[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.