OBJECTIVE: While higher therapeutic doses of toxic cardiac glycosides derived from Cerbera odollam are frequently employed in cases of suicide or homicide, ongoing research is investigating the potential anticancer properties of low-concentration extracts obtained from the fruits of C. odollam. The present study aimed to determine the enhanced anticancer effects and minimize potential side effects of combining extracts from C. odollam fruits from Thailand with sorafenib against HCT116 and HepG2 cells. METHODS: The dried powder of fresh green fruits of C. odollam was fractionated, and its phytochemical contents, including total cardiac glycosides, phenolics, flavonoids, and triterpenoids, were quantified. The cytotoxic effects of these fractions were evaluated against HCT116 and HepG2 cells using the MTT assay. The fractions showing the most significant response in HCT116 and HepG2 cells were subsequently combined with sorafenib to examine their synergistic effects. Apoptosis induction, cell cycle progression, and mitochondrial membrane potential (MMP) were then assessed. The underlying mechanism of the apoptotic effect was further investigated by analyzing reactive oxygen species (ROS) generation and the expression levels of antioxidant proteins. RESULTS: Phytochemical analysis showed that C. odollam-ethyl acetate fraction (COEtOAc) was rich in cardiac glycosides, phenolics, and flavonoids, while the dichloromethane fraction (CODCM) contained high levels of triterpenoids and saponins. Following 24 h treatment, HCT116 showed the most significant response to COEtOAc, while HepG2 responded well to CODCM with IC₅₀ values of (42.04 ± 16.94) μg/mL and (123.75 ± 14.21) μg/mL, respectively. Consequently, COEtOAc (20 μg/mL) or CODCM (30 μg/mL), both administered at sub-IC₅₀ concentrations, were combined with sorafenib at 6 μmol/L for HCT116 cells and 2 μmol/L for HepG2 cells, incubated for 24 h. This combination resulted in a significant suppression in cell viability by approximately 50%. The combination of treatments markedly enhanced apoptosis, diminished MMP, and triggered G₀/G₁ phase cell cycle arrest compared to the effects of each treatment administered individually. Concurrently, increased formation of ROS and decreased expression of the antioxidant enzymes superoxide dismutase 2 and catalase supported the proposed mechanism of apoptosis induction by the combination treatment. Importantly, the anticancer effect demonstrated a specific targeted action with a favorable safety profile, as evidenced by HFF-1 cells displaying IC₅₀ values 2-3 times higher than those of the cancer cells. CONCLUSION Utilizing sub-IC₅₀ concentrations of COEtOAc or CODCM in combination with sorafenib can enhance targeted anticancer effects beyond those achieved with single-agent treatments, while mitigating opposing side effects. Future research will focus on extracting and characterizing active constituents, especially cardiac glycosides, to enhance the therapeutic potential of anticancer compounds derived from toxic plants.

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.