Lycorine hydrochloride inhibits melanoma cell proliferation, migration and invasion via down-regulating p21Cip1/WAF1

Abstract

Lycorine hydrochloride (LH) is an active ingredient sourced from the medicinal herb Lycoris radiata. Previous studies have suggested that LH exerts tumor suppression activity in several human cancers. However, the anti-cancer effect of LH in melanoma and the potential molecular mechanisms still need to be further studied. p21^Cip1/WAF1, unlike its traditional cyclin-dependent kinase (CDK) inhibitor role, is believed to act as an oncogene under certain cellular conditions. In this research, an increased expression of p21^Cip1/WAF1 was found in human melanoma tissues and positively related to the tumor invasion depth. High level of p21^Cip1/WAF1 was found to correlate with bad outcomes of melanoma patients by Kaplan-Meier survival analysis. Functional experiments demonstrated that the proliferation, migration and invasion ability of A375 and MV3 melanoma cells was powerfully inhibited by LH through inducing S phase cell cycle arrest and regulating epithelial-mesenchymal transition (EMT). In NOD/SCID mice model, LH effectively inhibited the xenograft tumor growth and lung metastasis of A375 cells. Further research revealed that LH reduced p21^Cip1/WAF1 protein by accelerating its ubiquitination. Importantly, the LH-induced suppression of cell proliferation and metastasis was rescued by p21^Cip1/WAF1 overexpression, both in vitro an in vivo. Taken together, LH, which suppresses the proliferation and metastasis of melanoma cells via down-regulating p21^Cip1/WAF1, is expected to be developed as an effective medicine for melanoma therapy.

Keywords: Melanoma; cell proliferation; invasion; lycorine hydrochloride; migration; p21Cip1/WAF1.

Figure 1

Anti-proliferative effect of LH on melanoma cells. A. A375 and MV3 cells were treated with LH (0, 10, 20 and 40 μM) for 48 h and proliferation of the cells was observed. Cell percentage in 0 μM group is regarded as 100%. Scale bar, 100 μm. B. Under LH (0, 10, 20, and 40 μM) treatment, cell viability was measured by MTT assay on days 1, 3, 5 and 7. C. Percentage of Edu positive cells after LH (20 μM) treatment for 48 h. Scale bar, 50 μm. D. Cell cycle distribution of the cells after LH (20 μM) treatment for 48 h. E, F. The protein levels of CDK1, CDK2 and Cyclin E1 in LH treated cells were measured by western blotting. Mean ± SD; *P<0.05, **P<0.01, ***P<0.001.

Figure 2

LH inhibits the mobility of melanoma cells. A. The wounds on cell layers under the incubation of 20 μM LH or DMSO were observed at different times (0, 24, 48, 72 h) and the closure rates were calculated. Scale bar, 100 μm. B, C. Migration and invasion of the cells under LH (20 μM) or DMSO treatment. Scale bar, 100 μm. D, E. The protein levels of E-cadherin, N-cadherin and Vimentin in LH treated cells were detected by western blotting. Mean ± SD; **P<0.01, ***P<0.001.

Figure 3

LH suppresses melanoma growth and lung metastasis in NOD/SCID mice. A. After 3 weeks incubation with LH (20 μM) or DMSO, colonies formed by A375 and MV3 cells were imaged and counted. Scale bar, 1 mm. B. Under the treatment of LH (30 mg/kg/day) or DMSO, volume of A375 xenograft tumors was calculated every 5 days. C. After 25 days treatment with LH (30 mg/kg/day) or DMSO, the tumors were excised from the mice and weighed to analyze. D. Weight monitoring curve of the mice. E. Expression of Ki-67 in the xenograft tumors from LH or DMSO treated mice model. IHC; Scale bar, 100 μm. F. Tumors in the lung of mice that metastasized with A375 cells were observed after LH (30 mg/kg/day for 40 days) or DMSO treatment. H&E; Scale bar, 1 mm. Mean ± SD; **P<0.01, ***P<0.001; ns, not significant.

Figure 4

High p21 expression indicates poor prognosis of melanoma and LH promotes ubiquitination degradation of p21. A, B. Expression of p21 in A375 and MV3 cells was detected by western blotting after LH treatment. C. Expression of p21 in the xenograft tumors from LH or DMSO treated mice model. IHC; Scale bar, 100 μm. D. Expression of p21 in human cutaneous melanoma (n = 14) was detected by IHC staining, benign nevi (n = 10) was as control. Scale bar, 200 μm. E. The CDKN1A level in cutaneous melanoma tissues (n = 45) compared with benign nevi tissues (n = 18) from Oncomine database. F. The association of CDKN1A gene with clinical outcomes (OS and DFS) of melanoma patients were analyzed through GEPIA database. G. The effect of LH on the expression of p21 mRNA in melanoma cells. H. The LH and DMSO pre-treated A375 cells were incubated with 10 μM CHX and collected at 0, 1, 2 and 4 h, cell lysates were immunoblotted with anti-p21 antibody. I. Cells were cultured in the absence or presence condition of 20 μM LH, the p21 protein level was detected after 20 μM MG132 treatment. J. The ubiquitination of p21 was detected in LH treated 293-FT cells. Mean ± SD; *P<0.05, **P<0.01, ***P<0.001; ns, not significant.

Figure 5

Overexpressing p21 attenuates the anti-proliferative effect of LH. A. Viability of p21/vector-overexpressing A375 and MV3 cells under the treatment of 20 μM LH was measured by MTT assay. B. EdU staining of the p21/vector-overexpressing cells after LH (20 μM) treatment for 48 h. Scale bar, 50 μm. C. Cell cycle distribution of the p21/vector-overexpressing cells after LH (20 μM) treatment for 48 h. D, E. After being treated with LH (20 μM) or DMSO for 48 h, the protein levels of p21, CDK1, CDK2 and Cyclin E1 in p21/vector-overexpressing cells were detected by western blotting. Mean ± SD; *P<0.05, **P<0.01, ***P<0.001.

Figure 6

Overexpressing p21 partially counteracts the anti-migration/invasion effect of LH. A, B. Migration and invasion of p21/vector-overexpressing cells under LH (20 μM) or DMSO treatment. Scale bar, 100 μm. C, D. p21, E-cadherin, N-cadherin, and Vimentin proteins in p21/vector-overexpressing cells after LH (20 μM) or DMSO treatment for 48 h. Mean ± SD; **P<0.01, ***P<0.001.

Figure 7

Overexpressing p21 attenuates the LH-induced suppression on tumor growth and lung metastasis of melanoma cells. (A) After 3 weeks incubation with DMSO or 20 μM LH, colonies formed by p21/vector-overexpressing cells were imaged and counted. Scale bar, 1 mm. (B) Tumor volume of p21/vector-overexpressing A375 xenograft tumors in mice under LH (30 mg/kg/day for 25 days) or DMSO treatment was measured. (C) Tumors were excised from the mice after 25 days treatment. (D) Weight of the tumors in (C) was measured. (E) IHC staining for p21 and Ki67 was performed in the xenograft tumors. Scale bar, 100 μm. (F) Tumors in the lung of mice that metastasized with p21/vector-overexpressed A375 cells were observed after LH (30 mg/kg/day for 40 days) or DMSO treatment. H&E; Scale bar, 1 mm. Mean ± SD; *P<0.05, **P<0.01, ***P<0.001.