3.1 Melatonin inhibited colony formation, cell viability, and accelerated cell apoptosis.
The results showed that 24 hours of melatonin pretreatment reduced the ability of cell cloning dose-dependently (Figure 1A). The results of CCK-8 showed melatonin decreased cell viability dose dependently, and 2mmol/L of melatonin is the optimal concentration for further experiment (Figure 1B). Photographs of cell morphology showed that 2mmol/L of melatonin caused obvious cell shrinkage and loss of cellular attachment, as well as inhibited cell proliferative rate (Figure 1C). Photographs of immunofluorescence staining showed that 2mmol/L melatonin decreased the expression of CDK4 and showed cell shrinkage and lack of cellular adhesion(Figure 1D). Photographs of TUNEL assay showed higher proportion of cellular apoptosis in melatonin group rather than control group (Figure 1E). Therefore, it is suggested that melatonin inhibited NSCLC cell colony formation, cell viability, and accelerated apoptosis.
3.2 Inhibition of MEK activation particapated in melatonin-induced downregulation of CDK4.
Western blotting showed melatonin downregulated the level of proliferative proteins, CDK4 and PCNA, while upregulated the expression of apoptosis related protein, Bax (Figure 2A-D). qRT-PCR analysis showed the same trend as in western blotting (Figure 2E-G). Besides, melatonin decreased the phophorylation of ERK without altering the expression of total ERK (Figure 2H-K). We further found that a MEK inhibitor, PD98059, decreased the expression of CDK4 compared with control group. What’s more, combination of PD98059 and melatonin sinificantly downregulated the expression of CDK4 compared with PD98059 and melatonin alone group (Figure 2J). Therefore, it is suggested that inhibition of MEK activation particapated in melatonin-induced downregulation of CDK4.
3.3 Melatonin contributed to cell cycle arrest and apoptosis.
More cell accumulated in G1 phase after melatonin treatment while less cell accumulated in S phase, suggesting that melatonin blocked G1-to-S transition (Figure 3A, C-E). The proportion of apoptotic cells was significantly higher after melatonin treatment, especially in the early stage of apoptosis (Figure 3B, F). Therefore, it is suggested that melatonin contributed to cell cycle arrest and apoptosis.
3.4 CDK4 overexpression reversed anticancer actions of melatonin.
To further explore whether CDK4 is involved anticancer activities of melatonin, cells were transfected by recombinant lentivirus for upregulation of CDK4. CDK4 overexpression promoted the ability of colony formation. Interestingly, CDK4 overexpression reversed melatonin-induced inhibition of colony formation (Figure 4A). CDK4 and PCNA expressed higher CDK4 overexpression group. Besides, melatonin-induced suppression of CDK4 and PCNA were reversed by CDK4 overexpression. In contrary, the expression of Bax expressed lower in CDK4 overexpression group. Melatonin-induced increase of Bax was reversed by CDK4 overexpression (Figure 4B-E). qRT-PCR results showed the same trend as western blotting (Figure 4F-H). Therefore, it is suggested that CDK4 overexpression reversed anticancer actions of melatonin.
3.5 CDK4 knockdown enhanced the anticaner actions of melatonin.
Cells were transfected by lentivirus for downregulation of CDK4. CDK4 knockdown inhibited the ability of colony formation. What’ more, CDK4 knockdown enhanced melatonin-induced inhibition of colony formation (Figure 5A). CDK4 and PCNA expressed lower in CDK4 knockdown group. Besides, CDK4 and PCNA expressed the lowest in CDK4 knockdown and melatonin co-treatment group. On the contrary, Bax expressed higher in CDK4 knockdown group and expressed highest in CDK4 knockdown and melatonin co-treatment group (Figure 5B-E). qRT-PCR results showed the same trend as western blot (Figure 5F-H). Therefore, it is suggested that CDK4 knockdown enhanced anti-cancer actions of melatonin.
3.6 CDK4 overexpression weakened melatonin-induced cell cycle arrest and apoptosis.
Less cell accumulated in G1 phase while more cell accumulated in S phase in CDK4 overexpression group. Interestingly, CDK4 overexpression reversed melatonin-induced increase of cell proportion in G1 phase (Figure 6A, C-E). Besides, the proportion of apoptosis cells, especially in early stage, was much lower in CDK4 overexpression group. Moreover, CDK4 overexpression significantly weakened melatonin-induced cell apoptosis (Figure 6B, F). Therefore, it is suggested that CDK4 overexpression reversed melatonin-induced cell cycle arrest and apoptosis.
3.7 CDK4 knockdown enhanced melatonin-induced cell cycle arrest and apoptosis.
More cell accumulated in G1 phase while less cell accumulated in S phase in CDK4 knockdown group. What’s more, the maximum of cells distributed in G1 phase after CDK4 knockdown and melatonin co-treatment (Figure 7A, C-E). Cell apoptosis results showed that more cell distributed in apoptosis area in CDK4 knockdown group. Besides, the proportion of apoptotic cells is the highest after CDK4 knockdown and melatonin co-treatment (Figure 7B, F). Therefore, it is suggested that CDK4 knockdown enhanced melatonin-induced cell cycle arrest and apoptosis.
3.8 CDK4 overexpression reversed tumor growth inhibition of melatonin in vivo.
To further verify outcomes in vitro experiment, we established tumor model in BALB/C nude mice by subcutaneously injection of A549 cell suspension. All mice successfully formed subcutaneous tumors one week later. Tumor volume in CDK4 overexpression group was the largest while that in 40 mg/kg melatonin group was the smallest. Besides, CDK4 overexpression and melatonin co-treatment reversed the melatonin-induced inhibition of tumor growth (Figure 8A-C). However, the body weight of all nude mice in four groups had no significant difference (Figure 8D). Similarily, the result of western blotting showed that intraperitoneal injection of melatonin reduced CDK4 and PCNA level while raised Bax expression. Moreover, CDK4 overexpression weankened melatonin-induced decrease of CDK4 and PCNA as well as the increase of Bax (Figure 8E-H). Immunohistochemical assay also proved that introperitoneal of melatonin decreased the expression of CDK4 in tumor tissue (Figure 8I-J).Therefore, it is suggested that melatonin slowed down tumor growth, and CDK4 overexpression reversed melatonin induced inhibition of tumor growth in vivo.