Morusin shows potent antitumor activity for melanoma through apoptosis induction and proliferation inhibition

Abstract

Background: The discovery of new anti-melanoma drugs with low side effect is urgently required in the clinic. Recent studies showed that morusin, a flavonoid compound isolated from the root bark of Morus Alba, has the potential to treat multiple types of cancers, including breast cancer, gastric cancer, and prostate cancer. However, the anti-cancer effect of morusin on melanoma cells has not been investigated.

Methods: We analyzed the effects of morusin on the proliferation, cell cycle, apoptosis, cell migration and invasion ability of melanoma cells A375 and MV3, and further explored the effects of morusin on tumor formation of melanoma cell. Finally, the effects of morusin on the proliferation, cycle, apoptosis, migration and invasion of A375 cells after knockdown of p53 were detected.

Results: Morusin effectively inhibits the proliferation of melanoma cells and induces cell cycle arrest in the G2/M phase. Consistently, CyclinB1 and CDK1 that involved in the G2/M phase transition were down-regulated upon morusin treatment, which may be caused by the up-regulation of p53 and p21. In addition, morusin induces cell apoptosis and inhibits migration of melanoma cells, which correlated with the changes in the expression of the associated molecules including PARP, Caspase3, E-Cadherin and Vimentin. Moreover, morusin inhibits tumor growth in vivo with little side effect on the tumor-burden mice. Finally, p53 knockdown partially reversed morusin-mediated cell proliferation inhibition, cell cycle arrest, apoptosis, and metastasis.

Conclusion: Collectively, our study expanded the spectrum of the anti-cancer activity of morusin and guaranteed the clinical use of the drug for melanoma treatment.

Keywords: Apoptosis; Melanoma; Metastasis; Morusin; Proliferation; p53.

Fig. 1

Morusin inhibits cell growth and proliferation in melanoma cells. The IC50 of A A375 and B MV3 cells. C The cell morphology of A375 and MV3 cells after treating with indicated concentration of morusin for 24 h. DMSO was used as control. Scale bar was 100 μm. D The cell numbers of A375 and MV3 were counted and displayed. The DMSO treatment group was considered to be 100%. The viability of E A375 and F MV3 after treating with DMSO or morusin. G The image and quantification of BrdU staining cells of A375 and MV3 after treating with DMSO or morusin (5 μΜ for A375 and 10 μΜ for MV3) for 24 h. Scale bar was 100 μm. Each experiment was repeated three times. All data were shown as the mean ± SD and analyzed by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

Morusin induces cell cycle arrest at G2/M phase in melanoma cells. A The cell cycle of A375 and MV3 cells treated with DMSO or morusin (5 μΜ for A375 and 10 μΜ for MV3) for 24 h was analyzed by flow cytometry. B Percentage of indicated A375 and MV3 cells in different periods. C The expression levels of CyclinB1, p53, p21 and CDK1 in A375 and MV3 cells treated with morusin in different concentrations (2, 5, 10 μΜ for A375 and 5, 10, 15 μΜ for MV3) or DMSO for 24 h were determined by Western blot analysis. Tubulin was used as a control. D Densitometry of western blot in panel C. E The expression levels of CyclinB1, p53, p21 and CDK1 in A375 and MV3 cells treated with morusin (5 μΜ for A375 and 10 μΜ for MV3) in different times for 0, 12, 24, 36 h. Tubulin was used as a control. F Densitometry of western blot in panel E. Each experiment was repeated three times. All data were shown as the mean ± SD and analyzed by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

Morusin induces apoptosis in melanoma cells. A Apoptosis of A375 and MV3 cells treated with DMSO or morusin (2, 5 μΜ for A375 and 5, 10 μΜ for MV3) for 24 h was analyzed by flow cytometry. B Apoptosis rate of melanoma cells in Panel A. C The expression levels of PARP and Caspase3 in A375 and MV3 cells treated with morusin in different concentrations (2, 5, 10 μΜ for A375 and 5, 10, 15 μΜ for MV3) or DMSO for 24 h were determined by Western blot analysis. Tubulin was used as a control. D Densitometry of western blot in panel C. E The expression levels of PARP and Caspase3 in A375 and MV3 cells treated with morusin (5 μΜ for A375 and 10 μΜ for MV3) in different times for 0, 12, 24, 36 h. Tubulin was used as a control. F Densitometry of western blot in panel E. Each experiment was repeated three times. All data were shown as the mean ± SD and analyzed by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

Morusin inhibits cell migration and invasion in melanoma cells. A The migration of A375 and MV3 cells treated with morusin for the corresponding time was measured by wound-healing assay. Scale bar was 100 μm. B The effect of morusin on the healing of melanoma cells. The healing rate of 0 h was considered to be 100%. C Transwell migration assays of A375 and MV3 cells treated with DMSO or morusin (5 μΜ for A375 and 10 μΜ for MV3) for 24 h. Scale bar was 100 μm. The statistical analysis was presented in histograms, and cell migration rates were normalized by proliferation. D Transwell invasion assays of A375 and MV3 cells treated with DMSO or morusin (5 μΜ for A375 and 10 μΜ for MV3) for 72 h. Scale bar was 100 μm. Cell invasion rates were normalized by proliferation. E The expression levels of E-Cadherin and Vimentin in A375 and MV3 cells treated with morusin in different concentrations (2, 5,10 μΜ for A375 and 5, 10, 15 μΜ for MV3) or DMSO for 24 h. F Densitometry of western blot in panel E. G The expression levels of E-Cadherin and Vimentin in A375 and MV3 cells treated with morusin (5 μΜ for A375 and 10 μΜ for MV3) in different times for 0, 12, 24, 36 h. Tubulin was used as the control. H Densitometry of Western blot in panel G. Each experiment was repeated three times. All data were shown as the mean ± SD and analyzed by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

Morusin inhibits tumor growth in vitro and in vivo. A The clone formation ability of A375 and MV3 after treating with 5 and 10 μΜ morusin was detected by soft agar assay. Scale bar was 100 μm. B Colony numbers in panel A were quantified. C Body weight of mice treated with morusin or DMSO. D Tumor volume of mice treated with morusin (25 mg/kg) or DMSO. E Tumor weight of mice treated with morusin (25 mg/kg) or DMSO. F Photograph of tumors from indicated mice. G H&E and IHC staining of Ki67 in indicated tumors. Scale bar was 100 μm. H The positive cells of Ki67 in panel G. All data were shown as the mean ± SD and analyzed by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

Morusin-inducing cell proliferation inhibition, cell cycle arrest, apoptosis, and metastasis can be recovered by knocking down p53. A The expression level of p53 was detected by Western blot in A375 cells. B Growth curve of knock down p53 after treating with 5 μΜ morusin in A375 cells. DMSO and shGFP were used as control. C The image and quantification of BrdU positive cells in knockdown p53 A375 cells treatment with 5 μΜ morusin for 24 h. Scale bar was 100 μm. D Cell cycle was detected in knockdown p53 A375 cells after treating with 5 μΜ morusin for 24 h, and percentage of A375 cells in different phase. DMSO and shGFP were used as control. E Apoptosis of knockdown p53 A375 cells treated with 5 μΜ morusin for 24 h was analyzed by flow cytometry, and apoptosis rate of melanoma cells was quantified. DMSO and shGFP were used as control. F Transwell migration and invasion assays of knockdown p53 A375 cells treated with 5 μΜ morusin for 24 h and 72 h were analysed. Scale bar was 100 μm. DMSO and shGFP were used as control. Cell migration and invasion rates were normalized. G The expression of p53, p21, C-Caspase3 and E-Cadherin were checked in knockdown p53 A375 cells treated with 5 μΜ morusin for 24 h. DMSO and shGFP were used as control. Tubulin was used as the control. All data were shown as the mean ± SD and analyzed by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001