Dehydrocorydaline inhibits cell proliferation, migration and invasion via suppressing MEK1/2-ERK1/2 cascade in melanoma

Abstract

Purpose: Alkaloids are naturally occurring chemical compounds that are widely distributed in plants, and have pharmaceutical values and low toxicity. In recent years, some of them have been demonstrated to be promising therapeutic drug candidates for cancer treatment. Herein, we tried to explore the antitumor effect of dehydrocorydaline (DHC), a natural alkaloid isolated from Corydalis, on malignant melanoma. Methods: We treated two malignant metastatic melanoma cell lines, A375 and MV3, and a normal melanocyte cell line, PIG1, with various concentrations of DHC for set amounts of time, and detected cell proliferation, migration, and invasion by using MTT, BrdU, transwell, Western blot and soft agar assay in vitro and tumorigenicity in the xenografts in vivo. Results: Our results showed that DHC dramatically blocked cell proliferation and led to cell cycle arrest at G0/G1 phase and downregulated the expressions of cell cycle regulators CDK6 and Cyclin D1 in melanoma cells. However, DHC had little inhibitory effect on normal melanocyte cell line PIG-1. Meanwhile, DHC suppressed cell invasion and migration through modulating the epithelial-mesenchymal transition (EMT) markers including E-cadherin, vimentin, as well as β-catenin. In addition, DHC also significantly attenuated tumor growth in vivo. The expressions of cell cycle-related and metastasis-related proteins were further confirmed by immunohistochemical staining in the xenografts. Importantly, MEK1/2-ERK1/2 cascade was inactivated after DHC treatment and ERK activator t-butylhydroquinone (tBHQ) treatment rescued DHC-induced cell proliferation inhibition. Conclusions: Our results indicated that DHC inhibited cell proliferation and migration/invasion via inactivating MAPK signaling, and showed that DHC might be a potential novel drug to treat malignant melanoma.

Keywords: MAPK; cell cycle; dehydrocorydaline; melanoma; migration and invasion.

Figure 1

IC50 of dehydrocorydaline treatment for 48 h in the inhibition of cell proliferation in (A) A375 melanoma cells; (B) MV3 melanoma cells; and (C) PIG1 normal melanocytes determined by MTT assay.

Figure 2

Dehydrocorydaline inhibits cell growth and proliferation in human melanoma cells. (A) Cell morphology of A375 and MV3 melanoma cells after treating with DMSO or the indicated concentrations of DHC for 48 h. Scale bar, 100 μm. (B and C) The effect of DHC on the proliferation rates of A375 and MV3 cells determined by cell counting in the microscope. (D) The effect of different concentrations of DHC treatment for 48 h on the proliferation rate of PIG1 cells determined by MTT assay. (E and F) The effect of DHC on the viability of A375 and MV3 cells. (G) Images and quantifications of A375 and MV3 cells positive for BrdU staining after treating with DMSO or 40 μM DHC for 24 h. Scale bar, 100 μm. All data are shown as the mean ± SD. A two-tailed unpaired Student’s t-test was carried out. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Abbreviations: DHC, dehydrocorydaline; DMSO, dimethyl sulfoxide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide.

Figure 3

Dehydrocorydaline induces cell cycle arrest at G1 phase in human melanoma cells. (A and B) The cell cycle of A375 and MV3 cells was analyzed by flow cytometry after treating with DMSO or the indicated concentrations of DHC for 48 h. (C and D) Western blot assay was performed to assess the cell cycle-related protein levels in A375 and MV3 cells, respectively. Protein levels were calculated based on the grayscale value of protein bands and normalized with the grayscale value of GAPDH bands. Cells were treated with the indicated concentrations (0, 20, 40, 80 μM) of DHC for 48 h or with 40 μM DHC treatment for indicated times (0, 24, 48, 72 h) of DHC; GAPDH was used as a control. (E) The cell cycle of PIG1 cells was analyzed by flow cytometry after treating with DMSO or 40 μM DHC for 48 h. All data are shown as the mean ± SD. A two-tailed unpaired Student’s t-test was carried out. **p<0.01, ***p<0.001. Abbreviations: n.s.=not significant; DHC, dehydrocorydaline; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 4

Dehydrocorydaline inhibits cell migration and invasion in melanoma cells. (A) Cell migration rate detected by wound-healing assay of A375 and MV3 cells after treating with DMSO or 40 μM DHC for the indicated time. Scale bar, 100 μm. (B) The effect of 40 μM DHC on the wound closure in A375 and MV3 cells. (C) The effect of transwell migration assays in A375 and MV3 cells after treating with DMSO or 40 μM DHC for 24 h. Scale bar, 100 μm. Migration rates were normalized by proliferation. (D) The effect of transwell invasion assays in A375 and MV3 cells after treating with DMSO or 40 μM DHC for 72 h. Scale bar, 100 μm. Invasion rates were normalized by proliferation. (E and F) Western blot analysis of the metastasis-related protein levels in A375 and MV3 cells, respectively. Protein levels were calculated based on the grayscale value of protein bands and normalized with the grayscale value of GAPDH bands. Cells were treated with the indicated concentrations (0, 20, 40, 80 μM) of DHC for 48 h or with 40 μM DHC treatment for indicated times (0, 24, 48, 72 h) of DHC; GAPDH was used as a control. All data are shown as the mean ± SD. A two-tailed unpaired Student’s t-test was carried out. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Abbreviations: DHC, dehydrocorydaline; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 5

Dehydrocorydaline suppresses tumor growth in xenograft model of melanoma cells. (A and B) The colony formation was examined by soft agar assay (1,000 cells/well) in A375 or MV3 cells after treating with DMSO or 40 μM DHC for 14 days. Scale bar, 1 mm. (C and D) A375 and MV3 cells were injected into the flank of BALB/c nude mice. When tumors were palpable, mice were orally administrated with 100 mg/kg DHC or DMSO every day 12 times after the tumor plumped, and mice body weight was measured. (E and F) The tumor volume of xenograft tumors formed by the A375 and MV3 cells which were treated with DHC (100 mg/kg) or DMSO was measured. (G and H) The weight of the tumor was measured after DHC or DMSO treatment. All data are shown as the mean ± SD. A two-tailed unpaired Student’s t-test was carried out. *p<0.05, ***p<0.001, ****p<0.0001. Abbreviations: DHC, dehydrocorydaline; DMSO, dimethyl sulfoxide.

Figure 6

Dehydrocorydaline decreases the expression of cell cycle and metastatic markers in xenograft model of melanoma cells. The images of the H&E staining and immunohistochemistry analysis of d Ki-67, cyclin D1, and vimentin expression are presented. Abbreviations: DHC, dehydrocorydaline; DMSO, dimethyl sulfoxide.

Figure 7

Dehydrocorydaline downregulates MEK1/2-ERK1/2 cascade in melanoma cells. (A and B) Western blot analysis of the expression of MAPK cascade in A375 and MV3 cells, respectively. Protein phosphorylation levels were calculated based on the grayscale value of phosphorylated protein bands and normalized with the grayscale value of total protein bands. Cells were treated with the indicated concentrations (0, 20, 40, 80 μM) of DHC for 48 h or with 40 μM DHC treatment for indicated times (0, 24, 48, 72 h) of DHC; GAPDH was used as a control. (C) The effect of 40 μM DHC and 50 μM ERK1/2 activator t-butylhydroquinone (tBHQ) on the viability of A375 and MV3 cells, respectively. All data are shown as the mean ± SD. A two-tailed unpaired Student’s t-test was carried out. *p<0.05, **p<0.01, ***p<0.001. Abbreviations: DHC, dehydrocorydaline; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MAPK, mitogen-activated protein kinase; tBHQ, t-butylhydroquinone.

Figure 8

Overview of the molecular mechanism of dehydrocorydaline in the inhibition of cell proliferation, migration, and invasion of melanoma.