Transcriptome modulation following administration of luteolin to bleomycin-etoposide-cisplatin chemotherapy on rat LC540 tumor Leydig cells.

Leydig cell tumours represent 1%-3% of all cases of testicular tumours in men. Such tumours respond poorly to radiation or chemotherapy, including bleomycin-etoposide-cisplatin (BEP) combinatorial therapy. In this study, we investigated an alternative approach involving luteolin to improve the efficacy of chemotherapy. LC540 tumour Leydig cells were treated with BEP (bleomycin 40 µg/ml, etoposide 4 µg/ml, cisplatin 8 µg/ml) and/or luteolin 10 µM for comparison with DMSO-treated cells. We performed a transcriptome analysis using RNA-Seq to characterise changes in biological processes and signalling pathways. Treatments of LC540 tumour Leydig cells with luteolin significantly decreased the expression of genes involved in cholesterol biosynthesis, while increasing the expression of genes related to glutathione conjugation (p < .05). Genes being significantly upregulated in response to BEP treatment were involved in the response to toxic substances and transcriptional regulation. Oppositely, genes being significantly downregulated by BEP treatment were enriched for intracellular signal transduction, cell migration, cell adhesion, reproductive system development and cholesterol biosynthesis. BEP chemotherapy proved to be effective in increasing gene expression related to apoptosis of tumour Leydig cells. However, addition of luteolin to BEP treatment had no other effects on biological processes or pathways related to cancer treatment.

The transcription factors SF-1 and SOX8 cooperate to upregulate Cx43 expression in mouse TM4 sertoli cells.

Gap junctions made by connexins within the adult testis are essential for communication between Sertoli cells and for spermatogenesis. Sertoli cells play an important role in supporting germ cells differentiation and maturation into spermatozoa. Connexin43 (Cx43) is the most abundant and important connexin of the testis. We have shown previously that the expression of Cx43 is being regulated by SOX and AP-1 transcription factors in Sertoli cells. However, additional regulatory elements being able to recruit orphan nuclear receptors may be involved. Since SOX and SF-1 transcription factors have been shown to cooperate to regulate gene expression in Sertoli cells, we wondered if such mechanism could be involved in the activation of Cx43 expression. Thus, the activity of the Cx43 promoter was measured by co-transfections of luciferase reporter plasmid constructs with different expression vectors for transcription factors in the TM4 Sertoli cell line. The recruitment of SF-1 to the proximal region of the Cx43 promoter was evaluated by chromatin immunoprecipitation. Our results indicate that SOX8 and SF-1, as well as SOX9 and Nur77, cooperate to activate the expression of Cx43 and that SF-1 is being recruited to the -132 to -26 bp region of the Cx43 promoter. These results allow us to have a better understanding of the mechanisms regulating Cx43 expression and could explain some disturbances in communication between Sertoli cells responsible for impaired fertility.

Luteolin modulates gene expression related to steroidogenesis, apoptosis, and stress response in rat LC540 tumor Leydig cells.

In males, androgens are mainly produced by Leydig cells from the testis. A critical and highly regulated step of steroidogenesis involves the importation of cholesterol within the mitochondria by the steroidogenic acute regulatory (STAR) protein. During aging, STAR protein levels in Leydig cells gradually decrease, leading to a reduced entry of cholesterol into mitochondria and lower testosterone production. In addition to preserving its steroidogenic capacity, tumor Leydig cells can also be excellent models for evaluating the mechanisms of action of anticancer agents. In this study, we examined whether polyphenolics having structural similarities to luteolin could promote steroidogenic and cancer-related gene expressions within rat L540 tumor Leydig cells. In this cell model, luteolin activated Star expression and increased progesterone as well as testosterone productions. Interestingly, luteolin decreased gene expression related to cholesterol biosynthesis, possibly inhibiting membrane synthesis and cell proliferation. In addition, increased expression of genes such as Fas, Cdkn1a, Atp7b, and Tp53, as well as increased accumulation of cleaved caspase 3 and PARP, in response to luteolin treatment indicates that apoptosis is being activated. Luteolin also modulated the expression of genes involved in stress response, such as glutathione-S transferases Gsta1 and Gstt2, and the unfolded protein response. Thus, dietary luteolin may be effective in Leydig cell tumor chemoprevention and in maintaining steroidogenesis in aging males.