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.

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.

Effect of tamoxifen on ovarian reserve: A randomized controlled assessor-blind trial in a mouse model.

OBJECTIVE: To determine whether tamoxifen (TMX) exposure causes a permanent decrease in ovarian reserve. MATERIAL AND METHODS: A randomized controlled assessor-blind trial including 30 adult female inbred BALB/C mice. Fifteen mice in the TMX group were given a single 0.1-mg dose of TMX intraperitoneally. Fifteen mice in the control group were given a single dose of the vehicle at the same volume intraperitoneally. Two cycles later, blood samples were collected for determination of anti-Müllerian hormone (AMH) levels, and the mice were sacrificed. After gonadectomy, ovarian size was measured, and follicles were counted under light microscopy. RESULTS: Median serum AMH levels were 6.53 and 6.14 ng/ml in the control and TMX groups, respectively (p=0.03). Ovarian size was significantly decreased in the TMX group. While the number of primordial (9 vs 8), primary (6 vs 3), and secondary (4.5 vs 5) follicles were similar, there were significantly fewer preantral (11.5 vs 6, p<0.01) and antral (2 vs 1, p: 0.03) follicles, as well as corpora lutea (6 vs 3, p: 0.04), in the TMX group than in the control group. The number of atretic (2.5 vs 5, p: 0.048) follicles was increased in the TMX group. CONCLUSION: Tamoxifen administration leads to arrested growth of gonadotropin-sensitive follicles, while insensitive follicles can remain unaffected. TMX is merely an endocrine disruptor, and it does not cause a decrease in primordial follicle pool.

sFas levels increase in response to cisplatin-based chemotherapy in lung cancer patients.

The Fas/Fas Ligand (FasL) system and survivin have counteracting roles in cell survival. Therefore, we explored the role of circulating soluble Fas (sFas) and the tissue levels of Fas and survivin with regard to response to chemotherapy in lung cancer patients. Serum samples from 52 lung cancer patients and 54 control subjects (19 benign lung disease and 35 healthy control subjects) were collected prior to and 24 and 48 h after chemotherapy. sFas was statistically significantly higher in the cancer group than that in the control groups (p < 0.001). Baseline (before chemotherapy) sFas values showed a statistically significant inverse correlation with overall survival (r = -0.599, p < 0.001). There was a significant increase in serum sFas levels 24 h after treatment (p < 0.05). Contrarily, tissue levels of Fas and survivin were not changed following the chemotherapy (p > 0,05). In conclusion, increased sFas may be an indicator of poor outcome in lung cancer patients. However, cisplatin-based chemotherapy may not be effective via neither the Fas/FasL system nor survivin pathway. Indeed, larger sample size is required for further evaluation.

The effects of chronic low-dose capsaicin treatment on substance P levels.

Capsaicin, the pungent ingredient of red pepper, is consumed in varying amounts by many ethnic groups. It serves both therapeutically and as a specific tool to investigate sensory neurons. Although effects of high capsaicin doses are well-established, systemic effects of chronic low-dose capsaicin exposure are unknown. Sprague-Dawley rats (21-day old) were injected with capsaicin (0.5 mg/kg, ip) for 6 and 19 days. Changes in Substance P (SP) levels of lung and skin were measured. Two-step sequential acetic acid extraction was used to estimate neuronal and non-neuronal SP. Six-day, but not 19-day capsaicin treatment decreased SP levels in first as well as second extractions of both tissues. Because the cumulative dose used here was much lower than the neurotoxic doses of capsaicin, initial decrease of SP levels must be due to continuous release of SP from nerve endings as well as non-neuronal tissues. The fact that SP levels returned to control values at the end of 19-day treatment demonstrates that reactive increases in SP synthesis occurred. These findings suggest that systemic exposure to low-dose capsaicin enhances sensory nerve function and also increases SP in non-neuronal tissues. In addition, significantly decreased SP levels of both tissues were observed in 40-day, compared to 27-day old rats.

Histological investigations on the effects of feeding a diet containing red hot pepper on the reproductive organs of the chicken.

In the present study the growth and reproductive organ differences in chickens fed a diet containing 1% red hot pepper (10 g/kg diet) from the first day of age were investigated. In birds fed with the experimental diet it was observed that the abdominal fat content decreased. During the experiment the increase in weight gain in the treated group in the first 4 months was reversed in favour of the control group in month 5. Follicular development in the treated group was faster and laying started 11 days before the control group, and the epithelial and muscular development of the oviduct was always greater than that of the control group. The results indicated that red hot pepper consumed in lower concentrations during the development period in the chickens caused faster development of the reproductive system organs. Laying started 11 days earlier in chicks fed with the red hot pepper added diet, an important economic aspect for egg producers, but which may have implications for other animals. A decrease in abdominal fat content and disorders of lipid metabolism are still under investigation.