Cancer can initiate from mutations in specific genes, which induce constant cell proliferation, uncontrolled growth, tumor formation, and metastasis or recurrence (Bertram, 2000; Gibbs, 2003). Although diverse approaches have been used in attempts to unravel the complex mechanisms of oncogenesis and treat cancers, complete prevention or a perfect remedy remains unattainable (Baldwin, 2001).
Skin cancer is the most commonly diagnosed cancer in the United States, accounting for a third of all cancer diagnoses (Rager
Natural products of diverse origin are widely used in the treatment of cancer (Demain and Vaishnav, 2011; Rajesh
The JAK/STAT signaling pathway is involved in diverse biological processes such as embryonic development, stem cell maintenance, hematopoiesis, and inflammatory responses (Lopez-Onieva
Dulbecco’s Modified Eagle’s medium (DMEM), fetal bovine serum (FBS), phosphate buffered saline (PBS), penicillin and streptomycin (P/S) and 0.5% trypsin-EDTA were obtained from Thermo Fisher Scientific (Rockford, IL, USA). The primary antibodies against phospho (p)-STAT3 (Tyr 705), STAT3, Bcl-xl, Bcl-2, myeloid cell leukemia-1 (Mcl-1), Survivin, Cyclin-D1, p21, p27, p53, Bad, Bax, Bid and β-Actin, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against p-JAK2 (Tyr 1007/1008), JAK2, Caspase 3 and Poly ADP-ribose Polymerase (PARP), cleaved-Caspase 3, cleaved-PARP were obtained from Cell Signaling Inc (Danvers, MA, USA). 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), 4’-6-diamidino-2-phenylindole (DAPI) and cryptotanshinone (CTS), and S3I-201 were purchased from Sigma-Aldrich, Inc (St. Louis, MO, USA). LCH was synthesized and purified based on the synthetic method described elsewhere (Wang
Human melanoma A375 cells and human epidermoid carcinoma A431 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). These human skin cancer cells were cultured in DMEM containing 10% heat-inactivated FBS, and 100 U/mL each of penicillin and streptomycin at 37˚C in a humidified air with 5% CO2.
To confirm the viability of human skin cancer cells, 4.0×103 of A375 and 5.0×103 of A431 cells were seeded in 96-well microtiter plate. After a day of incubation, cells were treated with vehicle (for control), 5, 10, 20, and 30 µM of LCH. Cells were re-incubated for 24 and 48 h. After entire incubation, cell viabilities were measured with the MTS reagent (Abcam, Cambridge, MA, USA). Briefly, the dehydrogenase enzyme substrate MTS reagent was added to the respective wells and the plates were incubated at 37˚C with 5% CO2 for 2 h. The absorbance was measured at 490 nm using a microplate reader (Biotek, Winooski, VT, USA). The relative cell viability was calculated compared to the negative controls (0 µM LCH in DMSO). The data represent mean values obtained from three independent experiments.
A375 and A431 cells were seeded and treated with LCH (0, 10, 20, and 30 μM) for 48 h. After incubation, cells were harvested and fixed with 70% ethanol at –20°C for 2h. The cells were washed with 1×PBS, and stained with RNase A and propidium iodide (PI; BD Biosciences, Piscataway, NJ, USA). Finally, samples were incubated at 37˚C for 30 min in the dark. After reaction with reagent, they were analyzed using a fluorescence-activated cell sorting (FACS; BD Biosciences) according to the manufacturer’s instructions.
LCH (0-30 µM for 48 h)-treated A375 and A431 cells were harvested and washed with PBS. Proteins were then extracted using RIPA buffer (Thermo Fisher Scientific) containing protease inhibitor cocktail (Roche, Basel, Switzerland). Quantification of protein extracts was conducted with Pierce® BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein samples were resolved by 8~15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the resolved proteins were transferred to polyvinylidene-difluoride membranes. Membranes were blocked for 1 h at room temperature with 5% non-fat dried milk in TBS containing 0.1% Tween 20 (TBST), and incubated overnight at 4˚C with specific antibodies, followed by washing with TBST for 30 min and incubation with horseradish peroxidase-conjugated secondary antibody. The targeted protein bands were reacted using an ECL Plus Western Blotting Detection system from Santa Cruz Biotechnology and detected using ImageQuant LAS-4000 Mini (GE Healthcare Life Sciences, Buckinghamshire, UK) according to the manufacturer’s instructions.
Cells were treated with LCH (0, 10, 20, and 30 μM) for 48 h. Detached A375 and A431 cells were collected by centrifugation and combined with adherent cells. The cells were washed with cold 1×PBS, and stained with the FITC-Annexin-V Apoptosis Detection Kit (BD Biosciences) followed by analysis using a fluorescence activated cell sorter (FACS; BD Biosciences).
Results were presented as mean ± SD of at least three independent experiments performed in triplicate. Data were analyzed for statistical significance using one-way analysis of variance.
First, we determined if LCH (Fig. 1A) had an anti-proliferative effect in skin cancer cells, as the anticancer effect of LCH in skin cancer cells has not been studied yet. The MTS cell viability assay showed significant decreases in the viability of A375 and A431 cells in concentration- (0, 5, 10, 20, and 30 μM) and time- (24 and 48 h) dependent manners (Fig. 1B, 1C). The viability of A375 cells after treatment with 5, 10, 20, and 30 μM LCH for 24 h was 94%, 86%, 75%, and 56%, respectively, compared to 0 μM LCH. For 48 h treatment, the viability of A375 cells was 83%, 65%, 40%, and 22% in the 5, 10, 20, and 30 μM LCH-treated groups, respectively, compared to the controls. Likewise, we could observe the anti-proliferative effect of LCH in A431 cells in the similar manner. The viability of A431 cells after treatment with 5, 10, 20, and 30 μM LCH for 24 h was 94%, 88%, 74%, and 46%, respectively, compared to 0 μM LCH, and the viability after 48 h treatment went down to 93%, 74%, 57%, and 27% in the 5, 10, 20, and 30 μM LCH-treated groups, respectively, compared to the controls.
To investigate whether LCH induced apoptosis in skin cancer cells, the annexin V apoptosis assay with annexin V/PI double staining was used. The percentage of apoptotic cells (Fig. 2A, annexin V+/PI- cells and annexin V+/PI+, on the right side) after treatment with 0, 10, 20, and 30 μM LCH for 48 h was 4.02%, 25.25%, 56.57%, and 61.70%, respectively in A375 cells. Similarly, the percentage of apoptotic cells in A431 cells increased from 4.02% in the control group (0 μM LCH) to 24.37%, 55.76%, and 61.48% in the 10, 20, and 30 μM LCH-treated groups, respectively (Fig. 2A, 2B). These results suggested that skin cancer cell apoptosis was induced by LCH. To determine if LCH regulated the mitochondrial apoptosis pathway, we analyzed the protein expression levels of Bcl-2 family members by Western blots. When skin cancer cells were incubated with LCH, the expression of Bid, Bcl-xl, Bcl-2, and Mcl-1 decreased in a dose-dependent manner, whereas the levels of Bax and Bad increased. Treatment with LCH resulted in a decrease in survivin and an increase in the levels of cleaved caspase 3 and PARP in A375 and A431 cells (Fig. 2C).
To investigate if the suppression of cell proliferation by LCH was related to cell cycle arrest, we performed cell cycle analysis using PI staining. Incubating skin cancer cells with different LCH concentrations for 48 h led to a dose-dependent increase in the sub-G1 phase in both A375 and A431 cells. In A375 cells, the sub-G1 phase cell cycle population was 2.09%, 11.54%, 20.47%, and 36.60% at 0, 10, 20, and 30 μM LCH, respectively (Fig. 3A, 3B). Likewise, the sub-G1 phase cell cycle population in A431 cells increased from 2.78% in the control group (0 μM LCH) to 9.35%, 19.46%, and 42.96% in the 10, 20, and 30 μM LCH-treated groups, respectively (Fig. 3A, 3B). These results suggested G1 cell cycle arrest in human skin cancer cells by LCH treatment. We then assessed the protein levels of cyclin D1, CDK2, CDK6, p21, and p27 to elucidate the mechanism of LCH-induced cell cycle arrest in A375 and A431 cells. Western blot analysis revealed that the expression of cyclin D1 was downregulated, and the expression of p21, p27, and p53 was upregulated (Fig. 3C). These results indicated that cell cycle arrest may be associated with the anti-proliferative effect of LCH on A375 and A431 cells.
We performed Western blot assay to examine the effects of LCH on JAK2 and STAT3 signaling and the role of these pathways in the LCH-induced apoptosis of skin cancer cells, as the JAK2/STAT3 signaling controls the survival and proliferation of cancer cells (Bromberg
To further confirm the effects of LCH on the regulation of JAK2/STAT3 signaling, we compared the antiproliferative activity of LCH to those of pharmacological inhibitors of JAK2/STAT3 signaling (CTS and S3I-201). Expectedly, the levels of phosphorylated JAK2 and STAT3 assessed by Western blots were decreased in human skin cancer A375 and A431 cells treated with LCH, CTS, or S3I-201 for 48 h (Fig. 5A, 5B). Moreover, the MTS cell viability assay showed that decreases in JAK2 and STAT3 phosphorylation correlated with cell viability in both A375 and A431 cells, and the antiproliferative effects of CTS and S3I-201 were comparable to those of LCH (Fig. 5C).
Cancer accounts for a very high percentage of overall mortality. It ranks number two in the causes of death in the United States (Siegel
In addition, we detected LCH-mediated apoptosis in human skin cancer cells. The rate of apoptosis, determined by the annexin V apoptosis assay, increased significantly following LCH treatment. We observed a shift in the balance between pro-apoptotic and anti-apoptotic Bcl-2 proteins (Lee
To further elucidate the antiproliferative effect of LCH, we analyzed cell cycle distribution. Treatment with LCH increased the sub-G1 cell population of A375 and A431 cells in a dose-dependent manner (Fig. 3A, 3B). To understand the molecular mechanism of cell cycle arrest induced by LCH, we monitored the level of cyclin D1. Cyclin D is involved in G1 phase progression (Narasimha
Cancer cells survive by modulating several signaling pathways such as JAK/STAT, MAPK/ERK, and PI3K/Akt (Franceschelli
Based on our results, LCH induced cell cycle arrest and apoptosis in human skin cancer cells by modulating JAK2/STAT3 signaling. JAK2 and STAT3 phosphorylation was downregulated in A375 and A431 cells by treatment with LCH, and the inhibition of JAK2/STAT3 signaling resulted in cell cycle arrest and apoptosis. These findings suggest that LCH has the potential to treat human skin cancer.
We greatly appreciated using the Convergence Research Laboratory (established by the MNU Innovation Support Project in 2019) to conduct this research. This research was funded by the Basic Science Research Program of National Research Foundation Korea, grant number 2019R1A2C1005899. This work was carried out with the support of Cooperative Core Technology Development Project for Environmental Diseases Prevention and Management (2021003310003), funded by the Korea Ministry of Environment (MOE).
There are no conflicts of interest.