Effects of Progesterone and Selective Ligands of Membrane Progesterone Receptors in HepG2 Cells of Human Hepatocellular Carcinoma.

Progesterone exerts multiple effects in different tissues through nuclear receptors (nPRs) and through membrane receptors (mPRs) of adiponectin and progestin receptor families. The effect of progesterone on the cells through different types of receptors can vary significantly. At the same time, it affects the processes of proliferation and apoptosis in normal and tumor tissues in a dual way, stimulating proliferation and carcinogenesis in some tissues, suppressing them and stimulating cell death in others. In this study, we have shown the presence of high level of mPRß mRNA and protein in the HepG2 cells of human hepatocellular carcinoma. Expression of other membrane and classical nuclear receptors was not detected. It could imply that mPRß has an important function in the HepG2 cells. The main goal of the work was to study functions of this protein and mechanisms of its action in human hepatocellular carcinoma cells. Previously, we have identified selective mPRs ligands, compounds LS-01 and LS-02, which do not interact with nuclear receptors. Their employment allows differentiating the effects of progestins mediated by different types of receptors. Effects of progesterone, LS-01, and LS-02 on proliferation and death of HepG2 cells were studied in this work, as well as activating phosphorylation of two kinases, p38 MAPK and JNK, under the action of three steroids. It was shown that all three progestins after 72 h of incubation with the cells suppressed their viability and stimulated appearance of phosphatidylserine on the outer surface of the membranes, which was detected by binding of annexin V, but they did not affect DNA fragmentation of the cell nuclei. Progesterone significantly reduced expression of the proliferation marker genes and stimulated expression of the p21 protein gene, but had a suppressive effect on the expression of some proapoptotic factor genes. All three steroids activated JNK in these cells, but had no effect on the p38 MAPK activity. The effects of progesterone and selective mPRs ligands in HepG2 cells were the same in terms of suppression of proliferation and stimulation of apoptotic changes in outer membranes, therefore, they were mediated through interaction with mPRß. JNK is a member of the signaling cascade activated in these cells by the studied steroids.

Cytotoxic Effects of the Selective Ligands of Membrane Progesterone Receptors in Human Pancreatic Adenocarcinoma Cells BxPC3.

Progesterone and its synthetic analogues act on cells through different types of receptors, affecting proliferation and apoptosis. These compounds exert their effect through the nuclear receptors and the insufficiently studied membrane progesterone receptors (mPRs) belonging to the progestin and adiponectin Q receptor (PAQR) family. We have identified two selective ligands of mPRs that activate only this type of progesterone receptors - 19-hydroxypregn-4-en-20-one (LS-01) and 19-hydroxy-5ß-pregn-3-en-20-one (LS-02). The goal of this work is to study the effect of these compounds on proliferation and death of human pancreatic adenocarcinoma cells BxPC3 and involvement of the two kinases (p38 MAPK and JNK) in signaling pathways activated by progestins through mPRs. It was shown that progesterone and the compound LS-01 significantly (p < 0.05) inhibited the BxPC3 cell viability, with JNK serving as a mediator. The identified targets of these two steroids are the genes of the proteins Ki67, cyclin D1, PCNA, and p21. Progesterone and the compound LS-01 significantly (p < 0.05) stimulate DNA fragmentation, enhancing the cell death. The p38 mitogen-activated protein kinase (MAPK) is a key mediator of this process. The BCL2A1 protein gene was identified as a target of both steroids. The compound LS-02 significantly (p < 0.05) alters membrane permeability and changes the exposure of phosphatidylserine on the outer membrane leaflet, also enhancing the cell death. This compound acts on these processes by activating both kinases, JNK and p38 MAPK. The compound LS-02 targets the genes encoding the proteins HRK, caspase 9, and DAPK.

Progesterone inhibits proliferation and modulates expression of proliferation-Related genes in classical progesterone receptor-negative human BxPC3 pancreatic adenocarcinoma cells.

Recent studies suggest that progesterone may possess anti-tumorigenic properties. However, a growth-modulatory role of progestins in human cancer cells remains obscure. With the discovery of a new class of membrane progesterone receptors (mPRs) belonging to the progestin and adipoQ receptor gene family, it becomes important to study the effect of this hormone on proliferation of tumor cells that do not express classical nuclear progesterone receptors (nPRs). To identify a cell line expressing high levels of mPRs and lacking nPRs, we examined mRNA levels of nPRs and three forms of mPRs in sixteen human tumor cell lines of different origin. High expression of mPR mRNA has been found in pancreatic adenocarcinoma BxPC3 cells, while nPR mRNA has not been detected in these cells. Western blot analysis confirmed these findings at the protein level. We revealed specific binding of labeled progesterone in these cells with affinity constant similar to that of human mPR expressed in yeast cells. Progesterone at high concentration of 20 µM significantly reduced the mRNA levels of proliferation markers Ki67 and PCNA, as well as of cyclin D1, and increased the mRNA levels of cyclin dependent kinase inhibitors p21 and p27. Progesterone (1 µM and 20 µM) significantly inhibited proliferative activity of BxPC3 cells. These results point to anti-proliferative effects of the progesterone high concentrations on BxPC3 cells and suggest that activation of mPRs may mediate this action. Our data are a starting point for further investigations regarding the application of progesterone in pancreatic cancer.