Andrographolide inhibits proliferation and induces cell cycle arrest and apoptosis in human melanoma cells

Abstract

Andrographolide (Andro), a natural compound isolated from Andrographis paniculata, has been demonstrated to have anticancer efficacy in several types of tumors. In the present study, the anticancer effects and mechanism of Andro in human malignant melanoma were investigated. Cell viability analysis was performed using an MTT assay and the effect of Andro on the cell cycle and apoptosis of human malignant melanoma cells was determined by flow cytometry. Western blot analysis was performed to evaluate the protein expression levels of human malignant melanoma cells following treatment with Andro. The results revealed that Andro potently inhibited cell proliferation by inducing G2/M cell-cycle arrest in human malignant melanoma C8161 and A375 cell lines. In addition, treatment with Andro induced apoptosis, which was associated with the cleavage of poly(adenosine diphosphate-ribose) polymerase and activation of caspase-3. It was observed that Andro induced activation of the c-Jun N-terminal kinase and p38 signaling pathway, which may be connected with cell cycle arrest and apoptosis. In conclusion, the results demonstrated that Andro may be a promising and effective agent for antitumor therapy against human malignant melanoma.

Keywords: apoptosis; c-Jun N-terminal kinase; cell cycle arrest; melanoma.

Figure 1.

Andro inhibited cell proliferation in human melanoma cell lines. (A) A375 and (B) C8161 human melanoma cells were treated with different concentrations of Andro for 24 and 48 h, then a MTT assay was performed. Each experiment was repeated in triplicate. *P<0.05, **P<0.01 vs. the control group. Andro, andrographolide.

Figure 2.

Andro induced cell cycle arrest at G2/M phase in human melanoma cells. (A) Human melanoma cells were treated with dimethylsulphoxide or various concentrations of Andro for 24 h. The cells were collected, stained with PI and the percentage of cell cycle distribution was analyzed by flow cytometry. (B) The percentage of cell cycle distribution at G1, S, and G2/M phases is shown as the mean ± standard deviation from three independent experiments. *P<0.05, **P<0.01 vs. the control group. Andro, andrographolide; PI, propidium iodide.

Figure 3.

Andro induced apoptosis in A375 cells. (A) Apoptosis analysed by flow cytometry following Annexin V-FITC and PI staining. A375 cells were treated with Andro at the indicated doses for 24 h, labeled with Annexin V-FITC and PI, then analyzed by flow cytometer. (B) The histograms indicate that the percentage of total apoptosis. The percentage of apoptosis cells is shown as mean ± standard deviation from three independent experiments. *P<0.05, **P<0.01 vs. the control group. (C) Western blot analysis was used to detect the protein level of cleaved PARP and caspase-3. A375 cells were treated with Andro at the indicated doses for 24 h. Cell lysates were prepared and the levels of cleaved PARP and cleaved-caspase-3 were determined by western blot analysis. PI, propidium iodide; FITC, fluorescein isothiocyanate; Andro, andrographolide; PARP, poly(adenosine diphosphate-ribose) polymerase.

Figure 4.

Andro activates the JNK and p38 signaling pathway. A375 cells were treated with Andro at the indicated doses for 24 h. The expression of p-JNK, JNK, p-P38 and P38 were analyzed by western blotting. The amount of phosphorylation of JNK and p-P38 were increased in Andro treated cells. Andro, andrographolide; JNK, c-Jun N-terminal kinase; p, phosphorylated.