The molecular mechanism of action of superactive human leptin antagonist (SHLA) and quadruple leptin mutein Lan-2 on human ovarian epithelial cell lines

INTRODUCTION: A number of leptin receptor antagonists have been synthesised for therapeutic use, with pre-clinical tests suggesting their future use in anticancer therapy. To our knowledge, there are no data concerning the possible application of leptin receptor blockers in ovarian cancer. METHODS: In this study, we evaluated two leptin receptor antagonists: superactive human leptin antagonist (SHLA) and quadruple leptin mutein, Lan-2 (L39A/D40A/F41A/I42A), on cell proliferation (Alamar Blue test, BrdU assay), cell cycle gene (qPCR) and protein expression (Western blot) and cell signalling pathways (Western blot) in three different types of cell lines: OVCAR-3, CaOV-3 and HOSEpiC. RESULTS: Both receptor blockers had no effect on non-cancerous HOSEpiC cell line proliferation; however, both reversed the stimulatory effect of leptin on CaOV-3 cell line proliferation to control levels and to below control levels in OVCAR-3 cells. In metastatic carcinoma CaOV-3, both ObR antagonists had an inhibitory effect on the cdk2/cyclin D1 complex, while in serous carcinoma, OVCAR-3, they only had an effect on cdk2 and cdk4 protein expression. SHLA had an inhibitory effect on all investigated signalling pathways in OVCAR-3, while only on Stat3 in CaOV-3. Lan-2 had an inhibitory effect on Stat3 and ERK1/2 in CaOV-3, while in OVCAR-3 it only affected Akt protein phosphorylation. CONCLUSION: Based on these results, we conclude that SHLA and Lan-2 are promising leptin receptor inhibitors which could be used to block leptin activity, eliminating its negative effects on activities related to carcinogenesis. However, the selection of a specific antagonist should be related to tumour type

In Vitro

In our previous paper[1], we demonstrated that porcine follicles collected during the early stage of development are the most sensitive to the toxic action of polychlorinated biphenyl 153 (PCB 153). Follicles of this type were collected to test the effect of PCB 153 on cell steroidogenesis and viability. Cocultures of granulosa and theca cells were grown in M199 medium at 37ºC. Control cultures were maintained in that medium alone, while experimental ones were supplemented with PCB 153 at doses of 5, 10, 50, and 100 ng/ml. After 48, 96, and 144 h, media were collected for steroid analysis and cell viability was measured using an LDH (lactate dehydrogenase activity) cytotoxicity test. A 2-day exposure of follicular cells to all the investigated doses of PCB 153 caused a statistically significant decrease in progesterone (P4) secretion, while in doses of 50 and 100 ng/ml there was also a decrease in testosterone (T) secretion. No effect on estradiol (E2) secretion was observed. The observed decrease in P4 and T secretion, and lack of any statistically significant effect on E2 secretion by cells from small follicles exposed for 48 h to PCB, suggests that PCB 153 acts before P4 formation. Longer exposures caused an increase in P4 secretion, with a concomitant drastic decrease in T secretion and a tendency to decrease the E2 secretion, suggesting inhibition of P450 17α hydroxysteroid dehydrogenase, an enzyme that converts P4 to T. The observed PCB 153–induced increase in P4 secretion by cells collected from small antral follicles, with a concomitant decrease in E2 secretion, accounts for the induction of luteinization and, in this case, inhibition of aromatization process in the follicles. However, in all doses tested and at all times of exposure, PCB 153 had no effect on cell viability. These findings suggest different time of exposure–dependent action of PCB 153 on particular steps of steroidogenesis but not action on cell viability. These results should be considered preliminary, pending confirmation by other studies

Leptin stimulation of cell cycle and inhibition of apoptosis gene and protein expression in OVCAR-3 ovarian cancer cells

The OVCAR-3 cell line expressing the long (ObRb) and short (ObRt) isoforms of leptin receptor mRNA was used to analyze the effect of leptin on the expression of selected genes and proteins involved in the cell cycle and apoptosis. OVCAR-3 cells were exposed to 2, 20, 40, and 100 ng/ml of leptin. Cell proliferation was determined using the alamarBlue cell viability test and flow cytometry. Apoptosis was measured using a cellular DNA fragmentation ELISA kit. The expression of selected cell cycle and apoptosis genes was evaluated by real-time PCR and confirmed by western blot. The stimulatory action of leptin on cell proliferation was observed as an increase in cells in the S and G2/M phases. Up-regulation of genes responsible for inducing cell proliferation and suppression of genes responsible for inhibition of proliferation were noted. Western blots revealed increased expression of cyclins D and A and inhibition of p21WAF1/CIP1 protein expression by leptin. Inhibition of DNA fragmentation was observed under all leptin doses. Suppression of genes involved in the extrinsic and intrinsic apoptotic pathway was observed. Western blots illustrated decreased Bad, TNFR1, and caspase 6 protein expression in response to leptin treatment. Leptin promotes ovarian cancer cell line growth by up-regulating genes and proteins responsible for inducing cell proliferation as well as down-regulating pro-apoptotic genes and proteins in apoptotic pathways. Results of this study warrant examining the relationship between the risk of ovarian cancer and elevated leptin levels in obese women