Enhancement of apoptosis in HCT116 and HepG2 cells by Coix lacryma-jobi var. lacryma-jobi seed extract in combination with sorafenib

Abstract

Objective: Coix lacryma-jobi, a highly regarded Asian herb widely used in traditional Chinese medicine, is recognized for its dual benefits in promoting overall health and treating various diseases. While it exhibits moderate anticancer efficacy when used alone, this study investigated the enhanced anticancer potential of raw and cooked Coix lacryma-jobi var. lacryma-jobi (CL) seed extracts in combination with sorafenib against HCT116 and HepG2 cancer cell lines. The combination of sorafenib with other anticancer agents, including natural extracts, has garnered significant attention as a promising strategy for developing more effective cancer therapies.

Methods: Dry powders of raw (R) and cooked (C) CL seeds, obtained from a local commercial source in Thailand, were extracted and fractionated using ethanol (E), dichloromethane (D), ethyl acetate (A), and water (W) to produce eight fractions: CLRE, CLCE, CLRD, CLCD, CLRA, CLCA, CLRW, and CLCW. The coixol content in raw and cooked seed extracts was quantified and expressed as μg of coixol per gram of extract. The cytotoxic effects of these fractions were evaluated against HCT116 and HepG2 cells using the MTT assay. Fractions demonstrating the most significant cytotoxic responses were combined with sorafenib to evaluate their synergistic effects. Apoptosis induction and mitochondrial membrane potential (MMP) were assessed, and the underlying mechanism of apoptosis was explored by analyzing reactive oxygen species (ROS) generation and antioxidant protein expression levels. Additionally, the combination treatment's effect on the phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) pathway was investigated.

Results: One gram of CLCE and CLCD extracts contained higher coixol levels (7.02 μg and 9.69 μg, respectively) compared to CLRE and CLRD (2.66 μg and 5.96 μg, respectively). Coixol content in CLRA, CLRW, and CLCW fractions was undetectable under the study conditions. All extract fractions exhibited IC₅₀ values exceeding 1 mg/mL after 24- and 48-hour incubations with HCT116 and HepG2 cells, indicating limited cytotoxicity when used independently. CLRD and CLCD fractions were selected for combination studies at a concentration of 1 mg/mL, combined with sub-IC₅₀ concentrations of sorafenib to minimize its side effects. This combination significantly increased cytotoxicity, inducing apoptosis in HCT116 and HepG2 cells by elevating ROS levels and reducing the expression of superoxide dismutase 2 and catalase. Furthermore, the combination treatment downregulated the PI3K/AKT/mTOR pathway, indicating a targeted anticancer mechanism.

Conclusion: The combination of CLCD with sorafenib demonstrates significant potential as a strategy for future anticancer therapies. This CL seed extract, cultivated and commercially available in Thailand, shows promise as a natural supplement to enhance the efficacy of chemotherapy in upcoming clinical anticancer applications.

Keywords: Coix lacryma-jobi var. lacryma-jobi; HCT116; HepG2; PI3K/AKT/mTOR pathway; apoptosis; reactive oxygen species; seed extracts; sorafenib.

Fig. 1

Coixol contents in eight fractions of CL seed extracts (CLRE, CLRD, CLRA, CLRW, CLCE, CLCD, CLCA and CLCW).

Fig. 2

Cytotoxic effect of a combination of CL seed extracts of raw and cooked dichloromethane and ethyl acetate extract fractions (CLRD, CLRA, CLCD, and CLCA, respectively) and sorafenib on HCT116 and HepG2 cells. (A) A combination extracts at 1 000 µg/mL and sorafenib at 2 µmol/L and 6 µmol/L in HCT116 cells. (B) A combination of extracts at 1 000 µg/mL and sorafenib at 1 µmol/L and 4 µmol/L in HepG2 cells. The control vehicle comprised 0.8% DMSO. The analysis of mean ± SD from at least three different experiments was assessed with One-way ANOVA using Tukey’s HSD test. *P < 0.05 vs vehicle control, ^aP < 0.05 vs a single extract fraction treatment, ^bP < 0.05 vs a single sorafenib treatment at its same concentration.

Fig. 2

Cytotoxic effect of a combination of CL seed extracts of raw and cooked dichloromethane and ethyl acetate extract fractions (CLRD, CLRA, CLCD, and CLCA, respectively) and sorafenib on HCT116 and HepG2 cells. (A) A combination extracts at 1 000 µg/mL and sorafenib at 2 µmol/L and 6 µmol/L in HCT116 cells. (B) A combination of extracts at 1 000 µg/mL and sorafenib at 1 µmol/L and 4 µmol/L in HepG2 cells. The control vehicle comprised 0.8% DMSO. The analysis of mean ± SD from at least three different experiments was assessed with One-way ANOVA using Tukey’s HSD test. *P < 0.05 vs vehicle control, ^aP < 0.05 vs a single extract fraction treatment, ^bP < 0.05 vs a single sorafenib treatment at its same concentration.

Fig. 3

Cytotoxic effect of a combination of CLCD and sorafenib on IMR-90 cells. The control vehicle comprised 0.8% DMSO. The analysis of mean ± SD from at least three different experiments was assessed with One-way ANOVA using Tukey’s HSD test. *P < 0.05 vs vehicle control, ^aP < 0.05 vs a single CLCD fraction treatment, and ^bP < 0.05 vs a single sorafenib treatment at its same concentration.

Fig. 4

Combination of CLCD and sorafenib triggered apoptosis in HCT116 and HepG2 cells. (A) HCT116 cells treated for 48 h with a combination of CLCD at 1 000 µg/mL and sorafenib at 6 µmol/L. (B) Histogram depicting percentage of apoptotic HCT116 cells. (C) HepG2 cells treated for 48 h with a combination of CLCD at 1 000 µg/mL and sorafenib at 1 µmol/L. (D) Histogram illustrating proportion of apoptotic HepG2 cells. The control vehicle comprised 0.8% DMSO. The analysis of mean ± SD from at least three different experiments was assessed with One-way ANOVA using Tukey’s HSD test. *P < 0.05 vs vehicle control, ^aP < 0.05 vs CLCD fraction treatment, ^bP < 0.05 vs sorafenib treatment.

Fig. 5

Combination of CLCD and sorafenib triggered dissipation of MMP-dependent apoptosis in HCT116 and HepG2 cells. Flow cytometric measurement of MMP staining was done with JC-1 dye in (A) HCT116 cells treated for 48 h with a combination of CLCD at 1 000 µg/mL and sorafenib at 6 µmol/L. (B) Histogram displaying percentage of red fluorescence intensity of HCT116 cells. (C) HepG2 cells treated for 48 h with a combination of CLCD at 1 000 µg/mL and sorafenib at 1 µmol/L. (D) Histogram displaying percentage of red fluorescence intensity of HepG2 cells. The control vehicle comprised 0.8% DMSO. The analysis of mean ± SD from at least three different experiments was assessed with One-way ANOVA using Tukey’s HSD test. *P < 0.05 vs vehicle control, ^aP < 0.05 vs CLCD fraction treatment. ^bP < 0.05 vs sorafenib treatment.

Fig. 6

Combination of CLCD and sorafenib triggered dissipation of MMP-dependent apoptosis in HCT116 and HepG2 cells assessed by fluorescence microscopy. Representative fluorescent images of (A) HCT116 cells treated for 48 h with a combination of CLCD at 1 000 µg/mL and sorafenib at 6 µmol/L, and (B) HepG2 cells treated for 48 h with a combination of CLCD at 1 000 µg/mL and sorafenib at 1 µmol/L. Hoechst 33342 was used to stain apoptotic cell nuclei with DNA fragmentation. The control vehicle comprised 0.8% DMSO. Scale bar = 20 µm, under × 40 magnification, visualized by fluorescence microscopy.

Fig. 7

Combination of CLCD and sorafenib increased ROS formation in HCT116 and HepG2 cells by flow cytometry following. HCT116 cells treated for 48 h with a combination of CLCD at 1 000 µg/mL and sorafenib at 6 µmol/L (A−B). HepG2 cells treated for 48 h with a combination of CLCD at 1 000 µg/mL and sorafenib at 1 µmol/L (C−D). The control vehicle comprised 0.8% DMSO. The analysis of mean ± SD from at least three different experiments was assessed with One-way ANOVA using Tukey’s HSD test. *P < 0.05 vs vehicle control, ^aP < 0.05 vs CLCD fraction treatment. ^bP < 0.05 vs sorafenib treatment.

Fig. 8

Combination of CLCD and sorafenib decreased SOD2 and catalase expression in HCT116 and HepG2 cells by Western blotting. Relative expression levels compared to β-actin of HCT116 (A−C) and HepG2 (D−F) cells. The control vehicle comprised 0.8% DMSO. The analysis of mean ± SD from at least three different experiments was assessed with One-way ANOVA using Tukey’s HSD test. *P < 0.05 vs vehicle control, ^aP < 0.05 vs CLCD fraction treatment. ^bP < 0.05 vs sorafenib treatment.

Fig. 9

Significant role of ROS formation in inducing apoptosis in HCT116 and HepG2 cells treated with a combination of CLCD and sorafenib analyzed using flow cytometry after a 2-hour pre-treatment with 10 mmol/L N-acetylcysteine (NAC). (A and C) HCT116 cells treated for 48 h with a combination of CLCD at 1 000 µg/mL and sorafenib at 6 µmol/L, (B and D) HepG2 cells treated for 48 h with a combination of CLCD at 1 000 µg/mL and sorafenib at 1 µmol/L. The control vehicle comprised 0.8% DMSO. The analysis of mean ± SD from at least three different experiments was assessed with One-way ANOVA using Tukey’s HSD test. *P < 0.05 vs vehicle control, ^aP < 0.05 vs CLCD treatment. ^bP < 0.05 vs sorafenib treatment, ^cP < 0.05 vs a combination of CLCD and sorafenib treatment.

Fig. 10

Combination of CLCD and sorafenib involved downregulation of expression of phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway in HCT116 and HepG2 cells. Representative Western blotting images and bar graphs of relative expression levels of PI3K, Akt, and mTOR proteins following (A−C) HCT116 cells treated for 48 h with a combination of CLCD at 1 000 µg/mL and sorafenib at 6 µmol/L and (D−F) HepG2 cells treated for 48 h with a combination of CLCD at 1 000 µg/mL and sorafenib at 1 µmol/L. The control vehicle comprised 0.8% DMSO. The analysis of mean ± SD from at least three different experiments was assessed with One-way ANOVA using Tukey’s HSD test. *P < 0.05 vs vehicle control, ^aP < 0.05 vs CLCD treatment. ^bP < 0.05 vs sorafenib treatment.