Erianin induced tumor-specific inhibition in melanomas
To observe the efficiency of different concentrations of erianin in multiple melanoma cells, we first assessed the half-maximal inhibitory concentration (IC50) of erianin in normal pigment cells (PIG1) and multiple melanoma cells. As a result, the IC50 in melanoma cells ranged from approximately 50 nM to 200 nM, with the IC50 in normal pigmented cells approaching 35 µM (Fig. 1B). These data indicated that erianin promotes inhibition in a melanoma-specific manner.
Erianin inhibited melanoma cell proliferation and triggered melanoma cell death in vitro
To detect the antitumor impact of erianin, colony formation assays under different erianin concentrations were conducted. The results showed that the colonies formed by erianin-treated melanoma cells were fewer and smaller (Fig. 2A, B). Furthermore, a flow cytometry assay was performed to assess the cell cycle. As a result, erianin promoted melanoma cell cycle retardation, and the S phase was significantly prolonged (Fig. 2C, D), which indicated that erianin negatively affected DNA synthesis. To determine whether erianin induced melanoma cell death, we conducted an Annexin V-FITC double staining assay. Consequently, the number of dead melanoma cells after treatment with erianin significantly increased (Fig. 2E, F). Taken together, the data showed that erianin treatment impaired melanoma proliferation and induced cell death in a concentration-dependent manner.
Erianin suppressed melanoma cell proliferation in vivo
To further investigate the impact of erianin on melanoma cell growth in vivo, tumor-bearing models in nude mice were established (Fig. 3A). After xenografts were successfully established, tumors were treated with erianin (100 µM, 50 µL) or PBS (50 µL) every 3 days. After 3 weeks, the subcutaneous tumors were obtained. Notably, the tumors were markedly reduced in diameter after treatment with erianin (Fig. 3B). In addition, erianin-treated mice had tumors that were smaller in diameter and weighed less than those in mice in the control group (Fig. 3C, D). Moreover, HE staining suggested that the cells exhibited more roundness and smaller morphological features with deeper purple cell nuclei in the erianin-treated group (Fig. 3E, left). We next detected Ki-67 through immunohistochemistry analysis. The results indicated that Ki-67 was significantly reduced in melanoma cells treated with erianin (Fig. 3E, right). Taken together, these data implied that erianin suppressed melanoma cell growth in vivo.
Apoptosis Contributed To Erianin-induced Cell Death In Melanoma
To explore the mechanism underlying erianin in the regression of melanoma, high-throughput transcriptome sequencing was conducted. Importantly, thousands of genes were altered in melanoma cells (Figure S1, deposited in GSE217070). Next, we predicted the most likely targets of erianin using the online tool SwissTargetPrediction (http://swisstargetprediction.ch/). Consequently, we found that 55 genes could be the most likely targets of erianin in melanoma (Fig. 4A). KEGG pathway analysis of the differentially expressed genes suggested that 11 KEGG pathways were significantly enriched, including the cell cycle, apoptosis and P53 signaling pathways (Fig. 4B). GO analysis indicated that the main damage to melanoma cells caused by erianin was related to DNA repair (Figure S2, p value = 8.72E-21). Since melanoma cell death after treatment with erianin indicated apoptosis (Fig. 4B), the relevant genes were analyzed. The heatmap indicated that apoptosis-related genes were altered accordingly (Fig. 4C). The western blotting assay results suggested that the levels of apoptosis-related proteins (CASPASE9 and BAX) increased significantly (Fig. 4D). To further determine whether erianin induced melanoma cell apoptosis, cell death inhibitors were used. The results showed that the apoptosis inhibitor Z-VAD-FMK rescued erianin-treated melanoma cells the most compared with the ferroptosis inhibitor ferrostatin-1 and necroptosis inhibitor necrostatin-2 (Fig. 4E). Overall, the data suggested that erianin triggered apoptosis in melanoma cells.
Gsk3α Is A Candidate Target Of Erianin For Apoptosis Induction
To determine the critical regulator in erianin-induced apoptosis, we identified GSK3α as a target candidate of erianin. GSK3 is a highly conserved serine-threonine kinase encoded by two isoforms in mammals, termed GSK3α and GSK3β[1]. The role of GSK-3 in oncogenesis is paradoxical; GSK-3 acts as a tumor suppressor in some cancers and a tumor promotor in others[4]. Emerging evidence has shown that GSK3α plays a significant role in metastatic melanoma cells[10]. We confirmed that GSK3α in the erianin group was expressed at a lower level than that in the control group using Integrative Genomics Viewer (IGV, Fig. 5A). Moreover, to evaluate the affinity of erianin for GSK3α, we performed molecular docking analysis. It was observed that erianin docked into the three-dimensional structure of GSK3α (P49840, UniProt) with a low binding energy of -9.684 kcal/mol, indicating highly stable binding (Fig. 5B). Furthermore, the mRNA and protein levels of GSK3α after treatment with erianin were markedly reduced (Fig. 5C, D). Next, we overexpressed GSK3α exogenously. The CCK-8 assay results showed that the overexpression of GSK3α significantly rescued erianin-treated melanoma cell growth (Fig. 5E). In summary, these data indicated that erianin induced melanoma apoptosis by inhibiting GSK3α expression.
The Tumorigenic Role Of Gsk3α In Melanoma
Since GSK3α played an important role in erianin-induced apoptosis in melanoma cells and its nondeterminacy in melanoma, we investigated the role of GSK3α in the tumor progression of melanoma. First, we found that the expression level of GSK3α in various melanoma cell lines was higher than that in the normal cell line PIG1 (Fig. 6A, B). In addition, GEPIA (http://gepia.cancer-pku.cn/) analysis of the prognostic significance of GSK3α expression in melanoma patients indicated that high expression of GSK3α was associated with a worse prognosis in both uveal melanoma (p value = 0.036) and cutaneous melanoma (p value = 0.012, Fig. 6C, D). Importantly, the colonies formed by melanoma cells upon GSK3α downregulation were fewer and smaller than those formed by the control treatment (Fig. 6E, F). Additionally, upon GSK3α downregulation, A375 and MEL-290 cell lines grew more attenuately, as suggested by the CCK-8 assays (Fig. 6G). In brief, these results suggested that GSK3α played an arresting protumor role in melanoma.
Gsk3α Regulated The Noncanonical Nf-κb Signaling Pathway In Melanoma
It has been reported that GSK3α is required for promoting critical noncanonical NF-κB signaling in pancreatic cancer cells[2]. To determine whether the NF-κB signaling pathway in melanoma is influenced by erianin, we further assessed the alteration of the NF-κB effector p100-p52. The results showed that p52, but not p100, was expressed at lower levels in a time- and concentration-dependent manner (Fig. 7A, B). These data showed that GSK3α regulated the noncanonical NF-κB signaling pathway in melanoma.