Potent anti-cancer activity of Sphaerocoryne affinis fruit against cervical cancer HeLa cells via inhibition of cell proliferation and induction of apoptosis

Abstract

Background: Cervical cancer remains a significant global health issue, highlighting the need for effective therapeutic strategies. Given that Sphaerocoryne affinis (SA) has shown potential anti-cancer activity in several cancer types, herein, we investigate the effects of SA fruit (SAF) on human cervical cancer HeLa cells and their underlying mechanisms of action.

Methods: SAF extract cytotoxicity was assessed in various cancer cell lines. The effects of the hexane fraction (SAF-Hex) on HeLa cell viability, cell cycle protein expression, apoptosis, and DNA damage were evaluated using cytotoxicity assays, Western blotting, quantitative PCR, 4',6-diamidino-2-phenylindole (DAPI) staining, and a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay.

Results: SAF-Hex selectively inhibited HeLa cell viability with an IC50 of 4.20 ± 0.36 µg/mL and a selectivity index of 5.11 ± 0.58. The time-dependent cytotoxicity assay showed decreased cell survival after 48 h of treatment, accompanied by morphological changes and apoptotic bodies in HeLa cells. SAF-Hex also suppressed HeLa cell cycle proteins (Cyclin E, CDK2, and CDK1), reduced PCNA transcription, and diminished AKT and mTOR activation, thus inhibiting cell proliferation. The increased γH2AX expression, DNA fragmentation, and caspases-3 and -9 activation indicated SAF-Hex-induced DNA damage and apoptosis. However, the BAX/BCL-2 ratio remained unchanged, and BAX and BCL2 expression was attenuated.

Conclusion: SAF-Hex effectively inhibits HeLa cell proliferation and induces DNA damage in that cervical cancer cell line activating apoptosis through the intrinsic pathway. Interestingly, the BAX/BCL-2 ratio remained unchanged while BAX and BCL2 transcription was attenuated. Hence, further research is required to explore this unexpected finding and facilitate the development of novel therapies targeting cervical cancer HeLa cells.

Keywords: Anti-cancer; Apoptosis; Cervical cancer; DNA damage; HeLa cells; Sphaerocoryne affinis.

Fig. 1

Effects of SAF-Hex on cell cytotoxicity. Relative cell viability of various cell lines following treatment with A SAF-EA and B SAF-Hex. C Dose-dependent cytotoxicity of SAF-Hex, assessed at concentrations ranging from 0 to 100 and 400 µg/mL on HeLa (circle) and HEK293 (square) cells. Relative inhibition of cell survival was plotted against the logarithm of SAF-Hex concentrations (µg/mL). D Time-dependent cytotoxicity of SAF-Hex at IC50 (black square) on HeLa cells over 72 h. The control group was cultured without SAF-Hex (white circle). E Morphological changes in HeLa cells during the time-dependent cytotoxicity assay at 15 × magnification. F Alterations in the nuclei of HeLa cells following treatment with SAF-Hex at the IC50 concentration (4.2 µg/mL), as observed with DAPI staining at 60 × magnification. Scale bars represent 50 μm. IC50, half-maximal inhibitory concentration; SAF-EA, SAF-Hex, ethyl acetate, and hexane fractions of the SAF extract, respectively. Data are represented as mean ± SD; n = 3–4

Fig. 2

Effects of SAF-Hex on HeLa cell proliferation. HeLa cells were treated with SAF-Hex at concentrations of 0, 5, and 25 µg/mL for 36 h. A Western blotting was performed to assess the expression levels of proteins involved in the cell cycle and cell survival. Original images of blots are shown in Fig. S1. B Fold changes in expression of Cyclin E, CDK2, and CDK1. C Relative transcription levels of PCNA, as determined using quantitative PCR. SAF-Hex, hexane fraction of the SAF extract. Data are represented as mean ± SD and analyzed by one-way ANOVA; *P < 0.05, **P < 0.01, ***P < 0.001; n = 3–4

Fig. 3

SAF-Hex induces DNA damage in HeLa cells. DNA damage was assessed in HeLa cells treated with SAF-Hex at concentrations of 0, 5, and 25 µg/mL for 36 h using A Western blotting for γH2AX and B qPCR for P53 mRNA levels. Original images of Western blots are shown in Fig. S2. C TUNEL assay. DNA-fragmented positive cells appear brown. Images were captured at a magnification of 20 × , with scale bars indicating 50 μm. qPCR, quantitative PCR; SAF-Hex, hexane fraction of the SAF extract. Data are represented as mean ± SD and analyzed using one-way ANOVA; n = 3–4; ***P < 0.001

Fig. 4

SAF-Hex induces apoptotic cell death in HeLa cells. HeLa cells were treated with SAF-Hex at concentrations of 0, 5, and 25 µg/mL for 36 h. A Western blotting was used to measure apoptosis-related protein expression. Original images of blots are shown in Fig. S3. B Relative ratio of BAX and BCL-2, as determined using Western blot results. C qPCR was utilized to determine the relative transcription levels of BAX and BCL2. qPCR, quantitative PCR; SAF-Hex, hexane fraction of the SAF extract. Data are represented as mean ± SD and analyzed using one-way ANOVA; *P < 0.05, **P < 0.01; ns, not significant; n = 3–4