The serum shock process is one of the approved methods to reset cell rhythm, which regulates cell metabolism and circadian rhythm17. In our study, serum shock process was considered as the main variable for rearrangement of cellular metabolism. after the serum shock process and exclusion of U87-MG cells from the serum-containing medium for 8 hours, we saw an increase in rate of the proliferation in the shock-group compared to the control group. It is worth mentioning that, in shock group we observed less cellular damages compared to control group hence, it can be said that, the morphological changes confirm the difference between two groups in the rate of proliferation
Studies have shown that astrocyte cells in the cortex of rats and glioma cells are specifically capable of producing melatonin separately from pineal cells. 15 Melatonin synthesized from a tumor glioma, exerts an endocrine anti-proliferative effect; Thus, more aggressive gliomas synthesize or accumulate less melatonin. 16 After serum shock process We measure the concentration of melatonin in cell culture supernatant and cell layset at the same time. The concentration of melatonin in shock group was 2 times higher than control group. This finding may confirm the fact that, in the specific condition after the serum shock the U87-MG cells start to produce the melatonin which is help them to maintain their metabolic hemostasis.
One of the new findings in recent studies about melatonin is the presence of a transporter for this molecule in the cell. PEPT1 / 2 membrane transporter is the candidate transporter of melatonin in the cell. This protein is located both on the cell membrane and mitochondrial membrane, which is the main site of intercellular synthesis of melatonin. The function of this transporter is to regulate the concentration of melatonin both in intercellular and intracellular of the cell.18. The other finding of our study, was an approximately 10-time differences in the concentration of melatonin between intercellular and intracellular of the U87-MG cells. By considering our finding, the further studies should be design to prove the existence of this transporter on U87-MG cell lines.
The cellular changes observed after the serum shock process necessitated the measurement of the expression of certain genes that play pivotal roles in cellular pathways.
One of the genes that studied, is the TFAM gene, or mitochondrial transcription factor A. This gene is one of the main controllers of mitochondrial gene transcription and also plays an essential role in the preservation and stability of the mitochondrial genome. previous studies have shown that this gene can be one of the most important targets in the suppression of glioma tumors; Injection of exogenous melatonin into the U87-MG cell line reduced the expression of this gene, which in turn increased the instability in the mitochondrial genome, that eventually led to apoptosis of U87-MG cells.6 In our study, with increasing endogenous melatonin concentration after serum shock process, the expression of TFAM gene not only did not decrease but also showed a significant increase in the shock group compared to the control group. This increase in expression by considering the role of TFAM in the stability of the mitochondrial genome can be assumed as one of the factors involved in increasing the rate of proliferation in this study.
Another gene studied, is the PGC1-α gene. This gene is one of the key genes in regulating cell metabolism by regulating mitochondrial metabolic activity. PGC1-α plays a role by affecting the function of mitochondrial transcription factors. On the other hand, increased expression of this gene increases biogenesis in mitochondria. The results of previous studies have shown that in glioblastoma and especially in U87-MG cell line, increased expression of PGC1-α gene is significantly associated with pathogenesis and malignancy.19 In our study, the expression of PGC1-α gene was significantly increased, which along with the increase in TFAM expression could explain the maintenance the shock group of the U87-MG cell line.
The BMAL1-CLOCK protein complex promotes transcription of the ROR, Rev-erb families, and PGC-1α gene. On the other hand, Rev-erbα can directly participate in the regulation of PGC-1α expression, mitochondrial biogenesis and autophagy. In addition, circadian rhythm fluctuations, modulate mitochondrial dynamics by regulating Drp1 and ATP.20 These facts prompted us to measure BMAL1 gene expression after the serum shock process. The results obtained from the analysis of BMAL1 gene expression revealed that the expression of this gene also showed a significant increase along with TFAM and PGC-1α genes. This finding suggests that the process of serum shock and subsequent increase in endogenous melatonin concentration can activate different signaling pathways simultaneously.
Studies have shown that excessive increase or inhibition of PGC-1α gene expression impairs biogenesis and mitochondrial dynamics. However, a moderate increase in the expression of the PGC-1α gene can directly control the expression of the DRP1 gene by binding to its promoter and increase its expression, which results in an increase in stability in mitochondrial dynamics.21 This finding led us to measure the expression of DRP1 gene in our study after observing the increase in PGC-1α gene expression. Observing a significant increase in DRP1 gene expression, revealed that the serum shock process leads to the regeneration of pathways involved in cellular metabolism, in which the role of mitochondria is essential.
in conclusion, it can be said that U87-MG cells after serum shock produce melatonin, simultaneously in cooperation with elevation in the expression of the genes that control the metabolism in mitochondria and also circadian rhythm. The role of endogenous melatonin in the mention pathways should be studied because of the Extensive effect of this molecule in cellular hemostasis. Of course, it is worth noting that further studies to identify involved pathways in normal cells and other cancer cell lines are of great importance.