18E7 were examined by Western blotting. RA: Raddeanin A; RA+VD3: Raddeanin A combined with 100 nM/L VD3.

Figure 5.

The effect of RA and RA combined with 100 nM/L VD3 on PD-L1 and VDR in HeLa cells. HeLa cells treated with various doses of RA (2, 4 and 8 μM/L) and RA with the addition of 100 nM/L VD3 for 48 h. (a, b) Relative mRNA levels of PD-L1 and VDR in HeLa cells were detected by qRT-PCR. Data was presented as mean ± SD of three independent experiments. ** P < 0.01 versus the control groups (0 μM/L RA and 0 μM/L VD3). (c) The protein alterations of PD-L1 and VDR were examined by Western blotting. (C) The Western blotting analysis showed the alterations of Bax, Bcl-2, LC3 and Beclin-1 proteins in HeLa cells treated with RA alone, RA combined with 100 nM/L VD3 or RA combined with 100 nM/L VD3 under transfection with sh-PD-L1 and sh-VDR plasmids. RA: Raddeanin A; RA+VD3: Raddeanin A combined with 100 nM/L VD3.