Melatonin inhibits colon cancer RKO cell migration by downregulating Rho-associated protein kinase expression via the p38/MAPK signaling pathway

Abstract

Melatonin is predominately produced and secreted by the pineal gland, and inhibits cell growth in various cancer cell lines such as colorectal cancer. However, the precise mechanisms involved have not been fully elucidated. In the present study, the potential molecular mechanism underlying the efficacy of melatonin on migration in RKO colon cancer cells was investigated. The effects of melatonin and H-1152, a selective inhibitor of Rho-associated protein kinase (ROCK), on the migration of RKO cells were analyzed by an in vitro wound healing assay. The localization of zonula occludens-1 (ZO-1) and occludin were observed by immunofluorescence. Reverse transcription-quantitative polymerase chain reaction (qPCR) was performed to analyze the relative mRNA levels of ROCK, ZO-1 and occludin. In addition, western blot analysis was implemented to examine the expression of ROCK, phospho (p)-myosin phosphatase targeting subunit 1 (MYPT1), p-myosin light chains (MLC) and p-p38. The results revealed that the expression levels of ROCK2, p-MYPT1 and p-MLC in RKO cells were decreased, and the membrane protein expression of ZO-1 and occludin increased when the cells were treated with melatonin. qPCR demonstrated that melatonin downregulated ROCK2 gene expression, and upregulated the expression of the ZO-1 and occludin genes. The levels of ZO-1 and occludin localized in the tight junctions were markedly increased in the immunofluorescence assay. In addition, the phosphorylation levels of p38 were reduced when the cells were treated with melatonin, and treatment with H-1152 downregulated p38 phosphorylation. The results indicated that melatonin may inhibit the migration of RKO colon cancer cells by downregulating ROCK expression via the p38/mitogen-activated protein kinase signaling pathway.

The emergence of melatonin in oncology: Focus on colorectal cancer

“…Moreover, melatonin prevented the cytoskeletal disorganization of RKO cells, thereby inhibiting cell migration. Accordingly, the membrane expression of TJ‐structural proteins occludin and ZO‐1 has been reported upregulated on cell surface of RKO cells treated with melatonin (2.5 mM, up to 48 hours), thus inhibiting their migration and restraining their metastatic dissemination . Similar findings have been recently documented in human gastric carcinoma cells, in which millimolar melatonin (10 −4 M, 48 hours) relocated and concentrate occludin and ZO‐1 on their cell surface, thereby enhancing adhesion and inhibiting their migration and metastasis capacity .…”

“…Accordingly, the membrane expression of TJ-structural proteins occludin and ZO-1 has been reported upregulated on cell surface of RKO cells treated with melatonin (2.5 mM, up to 48 hours), thus inhibiting their migration and restraining their metastatic dissemination. 241 Similar findings have been recently documented in human gastric carcinoma cells, in which millimolar melatonin (10 −4 M, 48 hours) relocated and concentrate occludin and ZO-1 on their cell surface, thereby enhancing adhesion and inhibiting their migration and metastasis capacity. 124 Despite this, the mechanistic underlying the ability of melatonin to modulate TJ's functionality and inhibiting the invasive capacity of tumor cells from CRC and other neoplasias has been sparsely investigated and, consequently, a lot of work remains to be done.…”

“…In addition, the analysis of tumor propagation in an athymic nude mouse model has clearly shown that the combination of melatonin (25 mg/kg) and 5‐FU (20 mg/kg) potentiates the growth inhibition of xenografted CRC tumors with respect to that achieved with 5‐FU alone . Moreover, the p38 MAPK signaling, involved in CRC growth and resistance to 5‐FU and other chemotherapies, is downregulated along with the reinforcement of TJ proteins, in RKO colon cancer cells after treatment with 2.5 mM melatonin, as previously discussed regarding the effect of melatonin on the metastatic spreadout of CRC …”

“…318 Moreover, the p38 MAPK signaling, involved in CRC growth and resistance to 5-FU and other chemotherapies, 354 is downregulated along with the reinforcement of TJ proteins, in RKO colon cancer cells after treatment with 2.5 mM melatonin, as previously discussed regarding the effect of melatonin on the metastatic spreadout of CRC. 241 Even though the above summarized evidence proved that melatonin, per se or as a complementary adjunctive defense against CRC chemotherapy toxicity, is beneficial for relieving CRC patients from adverse side effects of therapeutic regimens, the oncostatic processes by which its works are far from being understood. Thus, for example, there is no unequivocal experimental basis to determine if the therapeutic synergy of melatonin combined with conventional chemotherapy drugs is due to its ability to enhance the oncostatic capacity of the chemotherapeutic agents, or vice versa (drugs amplifying the biological effectivity of melatonin).…”

Melatonin activates cell death programs for the suppression of uterine leiomyoma cell proliferation

“…The anticancer efficiency of melatonin has also been investigated over the past two decades . Melatonin has been reported to exert an anti‐proliferative and cytotoxic effect on prostate cancer and suppress cell migration and metastasis in colon cancer and lung cancer . Melatonin plays an important role in the anti‐proliferation of female reproductive cancers.…”