Calotropis procera extract inhibits prostate cancer through regulation of autophagy.

Current treatment options available for prostate cancer (PCa) patients have many adverse side effects and hence, new alternative therapies need to be explored. Anticancer potential of various phytochemicals derived from Calotropis procera has been studied in many cancers but no study has investigated the effect of leaf extract of C. procera on PCa cells. Hence, we investigated the effect of C. procera leaf extract (CPE) on cellular properties of androgen-independent PC-3 and androgen-sensitive 22Rv1 cells. A hydroalcoholic extract of C. procera was prepared and MTT assay was performed to study the effect of CPE on viability of PCa cells. The effect of CPE on cell division ability, migration capability and reactive oxygen species (ROS) production was studied using colony formation assay, wound-healing assay and 2',7'-dichlorodihydrofluorescein diacetate assay, respectively. Caspase activity assay and LDH assay were performed to study the involvement of apoptosis and necrosis in CPE-mediated cell death. Protein levels of cell cycle, antioxidant, autophagy and apoptosis markers were measured by western blot. The composition of CPE was identified using untargeted LC-MS analysis. Results showed that CPE decreased the viability of both the PCa cells, PC-3 and 22Rv1, in a dose- and time-dependent manner. Also, CPE significantly inhibited the colony-forming ability, migration and endogenous ROS production in both the cell lines. Furthermore, CPE significantly decreased NF-κB protein levels and increased the protein levels of the cell cycle inhibitor p27. A significant increase in expression of autophagy markers was observed in CPE-treated PC-3 cells while autophagy markers were downregulated in 22Rv1 cells after CPE exposure. Hence, it can be concluded that CPE inhibits PCa cell viability possibly by regulating the autophagy pathway and/or altering the ROS levels. Thus, CPE can be explored as a possible alternative therapeutic agent for PCa.

Triiodothyronine stimulates steroid and VEGF production in murine Leydig cells via cAMP-PKA pathway.

Thyroid hormones affect testicular development as well as functions like spermatogenesis and steroidogenesis, thereby influencing male fertility. Our group earlier showed that the stimulatory role of the thyroid hormone, T3 , on the production of vascular endothelial growth factor (VEGF) by murine Leydig cells is mediated by steroids and hypoxia-inducible factor-1 (HIF-1α). The current study further defines the signalling pathway(s) utilised by T3 to stimulate the production of steroids, VEGF and HIF-1α in mouse Leydig tumour cell line (MLTC-1). Specific inhibitors for different signalling molecules were used to study the role of cyclic AMP (cAMP), and its downstream mediators. Expression of VEGF and HIF-1α mRNA were measured by quantitative RT-PCR; VEGF secretion by ELISA; steroid secretion by radioimmunoassay and HIF-1α protein levels by western blotting. Inhibitors of adenylate cyclase (AC), protein kinase A (PKA), sarcoma kinase (SrcK), phosphoinositide 3-kinase (PI3K) and MAP kinase kinase (MEK1/2) abolished the T3 -induced increase in VEGF mRNA and protein levels. The same signalling molecules also mediated the increased production of steroids and HIF-1α protein in response to T3 . Therefore, it was concluded that T3 stimulates steroid secretion and HIF-1α protein in MLTC-1 cells through the AC-cAMP-PKA-PI3K-MEK pathway, which in turn stimulate VEGF production.

A Novel Antigonadotropic Role of Thyroid Stimulating Hormone on Leydig Cell-Derived Mouse Leydig Tumor Cells-1 Line.

Subclinical hypothyroid men characterized by a rise in only thyroid stimulating hormone (TSH) levels and normal thyroid hormone levels showed a fall in their serum progesterone and testosterone levels. This suggested a role of TSH in regulating Leydig cell steroidogenesis. Therefore, we investigated the direct role of TSH on steroid production and secretion using a mouse Leydig tumour cell line, MLTC-1. MLTC-1 cells were treated with different doses of TSH isolated from porcine pituitary as well as recombinant TSH. Steroid secretion was measured by radioimmunoassay. The mRNA levels of steroidogenic enzymes were quantitated by real time PCR whereas the corresponding protein levels were determined by Western blot. In MLTC-1 cells, pituitary TSH as well as recombinant TSH inhibited progesterone and testosterone secretion in a dose dependent manner. The inhibitory action of TSH on steroid secretion was unique and not mimicked by other anterior pituitary hormones including FSH and ACTH. Recombinant TSH showed no effect on StAR and CYP11A1, the enzymes catalysing the non-steroidogenic and steroidogenic rate-limiting steps of steroid synthesis respectively. Recombinant TSH was shown to inhibit steroidogenesis in MLTC-1 cells by inhibiting the 3ß hydroxy steroid dehydrogenase mRNA and protein levels, the enzyme that catalyses the conversion of pregnenolone to progesterone. This inhibitory effect of TSH is probably direct as both mRNA and protein of the TSH receptor were shown to be present in the MLTC-1 cells.

Triiodothyronine stimulates VEGF expression and secretion via steroids and HIF-1α in murine Leydig cells.

Leydig cells are the principal steroidogenic cells of the testis. Leydig cells also secrete a number of growth factors including vascular endothelial growth factor (VEGF) which has been shown to regulate both testicular steroidogenesis and spermatogenesis. The thyroid hormone, T3, is known to stimulate steroidogenesis in Leydig cells. T3 has also been shown to stimulate VEGF production in a variety of cell lines. However, studies regarding the effect of T3 on VEGF synthesis and secretion by the Leydig cells were lacking. Therefore, we investigated the effect of T3 on VEGF synthesis and secretion in a mouse Leydig tumour cell line, MLTC-1. The effect of T3 was compared with that of LH/cAMP and hypoxia, two known stimulators of Leydig cell functions. The cells were treated with T3, 8-Br-cAMP (a cAMP analogue), or CoCl2 (a hypoxia mimetic) and VEGF secreted in the cell supernatant was measured using ELISA. The mRNA levels of VEGF were measured by quantitative RT-PCR. In the MLTC-1 cells, T3, 8-Br-cAMP, and CoCl2 stimulated VEGF mRNA levels and the protein secretion. T3 also increased steroid secretion as well as HIF-1α protein levels, two well-established upstream regulators of VEGF. Inhibitors of steroidogenesis as well as HIF-1α resulted in inhibition of T3-stimulated VEGF secretion by the MLTC-1 cells. This suggested a mediatory role of steroids and HIF-1α protein in T3-stimulated VEGF secretion by MLTC-1 cells. The mediation by steroids and HIF-1α were independent of each other. ABBREVIATIONS: 8-Br-cAMP: 8-bromo - 3', 5' cyclic adenosine monophosphate; CoCl2: cobalt chloride; HIF-1α: hypoxia inducible factor -1α; LH: luteinizing hormone; T3: 3, 5, 3'-L-triiodothyronine; VEGF: vascular endothelial growth factor.

Thyroid and male reproduction.

Male reproduction is governed by the classical hypothalamo-hypophyseal testicular axis: Hypothalamic gonadotropin releasing hormone (GnRH), pituitary luteinizing hormone (LH) and follicle stimulating hormone (FSH) and the gonadal steroid, principally, testosterone. Thyroid hormones have been shown to exert a modulatory influence on this axis and consequently the sexual and spermatogenic function of man. This review will examine the modulatory influence of thyroid hormones on male reproduction.

Role of oxygen in the regulation of Leydig tumor derived MA-10 cell steroid production: the effect of cobalt chloride.

We have earlier shown that cobalt chloride (CoCl2)-induced hypoxia and second messenger 8-bromoadenosine 3', 5'-cyclic adenosine monophosphate (8-Br-cAMP) stimulates vascular endothelial growth factor (VEGF) production in Leydig tumor cell derived MA-10 cells. Both stimuli follow common signal transduction pathways including protein kinase A (PK-A), extracellular regulated kinase 1/2 (ERK1/2), and phosphatidyl inositol-3 kinase/akt (PI3-K/Akt) pathways in the stimulation of VEGF by MA-10 cells. In the present study we investigated the role of CoCl2 and 8-Br-cAMP on steroid production in MA-10 cells. The MA-10 cells were cultured in Waymouth MB 752/1 medium, supplemented with 15% heat inactivated horse serum. Progesterone was estimated by radioimmunoassay (RIA).We report that 8-Br-cAMP stimulated progesterone production by the MA-10 cells whereas CoCl2 inhibited the same. Also, 8-Br-cAMP stimulated steroidogenic acute regulatory protein (StAR) and cytochrome P450 side-chain cleavage enzyme (P450scc) mRNAs expression. However, CoCl2 had no effect on StAR mRNA. Cobalt chloride directly inhibited the expression of P450scc mRNA. The decrease in progesterone production could be attributed to three different mechanisms, (1) an increase in production of reactive oxygen species (ROS), (2) an increase in HIF-1α activity, and (3) ultimately a decrease in the level of cytochrome P450 side chain cleavage (CYT P450scc). Hypoxia has an action and mechanism of action similar to that of gonadotropins on VEGF production, whereas they have a contrasting effect on steroidogenesis. This study suggests that hypoxia could be as important as gonadotropins in regulating Leydig cell steroidogenesis.