Licochalcone H Induces Cell Cycle Arrest and Apoptosis in Human Skin Cancer Cells by Modulating JAK2/STAT3 Signaling

Abstract

Licochalcone H (LCH) is a phenolic compound synthetically derived from licochalcone C (LCC) that exerts anticancer activity. In this study, we investigated the anticancer activity of LCH in human skin cancer A375 and A431 cells. The 3-(4,5-dimethylthiazol- 2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell viability assay was used to evaluate the antiproliferative activity of LCH. Cell cycle distribution and the induction of apoptosis were analyzed by flow cytometry. Western blotting assays were performed to detect the levels of proteins involved in cell cycle progression, apoptosis, and the JAK2/STAT3 signaling pathway. LCH inhibited the growth of cells in dose- and time-dependent manners. The annexin V/propidium iodide double staining assay revealed that LCH induced apoptosis, and the LCH-induced apoptosis was accompanied by cell cycle arrest in the G1 phase. Western blot analysis showed that the phosphorylation of JAK2 and STAT3 was decreased by treatment with LCH. The inhibition of the JAK2/STAT3 signaling pathway by pharmacological inhibitors against JAK2/STAT3 (cryptotanshinone (CTS) and S3I-201) simulated the antiproliferative effect of LCH suggesting that LCH induced apoptosis by modulating JAK2/STAT3 signaling.

Keywords: Apoptosis; Human skin cancer; JAK2; Licochalcone H; STAT3.

Fig. 1

Growth inhibition of human skin cancer cells by LCH. (A) The chemical structure of licochalcone H (LCH). (B, C) The viability of skin cancer cell lines A375 and A431 treated with 0, 5, 10, 20, and 30 μM of LCH for 24 and 48 h was measured using the MTS reagent. The data represent mean percentages ± SD (n=3; *p<0.05, **p<0.01, ***p<0.001 compared to the control group).

Fig. 2

Effect of LCH on apoptosis induction in human skin cancer cells. (A, B) Quantitative detection of annexin-V-FITC and PI-positive cells via FACS analysis. A375 and A431 cells were treated with 0, 10, 20, and 30 μM of LCH, and apoptosis was analyzed after annexin-V-FITC and PI double staining. The data represent mean percentages ± SD (n=3; **p<0.01 compared to the control group). (C) A375 and A431 cells treated with 0, 10, 20, and 30 μM LCH for 48 h were harvested to prepare cell extracts. The cell extracts were subjected to SDS-PAGE and Western blot analysis to detect the levels of Bid, Bcl-xl, Bcl-2, Bax, Bad, Mcl-1, survivin, PARP, and caspase-3. β-Actin was used as the denominator to quantify relative protein expression levels.

Fig. 3

Effects of LCH on human skin cancer cell cycle distribution. (A, B) A375 and A431 cells were treated with 0, 10, 20, and 30 μM LCH for 48 h, stained with PI, and analyzed for DNA content by FACS analysis. The data represent mean percentages ± SD (n=3; **p<0.01, ***p<0.001 compared to the control group). (C) A375 and A431 cells treated with 0, 10, 20, and 30 μM LCH for 48 h were harvested to prepare cell extracts. The cell extracts were subjected to SDS-PAGE and Western blot analysis to detect the levels of p53, p27, p21, and cyclin-D1. β-Actin was used as the loading control.

Fig. 4

Effects of LCH on the modulation of JAK2/STAT3 signaling in human skin cancer cells. (A) A375 and A431 cells were treated with LCH (0, 10, 20, and 30 μM) for 48 h. Whole cell extracts were prepared, separated by SDS-PAGE, and subjected to Western blots using JAK2, p-JAK2, STAT3, and p-STAT3 antibodies. β-Actin was used as the loading control. (B) The percentage of p-JAK2 and p-STAT3 expression is presented. The data represent mean percentages ± SD (n=3; ***p<0.001). (C) Time-dependent effects of LCH on p-JAK2, p-STAT3, and cleaved-PARP were observed using A375 and A431 cells treated with LCH (30 μM) for 0, 12, 24, and 48 h.

Fig. 5

Effects of inhibitors of JAK2/STAT3 signaling in human skin cancer cells. (A) A375 and A431 cells were treated with the indicated concentrations of LCH, cryptotanshinone (CTS), and S3I-201 for 48 h. Western blot analysis to detect the levels of JAK2, p-JAK2, STAT3, and p-STAT3. β-Actin was used as the loading control. (B) The percentages of p-JAK2 and p-STAT3 expression is shown. The data represent mean percentages ± SD (n=3; ***p<0.001). (C) Viability of A375 and A431 skin cancer cell lines treated with the indicated concentrations of LCH, CTS, and S3I-201 for 48 h measured using the MTS reagent. The data represent mean percentages ± SD (n=3; ***p<0.001).