Endogenous Melatonin Concentration Along With the Expression of Mitochondrial Regulator Genes Is Elevated by the Serum Shock Process in the U87-MG Cell Line
Authors: Balkhi Shayan , Jazi Marie Saghaeian , Samaei Nader Mansour , Farahmand Mahtab 
Published: September 2021

Abstract
Glioblastoma, also known as the fourth grade in the development of astrocytoma according to the World Health Organization, is a tumor in the glial region confined to the central nervous system with high invasion capability to the parenchyma of the brain. Recent findings suggest that melatonin can be synthesized outside the pineal gland tissue. Mitochondria can produce melatonin independently but in coordination with cell demands which plays a critical role in regulating the cell cycle and cell metabolism. hence, we aimed to examine the relationship between cell metabolism and the induction of endogenous melatonin increase induced by the serum shock process, then, determine the percentage of cell proliferation.Background: glioblastoma is a highly invasive tumor of glial cell of brain tissue. Recently it was reported that melatonin can be produced in mitochondria organelle of the glioblastoma cells independent to pineal gland. Regarding the physiological function of melatonin released from pineal gland in regulation of rhythmicity, here we aimed to investigate if serum shock standard protocol known for cellular rhythm regulator can change the melatonin production ability of the glioblastoma cell.Material and methods: First, U87-MG glioblastoma cells were cultured in a DMEM medium containing 10% FBS and then cells were treated with a standard serum shock process (no FBS, 8h). The concentration of melatonin was measured using ELISA method in supernatant and cell extracts of Shock and control groups. The cell proliferation was measured by using BrdU staining and flow cytometry assessment. The gene expression levels of some mitochondria or circadian related genes including TFAM, BMAL1, PGC-1α, and DRP1 were measured, using qRT-PCR method.Results: our findings showed increased (two times) concentration of cellular and released endogenous melatonin in the FBS shock treated U87-MG glioblastoma cells compared to the control group. we found significant up-regulation of the mitochondria or circadian regulator genes (TFAM, BMAL1, PGC-1α, and DRP1) at mRNA level; in the FBS shock group compared to the control group (P <0.0002). Moreover, the percent of proliferative cell (Brdu positive) was also elevated in FBS shock group however it was not statistically significant. Conclusion: the serum shock process has a far effect on the cellular behavior of the U87-MG cell line. regard to the results of the study, it is worth mentioning that an increase in the concentration of endogenous melatonin affects many signaling pathways within the U87-MG cell line, and the elevated expression of the candidate genes was the proof of this fact.by considering the results of this study it also should be noted that detailed investigating the role of endogenous melatonin and its effects on cancer cells is pivotal and by comparing the results of the normal cells with cancer cells we can find the hotspots of the involved signaling pathways that could help better understanding the biology of glioblastoma.