Antitumor activity of resveratrol against human osteosarcoma cells: a key role of Cx43 and Wnt/β-catenin signaling pathway

Abstract

Osteosarcoma is a high-grade bone sarcoma with strong invasive ability. However, treatment with traditional chemotherapeutic drugs is limited by low tolerability and side effects. Resveratrol has been reported previously to have selective antitumor effect on various tumor cells while little is known about its effects and underlying mechanism in osteosarcoma biology. In this study, we found that resveratrol inhibits proliferation and glycolysis, induces apoptosis and reduces the invasiveness of U2-OS cells in vitro. After treatment with resveratrol, the expression of related Wnt/β-catenin signaling pathway target genes, such as β-catenin, c-myc, cyclin D1, MMP-2 and MMP-9, was downregulated and an increased E-cadherin level was observed as well. Additionally, the dual luciferase assay results also indicated that resveratrol suppressed the activity of Wnt/β-catenin signaling pathway. Interestingly, we noticed that the expression of connexin 43 (Cx43) increased with the prolongation of resveratrol treatment time. To further investigate the relationship between Cx43 and the Wnt/β-catenin signaling pathway in osteosarcoma, we used lentiviral-mediated shRNA to knockdown the expression of Cx43. Knockdown of Cx43 activated the Wnt/β-catenin signaling pathway, promoted proliferation and invasion, and inhibited apoptosis of U2-OS cells. Taken together, our results demonstrate that the antitumor activity of resveratrol against U2-OS cells in vitro occurs through up-regulating Cx43 and E-cadherin, and suppressing the Wnt/β-catenin signaling pathway. Moreover, Cx43 expression is negatively related to the activity of the Wnt/β-catenin pathway in U2-OS cells.

Keywords: Wnt/β-catenin signaling; antitumor activity; connexin 43; osteosarcoma; resveratrol.

Figure 1. Resveratrol inhibits the proliferation and glycolysis of U2-OS cells, and knockdown of Cx43 promotes the proliferation of U2-OS cells

(A) The inhibiting effect of resveratrol on U2-OS cell proliferation, mean ± SD, n = 3, ^*P<0.05, ^**P<0.01, vs. 0 μg/ml group. (B) Proliferation of U2-OS cells among the untreated, NTC and shCx43 groups, mean ± SD, n = 3, ^*P<0.05, ^**P<0.01, vs. untreated group (blank). ^#P<0.05 vs. NTC group. (C and D) A macrograph of U2-OS cell colony formation following treatment with resveratrol (C) and Cx43 knockdown (D). (E) Cloning efficiencies of U2-OS cells following addition with resveratrol, mean ± SD, n = 3, ^**P<0.01, vs. 0 μg/ml group. ^##P<0.01 vs. 6 μg/ml group. (F) Cloning efficiencies of Cx43 knockdown U2-OS cells, mean ± SD, n = 3, ^*P<0.05, vs. untreated group. ^#P<0.05 vs. NTC group. (G) Effect of resveratrol on glucose uptake of U2-OS cells, mean ± SD, n = 3, ^*P<0.05, ^**P<0.01, vs. 0 μg/ml group. ^#P<0.05 vs. 6 μg/ml group. (H) Effect of resveratrol on lactate production of U2-OS cells, mean ± SD, n = 3, ^*P<0.05, ^**P<0.01, vs. 0 μg/ml group. ^#P<0.05 vs. 6 μg/ml group.

Figure 2. Resveratrol alters the morphology of U2-OS cells and induces apoptosis, and knockdown of Cx43 reduces apoptosis

(A) Changes in cell morphology caused by increasing concentrations of resveratrol. (B and C) Analysis of apoptosis of U2-OS cells by flow cytometry, mean ± SD, n = 3, ^**P<0.01, vs. 0 μg/ml group. ^##P<0.01 vs. 6 μg/ml group. (D and E) Analysis of apoptosis of Cx43 knockdown U2-OS cells, mean ± SD, n = 3, ^**P<0.01, vs. untreated group. ^##P<0.01 vs. NTC group.

Figure 3. Resveratrol suppresses the migration and invasion of U2-OS cells, and knockdown of Cx43 enhances the migration and invasion of U2-OS cells

(A and B) Changes of U2-OS cell migration following treatment with resveratrol, mean ± SD, n = 3, ^**P<0.01, vs. 0 μg/ml group. ^##P<0.01 vs. 6 μg/ml group. (C and D) The changes of Cx43 knockdown U2-OS cell migration, mean ± SD, n = 3, ^**P<0.01, vs. untreated group. ^##P<0.01 vs. NTC group. (E and F) Changes of U2-OS cell invasion following treatment with resveratrol, mean ± SD, n = 3, ^**P<0.01, vs. 0 μg/ml group. ^##P<0.01 vs. 6 μg/ml group. (G and H) Changes of Cx43 knockdown U2-OS cell invasion, mean ± SD, n = 3, ^**P<0.01, vs. untreated group. ^##P<0.01 vs. NTC group.

Figure 4. Resveratrol suppresses the Wnt/β-catenin signaling pathway

(A) With or without pretreatment with CHIR-99021, the changes in TOP/FOP flash ratios of U2-OS cells following treatment with 0, 6 or 12 μg/ml resveratrol for 24 h, without pretreatment with CHIR-99021, mean ± SD, n = 3, ^*P<0.05, ^**P<0.01, vs. 0 μg/ml group. ^##P<0.01 vs. 6 μg/ml group; pretreatment with CHIR-99021, mean ± SD, n = 3, ^*P<0.05, ^**P<0.01, vs. 0 μg/ml + CHIR-99021 group. ^#P<0.05 vs. 6 μg/ml + CHIR-99021 group. (B) Changes in mRNA expression of Cx43, E-cadherin, β-catenin, c-myc, cyclin D1, MMP-2 and MMP-9 after treatment with 12 μg/ml resveratrol for 24 and 48 h, ^*P<0.05, ^**P<0.01, vs. 0 h group. ^##P<0.01 vs. 24 h group. (C and D) Changes in protein expression of Cx43, E-cadherin, β-catenin, c-myc, cyclin D1, MMP-2 and MMP-9 after treatment with 12 μg/ml resveratrol for 24 and 48 h, ^*P<0.05, ^**P<0.01, vs. 0 h group. ^#P<0.05, ^##P<0.01 vs. 24 h group.

Figure 5. Knockdown of Cx43 enhances Wnt/β-catenin pathway activity

(A) Fluorescence pictures of virus-infected U2-OS cells at different MOIs. (B) With or without treatment with XAV939, the changes of TOP/FOP flash ratios of Cx43 knockdown U2-OS cells, without treatment with XAV939, mean ± SD, n = 3,^**P<0.01, vs. untreated group. ^##P<0.01 vs. NTC group; treatment with XAV939, mean ± SD, n = 3, ^**P<0.01, vs. untreated group + XAV939. ^##P<0.01 vs. NTC group + XAV939. (C) Changes in mRNA expression of Cx43, E-cadherin, β-catenin, c-myc, cyclin D1, MMP-2 and MMP-9 in Cx43 knockdown U2-OS cells, mean ± SD, n = 3,^**P<0.01, vs. untreated group. ^##P<0.01 vs. NTC group. (D and E) Changes in protein expression of Cx43, E-cadherin, β-catenin, c-myc, cyclin D1, MMP-2 and MMP-9 in Cx43 knockdown U2-OS cells, mean ± SD, n = 3,^**P<0.01, vs. untreated group. ^##P<0.01 vs. NTC group.