MicroRNA-23b and microRNA-27b plus flutamide treatment enhances apoptosis rate and decreases CCNG1 expression in a castration-resistant prostate cancer cell line.

The acquisition of a castration-resistant prostate cancer phenotype by prostate cancer cells is the alteration that has the worst prognosis for patients. The aim of this study was to evaluate the role of the microRNAs-23b/-27b as well as the possible CCNG1 target gene in tissue samples from patients with localized prostate cancer that progressed to castration-resistant prostate cancer and in a castration-resistant prostate cancer cell line (PC-3). The microRNAs and target gene expression levels of the surgical specimens were analyzed by quantitative real-time polymerase chain reaction. The prostate cancer cell line, PC-3, was transfected with pre-miR-23b, pre-miR-27b, and their respective controls using Lipofectamine RNAiMAX and exposed or not to flutamide. After transfections, expression levels of both the microRNAs and the gene, CCNG1, were analyzed by quantitative real-time polymerase chain reaction. The apoptosis and cell cycle assays were performed on the mini MUSE cytometer. MicroRNAs-23b/-27b were underexpressed in surgical specimens of prostate cancer; however, their target gene, CCNG1, was overexpressed in 69% of the cases. After transfection with the microRNAs-23b/-27b and flutamide, we observed a reduction in gene expression compared with cells that were treated only with microRNAs or only with flutamide. In the apoptosis assay, we demonstrated cell sensitization following transfection with microRNAs-23b/-27b and potentiation when co-administered with flutamide. The number of cells in apoptosis was almost three times higher with the simultaneous treatments (miR + flutamide) compared with the control (p < 0.05). In the cell cycle assay, only flutamide treatment showed better results; a higher number of cells were found in the G0-G1 phase, and a lower percentage of cells completed the final phase of the cycle (p < 0.05). We conclude that microRNAs-23b/-27b are downexpressed in prostate cancer, and their target gene, CCNG1, is overexpressed. We postulated that microRNAs-23b/-27b sensitize the PC-3 cell line and that after the addition of flutamide in the apoptosis assay, we would observe synergism in the treatments between miR and flutamide. In the cell cycle assay, the use of flutamide was sufficient to decrease the number of cells in mitosis. Therefore, we postulate that microRNAs, along with other drugs, may become very useful therapeutic tools in the treatment of castration-resistant prostate cancer.

Estrogen and progesterone promote breast cancer cell proliferation by inducing cyclin G1 expression.

Breast cancer is the most common cause of cancer among women in most countries (WHO). Ovarian hormone disorder is thought to be associated with breast tumorigenesis. The present study investigated the effects of estrogen and progesterone administration on cell proliferation and underlying mechanisms in breast cancer MCF-7 cells. It was found that a single administration of estradiol (E2) or progesterone increased MCF-7 cell viability in a dose-dependent manner and promoted cell cycle progression by increasing the percentage of cells in the G2/M phase. A combination of E2 and progesterone led to a stronger effect than single treatment. Moreover, cyclin G1 was up-regulated by E2 and/or progesterone in MCF-7 cells. After knockdown of cyclin G1 in MCF-7 cells using a specific shRNA, estradiol- and progesterone-mediated cell viability and clonogenic ability were significantly limited. Additionally, estradiol- and progesterone-promoted cell accumulation in the G2/M phase was reversed after knockdown of cyclin G1. These data indicated that estrogen and progesterone promoted breast cancer cell proliferation by inducing the expression of cyclin G1. Our data indicated that novel therapeutics against cyclin G1 are promising for the treatment of estrogen- and progesterone-mediated breast cancer progression.

Estrogen and progesterone promote breast cancer cell proliferation by inducing cyclin G1 expression

Breast cancer is the most common cause of cancer among women in most countries (WHO). Ovarian hormone disorder is thought to be associated with breast tumorigenesis. The present study investigated the effects of estrogen and progesterone administration on cell proliferation and underlying mechanisms in breast cancer MCF-7 cells. It was found that a single administration of estradiol (E2) or progesterone increased MCF-7 cell viability in a dose-dependent manner and promoted cell cycle progression by increasing the percentage of cells in the G2/M phase. A combination of E2 and progesterone led to a stronger effect than single treatment. Moreover, cyclin G1 was up-regulated by E2 and/or progesterone in MCF-7 cells. After knockdown of cyclin G1 in MCF-7 cells using a specific shRNA, estradiol- and progesterone-mediated cell viability and clonogenic ability were significantly limited. Additionally, estradiol- and progesterone-promoted cell accumulation in the G2/M phase was reversed after knockdown of cyclin G1. These data indicated that estrogen and progesterone promoted breast cancer cell proliferation by inducing the expression of cyclin G1. Our data indicated that novel therapeutics against cyclin G1 are promising for the treatment of estrogen- and progesterone-mediated breast cancer progression.

Leukemia inhibitory factor induces DNA synthesis in Swiss mouse 3T3 cells independently of cyclin D1 expression through a mechanism involving MEK/ERK1/2 activation.

Leukemia inhibitory factor (LIF) and oncostatin M (OSM) induce DNA synthesis in Swiss 3T3 cells through common signaling mechanism(s), whereas other related cytokines such as interleukin-6 and ciliary neurotrophic factor do not cause this response. Induction of DNA replication by LIF or prostaglandin F2alpha (PGF2alpha) occurs, in part, through different signaling events. LIF and OSM specifically trigger STAT1 cytoplasmic to nuclear translocation, whereas PGF2alpha fails to do so. However, LIF and PGF2alpha can trigger increases in ERK1/2 activity, which are required for their mitogenic responses because U0126, a MEK1/2 inhibitor, prevents both ERK1/2 activation and induction of DNA synthesis by LIF or PGF2alpha treatment. PGF2alpha induces cyclin D expression and full phosphorylation of retinoblastoma protein. In contrast, LIF fails to promote increases in cyclin D mRNA/protein levels; consequently, LIF induces DNA synthesis without promoting full phosphorylation of retinoblastoma protein (Rb). However, both LIF and PGF2alpha increase cyclin E expression. Furthermore, LIF mitogenic action does not involve protein kinase C (PKC) activation, because a PKC inhibitor does not block this effect. In contrast, PKC activity is required for PGF2alpha mitogenic action. More importantly, the synergistic effect between LIF and PGF2alpha to promote S phase entry is independent of PKC activation. These results show fundamental differences between LIF- and PGF2alpha-dependent mechanism(s) that induce cellular entry into S phase. These findings are critical in understanding how LIF and other related cytokine-regulated events participate in normal cell cycle control and may also provide clues to unravel crucial processes underlying cancerous cell division.

A better cell cycle target for gene therapy of colorectal cancer: cyclin G.

The purpose of this study was to evaluate the overexpression of cyclin G in colorectal neoplasia, which may be a more frequent event than cyclin D1 during the cell cycle and thus may have a more enhanced therapeutic potential in treating colorectal cancer. Ninety formalin-fixed, paraffin-embedded human colon and rectal specimens were obtained from the Pathology Department of Norris Cancer Center/University of Southern California. The tissues had been obtained after surgical resection between 1995 and 2001, and had been processed by routine clinical histopathologic methods. Ninety-one percent of colorectal tumors had cyclin G overexpression. These cyclin-positive patients were evenly distributed between men and women, and between tumor locations, that is, 36% rectal tumors and 34% right-sided tumors. Thirty-two percent were well differentiated, and 66% were moderately differentiated. Thirty patients (38%) had stage I disease, 16 (20%) had stage II disease, 25 (32%) had stage III, and seven (9%) had stage IV disease. Eight patients (10%) in this group had recurrent disease during follow-up. There was no correlation between cyclin G overexpression and clinical and pathologic characteristics. Cyclin D1 overexpression was found to be present in only 42% of colorectal adenocarcinomas. There was no correlation between cyclin D1 overexpression and clinical and pathologic characteristics. The present study demonstrates that cyclin G overexpression is a frequent event in colorectal cancer. This frequent event in colorectal carcinogenesis may facilitate new therapeutic approaches acting as a target for gene therapy, possibly directed at downregulating cyclin G in colorectal cancer.

Effect of GDNF on neuroblast proliferation and photoreceptor survival: additive protection with docosahexaenoic acid.

PURPOSE: In a previous study, it was reported that docosahexaenoic acid (DHA) is essential to postpone apoptosis and to promote differentiation of rat retina photoreceptors in vitro. In the current study, the protective effects of GDNF on photoreceptor cells during development in vitro and its action when combined with DHA were investigated. METHODS: Rat retina neuronal cultures were incubated in a chemically defined medium, either without photoreceptor survival factors or supplemented with GDNF, DHA, or GDNF plus DHA. Evolution of survival, apoptosis, opsin expression, mitochondrial functioning, and cell proliferation were investigated at different times of development in vitro. RESULTS: Incubation with GDNF selectively increased the number of surviving photoreceptors, reduced their apoptosis, and augmented opsin expression. Proliferative cell nuclei antigen (PCNA) determination and addition of [(3)H]-thymidine or bromodeoxyuridine showed that GDNF promoted neuroblast proliferation during the first hours of development in vitro. The combined addition of GDNF and DHA enhanced opsin expression and photoreceptor survival in an additive manner. The advance of photoreceptor apoptosis in cultures without trophic factors correlated with an increased impairment in mitochondrial functionality. Addition of GDNF and DHA significantly diminished the loss of mitochondrial activity. CONCLUSIONS: These results show that GDNF stimulated the cell cycle progression, leading to neuroblast proliferation at early stages of development, and delayed the onset of apoptosis later on, improving differentiation and acting as a trophic factor for photoreceptors. The combination of GDNF with DHA had an additive effect both on photoreceptor survival and on opsin expression. Preservation of mitochondrial function may be involved in the antiapoptotic effect of both factors.