Abstract
p53, the major tumor suppressor, is frequently mutated in many cancers, and up to 84% of human melanomas harbor wild-type p53, which is considered to be an ideal target for melanoma therapy. Here, we evaluated the antitumor activity of a carbazole derivative, 9-ethyl-9H-carbazole-3-carbaldehyde (ECCA), on melanoma cells. ECCA had a selectively strong inhibitory activity against the growth of BRAF-mutated and BRAF-wild-type melanoma cells but had little effect on normal human primary melanocytes. ECCA inhibited melanoma cell growth by increasing cell apoptosis, which was associated with the upregulation of caspase activities and was significantly abrogated by the addition of a caspase inhibitor. In vivo assays confirmed that ECCA suppressed melanoma growth by enhancing cell apoptosis and reducing cell proliferation, and importantly ECCA did not have any evident toxic effects on normal tissues. RNA-Seq analysis identified several pathways related to cell apoptosis that were affected by ECCA, notably, activation of the p53 signaling pathway. Biochemical assays demonstrated that ECCA enhanced the phosphorylation of p53 at Ser15 in melanoma cells harboring wild-type p53, and importantly, the knockdown or deletion of p53 in those cells counteracted the ECCA-induced apoptosis, as well as senescence. Further investigations revealed that ECCA enhanced the phosphorylation of p38-MAPK and c-Jun N-terminal kinase (JNK), and treatment with either a p38-MAPK or a JNK inhibitor rescued the cell growth inhibition elicited by ECCA, which depended on the expression of the p53 gene. Finally, the combination of ECCA with a BRAF inhibitor significantly enhanced the growth inhibition of melanoma cells. In summary, our study demonstrates that the carbazole derivative, ECCA, induces melanoma cell apoptosis and senescence through the activation of p53 to significantly and selectively suppress the growth of melanoma cells without affecting normal human melanocytes, suggesting its potential to develop a new drug for melanoma therapy.
Highlights
Introduction The tumor suppressorTP53, discovered 40 years ago, is frequently mutated in human tumors, which disables its antitumor function and increases its tumor-promoting potential[1,2]
At 48 h after treatment, we found that ECCA significantly inhibited the growth of all melanoma cell lines tested at 1 μM no matter whether they harbored a wild-type or a mutated BRAF gene, and the inhibitory effect occurred in a dose-dependent manner (Fig. 1A), UACC62 cells were more sensitive to ECCA
In order to further determine the selective cellular toxicity of ECCA for melanoma cells, we treated normal human primary melanocytes with this compound, and interestingly, we found that ECCA had no severe cytotoxic effect on human primary melanocytes at low concentrations that significantly inhibit the growth of UACC62 cells (Fig. 1C)
Summary
Introduction The tumor suppressorTP53, discovered 40 years ago, is frequently mutated in human tumors, which disables its antitumor function and increases its tumor-promoting potential[1,2]. Wen et al Cell Death and Disease (2021)12:591 overexpression of the p53-negative regulators MDM2 or MDMX, as well as alterations in the expression of the p53 family member p73 isoform delta Np731,5,6. The reactivation of wild-type p53, which is able to induce cell cycle arrest, apoptosis, and cellular senescence in melanoma, has been successfully shown to play an antitumor function[3,4]. An impressive number of clinical trials of drugs targeting wild-type p53 to treat various tumors including melanoma have been carried out, most of them are still phase I/II studies[1]. The main approach developed for the pharmacological reactivation of p53 so far has been to target MDM2 or MDMX, which have been shown to play important roles in the regulation of various biological functions independent of p531. The exploration of new direct and efficient targets on tumor cells with little or no effect on normal cells would be beneficial for melanoma therapy in the future
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