P110ß Inhibition Reduces Histone H3K4 Di-Methylation in Prostate Cancer.

INTRODUCTION AND AIMS: Epigenetic alteration plays a major role in the development and progression of human cancers, including prostate cancer. Histones are the key factors in modulating gene accessibility to transcription factors and post-translational modification of the histone N-terminal tail including methylation is associated with either transcriptional activation (H3K4me2) or repression (H3K9me3). Furthermore, phosphoinositide 3-kinase (PI3 K) signaling and the androgen receptor (AR) are the key determinants in prostate cancer development and progression. We recently showed that prostate-targeted nano-micelles loaded with PI3 K/p110beta specific inhibitor TGX221 blocked prostate cancer growth in vitro and in vivo. Our objective of this study was to determine the role of PI3 K signaling in histone methylation in prostate cancer, with emphasis on histone H3K4 methylation. METHODS: PI3 K non-specific inhibitor LY294002 and p110beta-specific inhibitor TGX221 were used to block PI3 K/p110beta signaling. The global levels of H3K4 and H3K9 methylation in prostate cancer cells and tissue specimens were evaluated by Western blot assay and immunohistochemical staining. A synthetic androgen R1881 was used to stimulate AR activity in prostate cancer cells. A castration-resistant prostate cancer (CRPC) specific human tissue microarray (TMA) was used to assess the global levels of H3K4me2 methylation by immunostaining approach. RESULTS: Our data revealed that H3K4me2 levels were significantly elevated after androgen stimulation. With RNA silencing and pharmacology approaches, we further defined that inhibition of PI3 K/p110beta activity through gene-specific knocking down and small chemical inhibitor TGX221 abolished androgen-stimulated H3K4me2 methylation. Consistently, prostate cancer-targeted delivery of TGX221 in vivo dramatically reduced the global levels of H3K4me2 as assessed by immunohistochemical staining on tissue section of mouse xenografts from CRPC cell lines 22RV1 and C4-2. Finally, immunostaining data revealed a strong H3K4me2 immunosignal in CRPC tissues compared to primary tumors and benign prostate tissues. CONCLUSIONS: Taken together, our results suggest that PI3 K/p110beta-dependent signaling is involved in androgen-stimulated H3K4me2 methylation in prostate cancer, which might be used as a novel biomarker for disease prognosis and targeted therapy. Prostate 77:299-308, 2017. © 2016 Wiley Periodicals, Inc.

Hypoxia upregulates hypoxia inducible factor (HIF)-3alpha expression in lung epithelial cells: characterization and comparison with HIF-1alpha.

The role of the hypoxia-inducible factor (HIF) subunits 1alpha and 2alpha in response to hypoxia is well established in lung epithelial cells, whereas little is known about HIF-3alpha with respect to transcriptional and translational regulation by hypoxia. HIF-3alpha and HIF-1alpha are two similar but distinct basic helix-loop-helix-PAS proteins, which have been postulated to activate hypoxia responsive genes in response to hypoxia. Here, we used quantitative real time RT-PCR and immunoblotting to determine the activation of HIF-3alpha vs. HIF-1alpha by hypoxia. HIF-3alpha was strongly induced by hypoxia (1% O2) both at the level of protein and mRNA due to an increase in protein stability and transcriptional activation, whereas HIF-1alpha protein and mRNA levels enhanced transiently and then decreased because of a reduction in its mRNA stability in A549 cells, as measured on mRNA and protein levels. Interestingly, HIF-3alpha and HIF-1alpha exhibited strikingly similar responses to a variety of activating or inhibitory pharmacological agents. These results demonstrate that HIF-3alpha is expressed abundantly in lung epithelial cells, and that the transcriptional induction of HIF-3alpha plays an important role in the response to hypoxia in vitro. Our findings suggest that HIF-3alpha, as a member of the HIF system, is complementary rather than redundant to HIF-1alpha induction in protection against hypoxic damage in alveolar epithelial cells.

[Quantitative analysis on Radix Salviae miltiorrhizae by NIR].

OBJECTIVE: Rapid nondestructive determination of salvianolic acid B and tanshinone IIA in Radix Salviae Miltiorrhizae with near-infrared reflectance spectroscopy. METHOD: A quantitive model was built up with near-infrared diffuse reflectance spectroscopy. RESULTS: The RMSEP in quantitative calibration model for salvianolic acid B and tanshinone IIA were 0.259 and 0.0232 respectively. CONCLUSION: NIR technique can dispose the samples without complicated pretreatment. You can achieve the results rapidly and correctly. It owns many remarkable advantages that cannot be displayed by traditional analysis methods. It is qualified to rapidly analyze traditional Chinese medicine whose components are complex. NIR can control the quality in production process of traditional Chinese medicine.

[The clinical study on chronic hepatitis B treated by the four-step therapeutics of Traditional Chinese Medicine].

OBJECTIVE: To observe the clinical curative effect of chronic hepatitis B treated by the four-step therapeutics of Traditional Chinese Medicine (TCM). METHODS: 120 patients with mild or moderate Chronic Hepatitis B (CHB) were randomly divided into two groups: 80 patients in treatment group and 40 in control group. All enrolled cases accorded with the enroll standard. In treatment group, the patients were divided into mild moderate and severe degree of immune intervention based on the ALT level and treated with four-step therapeutics according to the dialectical theory. In control group, all patients were administered 100mg Lamivudine orally daily for two years. RESULTS: The loss rates of HBeAg, HBV-DNA, precore mutation were 58.9%, 78.9% in treatment group respectively, and 33.3%, 38.9% in control group. There were significant defferences between them. The total effectiveness ratio of two groups has no significant difference. After the treatment, the value of HA, PCIII, IV. C,LN decreased dramatically in treatment group and the antihepatic fibrosis results of treatment group were superior to those of control group. The four-step therapeutics of TCM could improve the ALT value and the ALT value declined to normal after the virus indexes' loss. The response rate in treatment group of ALT-elevating patients was higher than those of no ALT- elevating patients. CONCLUSION: The four-step therapeutics of TCM is effective in treating the CHB patients.

Addition and Subtraction Theory of TCM Using Xiao-Chaihu-Decoction and Naturopathy in Predicting Survival Outcomes of Primary Liver Cancer Patients: A Prospective Cohort Study.

To investigate the therapeutic effect of combined Xiao-Chaihu-Decoction and naturopathic medicine therapy on survival outcomes of patients' PLC. In XCHD group (n = 76), patients were treated with Xiao-Chaihu-Decoction in accordance with the addition and subtraction theory of TCM; in NM group (n = 89), patients were managed by naturopathic medicine; in combined group (n = 70), the same volume of Xiao-Chaihu-Decoction combined with naturopathic medicine procedures was applied. There were no evident statistical differences of age, gender, KPS score, body weight, smoking status, AFP levels, HbsAg status, TBIL levels, tumor diameters, and numbers among different groups, showing comparability among groups. No significant difference was found regarding the total remission rate and stability rate of tumors in patients treated by Xiao-Chaihu-Decoction and naturopathic medicine, except the combined therapy. KPS scores were significantly improved after treatment among groups. After treatment, 52.8% cases maintained a stable or slight increase in weight, of which 42.1%, 48.3%, and 70.0% cases maintained weight stably in the XCHD group, NM group, and combined treatment group, respectively. Xiao-Chaihu-Decoction associated with naturopathy may predict improved prognostic outcomes in PLC patients, along with improved remission and stability rates, increased KPS scores, and stable weight maintenance.

Protective role of adiponectin in a rat model of intestinal ischemia reperfusion injury.

AIM: To determine the potential protective role of adiponectin in intestinal ischemia reperfusion (I/R) injury. METHODS: A rat model of intestinal I/R injury was established. The serum level of adiponectin in rats with intestinal I/R injury was determined by enzyme-linked immunosorbent assay (ELISA). The serum levels of interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α were also measured by ELISA. Apoptosis of intestinal cells was detected using the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. The production of malondialdehyde (MDA) and superoxide dismutase (SOD) and villous injury scores were also measured. RESULTS: Adiponectin was downregulated in the serum of rats with intestinal I/R injury compared with sham rats. No significant changes in the expression of adiponectin receptor 1 and adiponectin receptor 2 were found between sham and I/R rats. Pre-treatment with recombinant adiponectin attenuated intestinal I/R injury. The production of pro-inflammatory cytokines, including IL-6, IL-1ß, and TNF-α, in rats with intestinal I/R injury was reduced by adiponectin pre-treatment. The production of MDA was inhibited, and the release of SOD was restored by adiponectin pre-treatment in rats with intestinal I/R injury. Adiponectin pre-treatment also inhibited cell apoptosis in these rats. Treatment with the AMP-activated protein kinase (AMPK) signaling pathway inhibitor, compound C, or the heme oxygenase 1 (HO-1) inhibitor, Snpp, attenuated the protective effects of adiponectin against intestinal I/R injury. CONCLUSION: Adiponectin exhibits protective effects against intestinal I/R injury, which may involve the AMPK/HO-1 pathway.

Up-regulation of hypoxia inducible factor 1alpha by isoflurane in Hep3B cells.

BACKGROUND: The volatile anesthetic isoflurane induces hypoxia inducible factor (HIF)-1-responsive genes heme oxygenase 1, inducible nitric oxide synthase, and vascular endothelial growth factor (VEGF) expression. Little is known about the extent to which induction of HIF-1alpha is affected by isoflurane. METHODS: Hep3B cells were exposed to isoflurane at various concentrations (0.5-4%) or for different time periods (2-8 h) at 37 degrees C. HIF-1alpha gene expression and transcriptional activity, heme oxygenase 1, inducible nitric oxide synthase, and VEGF gene expression were quantified. RESULTS: Isoflurane induced a time- and concentration-dependent increase in HIF-1alpha protein but not for HIF-1alpha messenger RNA (mRNA) in Hep3B cells. The maximal increase was induced by 2% isoflurane, and the cells incubated with 2% isoflurane for 4-8 h expressed the highest protein. Similarly, HIF-1alpha transcriptional activity was higher in Hep3B cells exposed to 2% isoflurane for 16 h than that in control cells. The combination of 2% isoflurane and desferrioxamine, a hypoxia mimetic, caused a higher level of HIF-1alpha protein than that induced by 2% isoflurane alone. Reoxygenation and inhibitor of proteasome pathway MG132 did not affect the isoflurane-induced HIF-1alpha protein accumulation. Cycloheximide, an inhibitor for protein synthesis, completely abrogated the induction of HIF-1alpha protein by isoflurane. Isoflurane stimulated heme oxygenase 1, inducible nitric oxide synthase, and VEGF mRNA expression in a concentration-dependent manner, and inactivation of HIF-1alpha attenuated the induction of VEGF mRNA by isoflurane. CONCLUSION: Isoflurane can up-regulate HIF-1alpha and enhance HIF-1-responsive genes heme oxygenase 1, inducible nitric oxide synthase, and VEGF mRNA expression in Hep3B cells. The induction of HIF-1alpha by isoflurane does not involve protein degradation but depends on translation pathway.