Expression of dual-specificity phosphatases in TGFß1-induced EMT in SKOV3 cells.

BACKGROUND: The study aims to profile the dual-specificity phosphatases (DUSP) expression in response to Transforming growth factor ß1 (TGFß1)-induced epithelial- mesenchymal transition (EMT) in ovarian adenocarcinoma cells. METHODS: The ovarian adenocarcinoma cell line SKOV3 was used as a TGFß1-induced EMT model. Cells were incubated with 5 ng/mL TGFß1 to induce EMT. EMT was confirmed with real-time qPCR, western blot, and immunofluorescence analyses of various EMT markers. Western blot was used to analyze phospho- and total MAPK protein levels. Typical and atypical DUSPs mRNA expression profile was determined by real-time qPCR. RESULTS: The epithelial marker E-cadherin expressions were decreased and mesenchymal EMT markers Snail and Slug expression levelswere increased after TGFß1 induction. Phosphorylation of ERK1/2 and p38 MAPK were enhanced in response to TGFß1 treatment. The expression of DUSP2, DUSP6, DUSP8, DUSP10, and DUSP13 were decreased while DUSP7, DUSP16, DUSP18, DUSP21, and DUSP27 were increased by TGFß1. DISCUSSION: TGFß1 induced EMT which was accompanied by increased activity of MAPKs, and led to marked changes in expressions of several DUSPs in SKOV3 cells.

Upregulation of dual-specificity phosphatase-26 is required for transforming growth factor ß1(TGFß1)-induced Epithelial-mesenchymal transition in A549 and PANC1 cells.

BACKGROUND: Transforming Growth Factor ß (TGFß) proteins are potent inducers of the epithelial-mesenchymal transition (EMT) in tumor cells. Although mitogen-activated protein kinase (MAPK) family has been shown to be involved in TGFß-induced EMT, role of Dual Specificity Phosphatases (DUSP), key regulators of MAPK activity, in TGFß-induced EMT is largely unkonwn. METHODS AND RESULTS: Real-time qPCR analyses were performed to determine the effect of TGFß1 on expression of EMT genes and DUSP proteins in the non-small cell lung cancer model A549 and pancreatic adenocarcinoma model PANC1 cells. Western blot analyses were conducted to study the changes in protein levels of EMT proteins and select DUSP proteins, as well as phosphorylations of MAPK proteins upon TGFß1 stimulation. Small interfering RNA (siRNA) was utilized to reduce expressions of DUSP genes. We observed that the EMT phenotype coincided with increases in phosphorylations of the MAPK proteins ERK1/2, p38MAPK, and JNK upon TGFß1 stimulation. Real-time qPCR analysis showed increases in DUSP15 and DUSP26 mRNA levels and Western blot analysis confirmed the increase in DUSP26 protein levels in both A549 and PANC1 cells treated with TGFß1 relative to control. Silencing of DUSP26 expression by siRNA markedly suppressed the effect of TGFß1 on E-cadherin and mesenchymal genes in the cells. CONCLUSIONS: Data provided suggest that TGFß1 modulates the expression of DUSP genes and that upregulation of DUSP26 may be required for TGFß1-promoted EMT in A549 and PANC1 cells. Further studies should be carried out to elucidate the requirement of individual DUSPs in TGFß1-associated EMT in tumor cells.

Overexpression of dual-specificity phosphatases 4 and 13 attenuates transforming growth factor ß1-induced migration and drug resistance in A549 cells in vitro.

Transforming growth factor-beta (TGFß) proteins induce an epithelial-mesenchymal transition (EMT) programme that is associated with increased invasive and drug-resistant phenotype of carcinoma cells. In addition to the canonical pathway involving SMAD proteins, the mitogen-activated kinase (MAPK) pathway via extracellular signal-regulated kinases ½ (ERK1/2) is also involved in promoting and maintaining a mesenchymal phenotype by tumor cells following TGFß signal activation. As dual-specificity phosphatases (DUSPs) regulate ERK1/2 activity by dephosphorylation, we aimed to examine DUSPs' expression upon TGFß stimulation and whether DUSPs play a role in the EMT and related phenotypes promoted by TGFß1 in A549 cells. We found that TGFß1 stimulation led to marked changes in several DUSP proteins, including significant decreases in DUSP4 and DUSP13 expressions. We then showed that the ectopic co-expression of DUSP4/13 suppresses TGFß1-induced ERK1/2 phosphorylation and protein levels of the EMT transcription factors Snail and Slug proteins. We then demonstrated that DUSP4/13 co-expression partially inhibited TGFß1-promoted migration, invasion, and chemoresistance in A549 cells. Collectively, this report provides data for the involvement of DUSP4/13 in malignant phenotypes regulated by TGFß1 in A549 cells.

Simultaneous inhibition of PFKFB3 and GLS1 selectively kills KRAS-transformed pancreatic cells.

Activating mutations of the oncogenic KRAS in pancreatic ductal adenocarcinoma (PDAC) are associated with an aberrant metabolic phenotype that may be therapeutically exploited. Increased glutamine utilization via glutaminase-1 (GLS1) is one such feature of the activated KRAS signaling that is essential to cell survival and proliferation; however, metabolic plasticity of PDAC cells allow them to adapt to GLS1 inhibition via various mechanisms including activation of glycolysis, suggesting a requirement for combinatorial anti-metabolic approaches to combat PDAC. We investigated whether targeting the glycolytic regulator 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) in combination with GLS1 can selectively prevent the growth of KRAS-transformed cells. We show that KRAS-transformation of pancreatic duct cells robustly sensitizes them to the dual targeting of GLS1 and PFKFB3. We also report that this sensitivity is preserved in the PDAC cell line PANC-1 which harbors an activating KRAS mutation. We then demonstrate that GLS1 inhibition reduced fructose-2,6-bisphosphate levels, the product of PFKFB3, whereas PFKFB3 inhibition increased glutamine consumption, and these effects were augmented by the co-inhibition of GLS1 and PFKFB3, suggesting a reciprocal regulation between PFKFB3 and GLS1. In conclusion, this study identifies a novel mutant KRAS-induced metabolic vulnerability that may be targeted via combinatorial inhibition of GLS1 and PFKFB3 to suppress PDAC cell growth.

PFKFB2 regulates glycolysis and proliferation in pancreatic cancer cells.

Tumor cells increase glucose metabolism through glycolysis and pentose phosphate pathways to meet the bioenergetic and biosynthetic demands of rapid cell proliferation. The family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) are key regulators of glucose metabolism via their synthesis of fructose-2,6-bisphosphate (F2,6BP), a potent activator of glycolysis. Previous studies have reported the co-expression of PFKFB isozymes, as well as the mRNA splice variants of particular PFKFB isozymes, suggesting non-redundant functions. Majority of the evidence demonstrating a requirement for PFKFB activity in increased glycolysis and oncogenic properties in tumor cells comes from studies on PFKFB3 and PFKFB4 isozymes. In this study, we show that the PFKFB2 isozyme is expressed in tumor cell lines of various origin, overexpressed and localizes to the nucleus in pancreatic adenocarcinoma, relative to normal pancreatic tissue. We then demonstrate the differential intracellular localization of two PFKFB2 mRNA splice variants and that, when ectopically expressed, cytoplasmically localized mRNA splice variant causes a greater increase in F2,6BP which coincides with an increased glucose uptake, as compared with the mRNA splice variant localizing to the nucleus. We then show that PFKFB2 expression is required for steady-state F2,6BP levels, glycolytic activity, and proliferation of pancreatic adenocarcinoma cells. In conclusion, this study may provide a rationale for detailed investigation of PFKFB2's requirement for the glycolytic and oncogenic phenotype of pancreatic adenocarcinoma cells.

Effect of high-fat diet on the various morphological parameters of the ovary / 대한해부학회지

Increased food consumption rich in fat and carbohydrate and sedentary lifestyle have seriously increased the rates of obesity and obesity-associated diseases in developed countries. Female mice with diet-induced obesity exhibit infertility and thus can serve as a model for human polycystic ovary syndrome. The aim of the present study was to examine how ovary is affected by diet-induced obesity. The effects of high-fat diet (HFD) on ovary morphology in mice fed with HFD were investigated using unbiased stereological methods. The ovary of mice fed with HFD (n=8, C1090-60, Altromine) for 9 weeks, were compared with that of mice fed with standard chow diet (n=8, C1090-10, Altromine). Stereological parameters were obtained in diestrus cycle. The samples were processed through routine and standard paraffin embedding and were serially sectioned in 5-μm thickness then, every 10th section was saved, stained with Crossman’s triple stain for counting and measuring. In all sampled sections mean follicle numbers, diameters, total ovarian volume cortex to medulla ratio (Vv), ovum to cell ratio in secondary follicle were examined in all sampled sections. The present results showed that weight of ovarian and amount of intraperitoneal adipose tissue and the body weight markedly increased in obese mice when compared with control groups. Moreover, follicle numbers (except primordial follicles) and diameters were significantly increased in obese mice. Cortex to medulla ratio (Vv) and ovum to cell ratio in secondary follicle were also considerably different between experimental and the control groups. The present findings indicate that obesity adversely affects overall ovarian morphology.

Effects of capsaicin on ovarian granulosa cell proliferation and apoptosis.

Capsaicin is the pungent ingredient in red peppers. Due to the effects on the sensory nerve fibers, capsaicin has been used to treat pain and inflammation associated with a variety of diseases including rheumatoid arthritis and diabetic neuropathy, obesity, and cardiovascular and gastrointestinal conditions. Despite the extensive publications on different systems, the studies of the effects on the ovary are very limited. The present study was conducted to examine the possible proliferative and/or apoptotic effects of various doses of capsaicin on primarily derived granulosa cells. In accordance with this purpose, ovarian granulosa cells were exposed to different doses of capsaicin for 24 and 48 h. The proliferative effects of capsaicin were examined by immunocytochemistry, immunofluorescence, and western blot using an antibody against proliferating cell nuclear antigen (PCNA) and cell viability assay (MTT). The effects on apoptosis were determined by immunocytochemistry and immunofluorescence using antibodies against cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP). We showed that the number of apoptotic cells increased in a capsaicin dose and time-dependent manners. We found that a low dose of CAP in 24 h administration was more effective on granulosa cell proliferation. Our results suggest that low-dose and short-term administration of CAP may have a positive effect on ovarian folliculogenesis.