Eicosapentaenoic Acid Inhibits KRAS Mutant Pancreatic Cancer Cell Growth by Suppressing Hepassocin Expression and STAT3 Phosphorylation.

BACKGROUND: The oncogenic Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation was reported to be the signature genetic event in most cases of pancreatic ductal adenocarcinoma (PDAC). Hepassocin (HPS/FGL1) is involved in regulating lipid metabolism and the progression of several cancer types; however, the underlying mechanism of HPS/FGL1 in the KRAS mutant PDAC cells undergoing eicosapentaenoic acid (EPA) treatment remains unclear. METHODS: We measured HPS/FGL1 protein expressions in a human pancreatic ductal epithelial (HPNE) normal pancreas cell line, a KRAS-wild-type PDAC cell line (BxPC-3), and KRAS-mutant PDAC cell lines (PANC-1, MIA PaCa-2, and SUIT-2) by Western blot methods. HEK293T cells were transiently transfected with corresponding KRAS-expressing plasmids to examine the level of HPS expression with KRAS activation. We knocked-down HPS/FGL1 using lentiviral vectors in SUIT-2 cells and measured the cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenicity assays. Furthermore, a lipidomic analysis was performed to profile changes in lipid metabolism after HPS/FGL1 knockdown. RESULTS: We found that the HPS/FGL1 level was significantly upregulated in KRAS-mutated PDAC cells and was involved in KRAS/phosphorylated (p)-signal transduction and activator of transcription 3 (STAT3) signaling, and the knockdown of HPS/FGL1 in SUIT-2 cells decreased cell proliferation through increasing G2/M cell cycle arrest and cyclin B1 expression. In addition, the knockdown of HPS/FGL1 in SUIT-2 cells significantly increased omega-3 polyunsaturated fatty acids (PUFAs) and EPA production but not docosahexaenoic acid (DHA). Moreover, EPA treatment in SUIT-2 cells reduced the expression of de novo lipogenic protein, acetyl coenzyme A carboxylase (ACC)-1, and decreased p-STAT3 and HPS/FGL1 expressions, resulting in the suppression of cell viability. CONCLUSIONS: Results of this study indicate that HPS is highly expressed by KRAS-mutated PDAC cells, and HPS/FGL1 plays a crucial role in altering lipid metabolism and increasing cell growth in pancreatic cancer. EPA supplements could potentially inhibit or reduce ACC-1-involved lipogenesis and HPS/FGL1-mediated cell survival in KRAS-mutated pancreatic cancer cells.

1,25-Dihydroxyvitamin D3 increases testosterone-induced 17beta-estradiol secretion and reverses testosterone-reduced connexin 43 in rat granulosa cells.

BACKGROUND: Aromatase converts testosterone into 17beta-estradiol in granulosa cells, and the converted 17beta-estradiol contributes to follicular maturation. Additionally, excessive testosterone inhibits aromatase activity, which can lead to concerns regarding polycystic ovary syndrome (PCOS). Generally, 1,25-dihydroxyvitamin D3 (1,25D3) supplements help to improve the symptoms of PCOS patients who exhibit low blood levels of 1,25D3. Therefore, this study investigated the interaction effects of 1,25D3 and testosterone on estrogenesis and intercellular connections in rat granulosa cells. METHODS: Primary cultures of granulosa cells were treated with testosterone or testosterone plus 1,25D3, or pre-treated with a calcium channel blocker or calcium chelator. Cell lysates were subjected to western blot analysis to determine protein and phosphorylation levels, and 17beta-estradiol secretion was examined using a radioimmunoassay technique. Cell viability was evaluated by MTT reduction assay. Connexin 43 (Cx43) mRNA and protein expression levels were assessed by qRT-PCR, western blot, and immunocytochemistry. RESULTS: Testosterone treatment (0.1 and 1 microg/mL) increased aromatase expression and 17beta-estradiol secretion, and the addition of 1,25D3 attenuated testosterone (1 microg/mL)-induced aromatase expression but improved testosterone-induced 17beta-estradiol secretion. Furthermore, testosterone-induced aromatase phosphotyrosine levels increased at 10 min, 30 min and 1 h, whereas 1,25D3 increased the longevity of the testosterone effect to 6 h and 24 h. Within 18-24 h of treatment, 1,25D3 markedly enhanced testosterone-induced 17beta-estradiol secretion. Additionally, pre-treatment with a calcium channel blocker nifedipine or an intracellular calcium chelator BAPTA-AM reduced 1,25D3 and testosterone-induced 17beta-estradiol secretion. Groups that underwent testosterone treatment exhibited significantly increased estradiol receptor beta expression levels, which were not affected by 1,25D3. Neither testosterone nor 1,25D3 altered 1,25D3 receptor expression. Finally, at high doses of testosterone, Cx43 protein expression was decreased in granulosa cells, and this effect was reversed by co-treatment with 1,25D3. CONCLUSIONS: These data suggest that 1,25D3 potentially increases testosterone-induced 17beta-estradiol secretion by regulating aromatase phosphotyrosine levels, and calcium increase is involved in both 1,25D3 and testosterone-induced 17beta-estradiol secretion. 1,25D3 reverses the inhibitory effect of testosterone on Cx43 expression in granulosa cells.

Primary and repeated surgeries for ectopic pregnancies and distribution by patient age, surgeon age, and hospital levels: an 11-year nationwide population-based descriptive study in Taiwan.

STUDY OBJECTIVE: To describe the changing trend, repeat operation rate, and distribution of laparoscopy, as compared with laparotomy, in treating ectopic pregnancy, according to patient age, preoperative conditions, surgeon age, and hospital accreditation level, in Taiwan over 11-years. DESIGN: Retrospective cohort study (Canadian Task Force classification II-2). SETTING: Population-based nationwide insurance database. PATIENTS: Women who underwent either laparotomy or laparoscopy because of ectopic pregnancy. INTERVENTIONS: Women who had National Health Insurance (NHI) underwent various surgical procedures to treat ectopic pregnancy. Data for this study were obtained from the Inpatient Expenditures by Admissions files of the NHI Research Database, released by the NHI program in Taiwan between 1997 and 2007. MEASUREMENTS AND MAIN RESULTS: A total of 43 170 women with 44 928 operations were identified. Only the primary surgeries, via either laparotomy or laparoscopy, performed because of ectopic pregnancy were included for analysis. The annual number of procedures to treat ectopic pregnancies decreased in the later years of the 11-year study. Laparotomy decreased significantly, from 81.2% in 1997 to 26.2% in 2007, whereas laparoscopic procedures increased significantly, from 18.8% in 1997 to 73.8% in 2007, as evidenced at log-linear regression analysis (p < .001). The rate of repeat operations because of persistent ectopic pregnancy was higher in the laparoscopy group than in the laparotomy group (0.38% vs 0.14 %; p < .001). Patients were more likely to undergo the same type of operation for the repeated surgery (i.e., laparotomy to laparotomy in 73.1% or laparoscopy to laparoscopy in 80.2%; p = 0.43). Use of laparoscopy (58.1%) and laparotomy (41.9%) differed according to patient age, preoperative comorbidities, surgeon age, and hospital accreditation level and ownership type. With older patients, those with preoperative anemia or shock, and elder surgeons, there was a greater chance that laparotomy would be performed. The probability of undergoing laparotomy was greater in patients in regional hospitals, local hospitals, and office-based clinics compared with those in medical centers. CONCLUSIONS: There has been considerable change in the type of surgical approach used to treat ectopic pregnancy in Taiwan over the past 11 years. Laparoscopy is preferred to laparotomy, and has become the standard surgical approach to treating ectopic pregnancies in Taiwan. However, laparoscopy is associated with a higher rate of repeat operations. The laparoscopic approach signifies a profound change in treating ectopic pregnancies among patients, surgeons, and hospital types.