Nutr Res Pract.  2025 Feb;19(1):131-142. 10.4162/nrp.2025.19.1.131.

Orostachys japonicus induce caspase-dependent apoptosis in HeLa human cervical cancer cells

• Kim SH
• Lee DS

• KMID: 2565390
• DOI: http://doi.org/10.4162/nrp.2025.19.1.131

Abstract

BACKGROUND/OBJECTIVES
Orostachys japonicus A. Berger (O. japonicus) is a perennial herb belonging to the Crassulaceae family that has been traditionally used to treat inflammation, fever, and poisoning. Although studies on the anticancer activity of O. japonicus have been conducted, its effect on virus-induced cancers has yet to be elucidated.
MATERIALS/METHODS
In the present study, we investigated the effects and mechanisms of action of the ethyl acetate fraction of O. japonicus extract (E-OJ) on the viability and apoptosis of HeLa cervical cancer cells.
RESULTS
The effect of E-OJ on HeLa cells was compared to that of kaempferol, quercetin, and gallic acid, which are components of O. japonicus. Treatment with E-OJ induced a concentration-dependent decrease in cell viability, as confirmed by MTS assay. Pretreatment with a broad-spectrum caspase inhibitor resulted in the recovery of cell viability. Western blot analysis was conducted to determine whether the induction of apoptosis was caspasedependent. E-OJ induced apoptosis by increasing Bax/Bcl-2 ratio. Furthermore, it modulated the levels of cleaved caspase-3, -8, and -9, indicative of an impact on both the intrinsic and extrinsic pathways of apoptosis. Pretreatment with caspase inhibitors reduced caspase activity.
CONCLUSION
These results suggest that the anticancer activity of O. japonicus is mediated by caspases, resulting in a decrease in the viability of HeLa cells.

Keyword

Extract; apoptosis; caspase; Bcl-2; Bax; HeLa cells

Figure

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  Fig. 1 Effect of O. japonicus on HeLa cell viability. HeLa cells were treated with the various concentrations of the EtOAc fraction of O. japonicus (E-OJ) for 12 and 24 h. Cell viability was measured using MTS assay. The results are presented as mean ± SD.E-OJ, the ethyl acetate fraction of O. japonicus extract.*P < 0.05; ***P < 0.001.

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  Fig. 2 Effect of general caspase inhibitor on viability in HeLa cells after treatment with E-OJ. HeLa cells were treated with 10 μg/mL E-OJ, 80 μM kaempferol (K), quercetin (Q), and gallic acid (G) for 12 h, followed by treatment with or without Z-VAD-FMK (a general caspase inhibitor). Cell viability was determined using the MTS assay. Values are expressed as mean ± SD.E-OJ, the ethyl acetate fraction of O. japonicus extract.***P < 0.001.

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  Fig. 3 Alterations in the expression of Bcl-2 and Bax proteins upon treatment with E-OJ, kaempferol, quercetin, and gallic acid. HeLa cells were treated with E-OJ, kaempferol (K), quercetin (Q), or gallic acid (G) for 12 h, respectively. (A) Western blotting analysis was performed to analyze the expression of Bcl-2 and Bax proteins, with GAPDH serving as an internal control. (B) The ratio of Bax/Bcl-2 was calculated. The band intensities were measured using densitometry in three separate experiments with comparable results. The data are expressed as mean ± SD.E-OJ, the ethyl acetate fraction of O. japonicus extract.*P < 0.05; **P < 0.01; ***P < 0.001.

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  Fig. 4 Regulation of caspase-3, -8 and -9 levels in HeLa cells following treatment with E-OJ. HeLa cells were treated with different concentration of E-OJ, 80 μM of kaempferol (K), quercetin (Q), or gallic acid (G) for 12 h. The level of pro-caspase-3, cleaved caspase-3(A), pro-caspase-8, cleaved caspase-8 (B), pro-caspase-9, and cleaved caspase-9 (C) were analyzed using western blotting analysis. The band density was quantified and presented in a bar graph with GAPDH as an internal control. The results were obtained from three separate experiments and expressed as mean ± SD.E-OJ, the ethyl acetate fraction of O. japonicus extract.*P < 0.05; **P < 0.01; ***P < 0.001.

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  Fig. 5 Effect of caspase inhibitors on apoptosis of HeLa cells treated with E-OJ. Hela cells were pretreated with Z-VAD-FMK (pan-caspase inhibitor), Z-DEVD-FMK (caspase-3 inhibitor), Z-IETD-FMK (caspase-8 inhibitor), and Z-LEHD-FMK (caspase-9 inhibitor) and then treated with 10 μg/mL E-OJ, 80 μM kaempferol (K), quercetin (Q), and gallic acid (G) for 12 h. The levels of pro-caspase-3, cleaved caspase-3 (A), pro-caspase-8, cleaved caspase-8 (B), pro-caspase-9, and cleaved caspase-9 (C) were analyzed using western blotting analysis with GAPDH as an internal control. Values are expressed as mean ± SD. Significance compared to the cells treated with E-OJ only.E-OJ, the ethyl acetate fraction of O. japonicus extract.*P < 0.05; **P < 0.01; ***P < 0.001.

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  Fig. 6 Mechanisms of caspase-dependent apoptosis induced OJ.OJ, Orostachys japonicus.

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