[Blueberry anthocyanins combined with oxaliplatin regulate the GSK-3β pathway to enhance chemosensitivity in colon cancer cells].

OBJECTIVE: To investigate the effect and potential mechanism of blueberry anthocyanins(BA) in enhancing the chemosensitivity of colon cancer cells to oxaliplatin(OXA).

METHODS: Colon cancer HCT116 and LOVO cells were treated with varying concentrations of BA(0, 10, 25, 50, 100, 500, 750 and 1000 μg/mL). HCT116 cells were treated with different concentrations of OXA(0, 0.8, 1.6, 3.125, 6.25, 12.5, 25 and 50 μmol/L), and LOVO cells were treated with OXA(0, 0.1, 0.2, 0.4, 0.8, 1.6, 3.125 and 6.25 μmol/L). Cell viability was assessed using the Methylene Blue(MB) assay. HCT116 cells were treated with 20 μg/mL BA and 0.15 μmol/L OXA alone or in combination, while LOVO cells were treated with 50 μg/mL BA and 0.10 μmol/L OXA alone or in combination. Cell viability was measured by the MB or CCK-8 assay, and colony formation was examined. Cell cycle distribution was analyzed by flow cytometry. Western blot was performed to detect the expression of cell cycle-related proteins(Cyclin D1, Cyclin B1, CDK4), proliferating cell nuclear antigen(PCNA), and glycogen synthase kinase-3β(GSK-3β). After pretreatment with 50 μmol/L lithium chloride(LiCl) for 6 hours, HCT116 cell viability was assessed by CCK-8 assay, and GSK-3β protein expression was measured by Western blot. A xenograft tumor model was established by subcutaneously injecting 0.2 mL of a cell suspension containing 2.0×10~6 HCT116 cells into the right shoulder of 4-week-old female BALB/c nude mice. The mice were divided into four groups(n=5 per group): control group, BA group, OXA group, and BA+OXA combination group. Starting from the second day after HCT116 cell inoculation, the mice were intraperitoneally injected with the test substances every other day. The control group received 10% dimethyl sulfoxide(DMSO). BA was dissolved in physiological saline containing 10% DMSO and administered at a dose of 60 mg/kg. OXA was dissolved in physiological saline and administered at a dose of 2.0 mg/kg. The combination group received both 60 mg/kg BA and 2.0 mg/kg OXA. Administration continued for 35 days. Body weight was measured every other day between 8:00 and 10:00 AM. Starting from day 13 after HCT116 cell inoculation, tumor diameter was measured every other day using a caliper. PCNA protein expression in the xenograft tumors was detected by immunohistochemistry. The basic morphological structure, pathological features, and drug-induced histological changes of the xenograft tumors were examined by hematoxylin and eosin(HE) staining. The expression of GSK-3β protein and cell cycle-related proteins was detected by Western blot.

RESULTS: Both BA and OXA inhibited the viability of HCT116 and LOVO cells in a dose-and time-dependent manner(P<0.01). The 72-hour median effect concentration(EC_(50)) for BA and OXA in HCT116 cells was(194.23±60.96) μg/mL and(1.54±0.64) μmol/L, respectively. In LOVO cells, the EC_(50) was(176.13±25.26) μg/mL and(0.32±0.03) μmol/L, respectively. Analysis of the combination treatment showed that the combination index(CI) for 20 μg/mL BA combined with 0.15 μmol/L OXA in HCT116 cells was 0.67, and the CI for 50 μg/mL BA combined with 0.10 μmol/L OXA in LOVO cells was 0.73. The combination of BA and OXA significantly inhibited the proliferation and colony formation of HCT116 and LOVO cells(P<0.01) and markedly suppressed tumor growth in the nude mouse xenograft model(P<0.05). HE staining revealed that compared with the control group, all treatment groups exhibited increased structural disorganization and necrosis in tumor tissues. Specifically, the BA group showed significant necrotic areas; the OXA group also displayed some necrosis; the combination group exhibited a significant reduction in cancer cell density, extensive necrotic areas, and more pronounced lymphocyte infiltration. Immunohistochemical detection showed the number of PCNA-positive cells in the xenograft tissues per field: control group(180±15), BA group(103±51), OXA group(83±11), and BA+OXA group(35±3). The number of PCNA-positive cells in the BA+OXA group was significantly lower than that in the control group(P<0.01). Furthermore, BA combined with OXA arrested the cell cycle of HCT116 cells in the G_2/M phase and that of LOVO cells in the G_0/G_1 phase(P<0.01), and decreased the expression of PCNA, Cyclin D1, Cyclin B1, CDK4, and pGSK-3β proteins in HCT116 cells and nude mouse xenograft tumor tissues(P<0.01). Compared with the non-pretreated group, pretreatment with LiCl significantly increased the viability of HCT116 cells and the expression of pGSK-3β protein(P<0.01).

CONCLUSION: BA enhances the chemosensitivity of colon cancer cells to OXA by modulating the GSK-3β signaling pathway.