Nutr Res Pract.  2024 Feb;18(1):62-77. 10.4162/nrp.2024.18.1.62.

Combination of oxaliplatin and β-carotene suppresses colorectal cancer by regulating cell cycle, apoptosis, and cancer stemness in vitro

• Lee J
• Heo SC
• Kim Y

• KMID: 2552280
• DOI: http://doi.org/10.4162/nrp.2024.18.1.62

Abstract

BACKGROUND/OBJECTIVES
Colorectal cancer (CRC) is the third most common cancer worldwide with a high recurrence rate. Oxaliplatin (OXA) resistance is one of the major reasons hindering CRC therapy. β-Carotene (BC) is a provitamin A and is known to have antioxidant and anticancer effects. However, the combined effect of OXA and BC has not been investigated. Therefore, this study investigated the anticancer effects and mechanism of the combination of OXA and BC on CRC.
MATERIALS/METHODS
In the present study, the effects of the combination of OXA and BC on cell viability, cell cycle arrest, and cancer stemness were investigated using HCT116, HT29, OXA-resistant cells, and human CRC organoids.
RESULTS
The combination of OXA and BC enhanced apoptosis, G _{2,/sub> /M phase cell cycle arrest, and inhibited cancer cell survival in human CRC resistant cells and CRC organoids without toxicity in normal organoids. Cancer stem cell marker expression and self-replicating capacity were suppressed by combined treatment with OXA and BC. Moreover, this combined treatment upregulated apoptosis and the stem cell-related JAK/STAT signaling pathway.
CONCLUSIONS
Our results suggest a novel potential role of BC in reducing resistance to OXA, thereby enhances the anticancer effects of OXA. This enhancement is achieved through the regulation of cell cycle, apoptosis, and stemness in CRC.}

Keyword

Beta-carotene; oxaliplatin; colorectal neoplasm; organoids; cell cycle

Figure

• 

  Fig. 1 OXA and BC decrease cell viability of HCT116, HT29, and HCT116/OXA cells. MTT assay was performed to assess cell viabilities of (A) HCT116, (B) HT29, and (C) HCT116/OXA. HCT116, HT29, and HCT116/OXA cells were treated with OXA (0, 0.5, 1, 2, 4, 8, and 16 µM) and BC (0, 10, 20, 40, 80, and 160 µM) for 48 h. (D) HCT116, HT29, HCT116/OXA cells were treated with IC50 doses of OXA (1.57 µM for HCT116, 2.62 µM for HT29, and 4.15 µM for HCT116/OXA), or BC (32.51 µM for HCT116, 32.70 µM for HT29, and 32.51 µM for HCT116/OXA) for 48 h. Data are presented as means ± SEM from at least 3 independent experiments. The analysis was performed by one-way analysis of variance, followed by the Newman-Keuls post-test for multiple comparisons. Statistically significant differences between groups are indicated by different alphabetical letters (P < 0.05).O.D., optical density; CTRL, control; OXA, oxaliplatin; BC, β-carotene; Combi, combination; IC50, half maximal inhibitory concentration.

• 

  Fig. 2 OXA, BC, or their combination induces G2 phase cell cycle arrest in HCT116 and HCT116/OXA cells. HCT116: CTRL, Control; OXA, 1.57 µM OXA; BC, 32.51 µM BC; Combi, 1.57 µM OXA + 32.51 µM BC. HCT116/OXA: CTRL, Control; OXA, 4.15 µM OXA; BC, 31.52 µM BC; Combi, 4.15 µM OXA + 31.52 µM BC. HCT116 and HCT116/OXA cells were treated with OXA, BC, or their combination for 48 h, and cell cycle analysis was conducted using FACS. Comparison of each cell cycle phase ratio in HCT116 and HCT116/OXA cells was shown. Data are presented as means ± SEM from at least 3 independent experiments. The analysis was performed by one-way analysis of variance, followed by the Newman-Keuls post-test for multiple comparisons. Statistically significant differences between groups are indicated by different alphabetical letters (P < 0.05). CTRL, control; OXA, oxaliplatin; BC, β-carotene; HCT116/OXA, oxaliplatin resistant HCT116 cell line; Combi, combination; FACS, fluorescence-activated cell sorting.

• 

  Fig. 3 OXA, BC, or their combination induces apoptosis in human CRC cells and OXA -resistant HCT116/OXA cells. HCT116: CTRL; OXA, 1.566 µM OXA; BC, 32.51 µM BC; Combi, 1.566 µM OXA + 32.51 µM BC. HT29: CTRL; OXA, 2.624 µM OXA; BC, 32.7 µM BC; Combi, 2.624 µM OXA + 32.7 µM BC. HCT116/OXA: CTRL; OXA, 4.15 µM OXA; BC, 31.52 µM BC; Combi, 4.15 µM OXA + 31.52 µM BC. Human CRC HCT116, HT29, and HCT116/OXA cells were treated with OXA, BC, or their combination for 48 h. Western blot assay was performed to detect the protein expressions of cleaved caspase-3, Bax, and Bcl-2, with alpha-tubulin as a loading control. The results are quantified by the ImageJ quantification program. Representative blots. Quantification of cleaved caspase-3, Bax, and Bcl-2 levels normalized to alpha-tubulin in HCT116, HT29, and HCT116/OXA cells are presented as means ± SEM from at least 3 independent experiments. The analysis was performed by 1-way analysis of variance, followed by the Newman-Keuls post-test for multiple comparisons. Statistically significant differences between groups are indicated by different alphabetical letters (P < 0.05).CTRL, control; OXA, oxaliplatin; BC, β-carotene; Combi, combination; HCT116/OXA, oxaliplatin resistant HCT116 cell line; CRC, colorectal cancer.

• 

  Fig. 4 OXA, BC, or their combination decreases stemness of cancer cells. HCT116: CTRL; OXA, 1.57 µM OXA; BC, 32.51 µM BC; Combi, 1.57 µM OXA + 32.51 µM BC. HCT116/OXA: CTRL; OXA, 4.15 µM OXA; BC, 31.52 µM BC; Combi, 4.15 µM OXA + 31.52 µM BC. (A) HCT116 and HCT116/OXA cells were treated with OXA, BC, or their combination in 6-well plates for 7–10 day. Colonies were stained and imaged. The number of colonies containing ≥ 50 cells was counted, and plating efficiency (percentage of colonies per input ± SEM) was calculated. (B) HCT116 and HCT116/OXA cells were cultured in sphere medium in poly-HEMA coated 6-well plates and treated with OXA and BC individually or in combination. Sphere formation was visualized (magnification, ×100). The number of spheres was counted, and (C) mRNA expressions of CD133, SOX2, and OCT4 in HCT116 and HCT116/OXA cells were assessed by quantitative RT-PCR, with GAPDH as a loading control. (D) CD133, Sox2, Oct4, p-JAK1, JAK1, p-STAT3, and STAT3 protein expressions in HCT116 and HCT116/OXA cells were also analyzed by Western blot assay, with alpha-tubulin as a loading control. Representative Western blots are shown. Quantification graph of CD133, Sox2, and Oct4 levels normalized to alpha-tubulin in HCT116 cells and HCT116/OXA cells. The results are quantified by the ImageJ quantification program. Data are presented as means ± SEM from at least 3 independent experiments. The analysis was performed by one-way analysis of variance, followed by the Newman-Keuls post-test for multiple comparisons. Statistically significant differences between groups are indicated by different alphabetical letters (P < 0.05).CTRL, control; OXA, oxaliplatin; BC, β-carotene; Combi, combination; HCT116/OXA, oxaliplatin resistant HCT116 cell line.

• 

  Fig. 5 JAK/STAT pathway is regulated in the anti-cancer effect of OXA, BC, or their combination in CRC. HCT116: CTRL; OXA, 1.57 µM OXA; BC, 32.51 µM BC; Combi, 1.57 µM OXA + 32.51 µM BC. HCT116/OXA: CTRL; OXA, 4.15 µM OXA; BC, 31.52 µM BC; Combi, 4.15 µM OXA + 31.52 µM BC. Representative Western blots and relative quantification of p-JAK1 levels to JAK1 and p-STAT3 levels to STAT3 in HCT116 and HCT116/OXA cells were shown. The results are quantified by the ImageJ quantification program. Data are presented as means ± SEM from at least 3 independent experiments. The analysis was performed by one-way analysis of variance, followed by the Newman-Keuls post-test for multiple comparisons. Statistically significant differences between groups are indicated by different alphabetical letters (P < 0.05).CTRL, control; OXA, oxaliplatin; BC, β-carotene; Combi, combination; HCT116/OXA, oxaliplatin resistant HCT116 cell line.

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  Fig. 6 OXA, BC, or their combination reduces cell viability and tumor differentiation in human colon organoids. Human colon organoids were treated with IC50 doses of OXA (1.57 µM), BC (32.51 µM), or Combi (OXA 1.57 µM and BC 32.51 µM) for 48 h. (A) Cell viability was assessed using the MTT assay. (B) The size and number of human normal and tumor organoids were measured and imaged using a phase-contrast microscope (magnification, ×100). (C) Immunofluorescence staining for beta-catenin (green) was performed on human colon tumor organoids (magnification, ×100). Data are presented as means ± SEM from at least 3 independent experiments. The analysis was performed by one-way analysis of variance, followed by the Newman-Keuls post-test for multiple comparisons. Statistically significant differences between groups are indicated by different alphabetical letters (P < 0.05). O.D., optical density; CTRL, control; BC, β-carotene; OXA, oxaliplatin; Combi, combination; IC50, half maximal inhibitory concentration.

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