Overexpression of von Hippel-Lindau tumor suppressor protein and antisense HIF-1α eradicates gliomas
The von Hippel–Lindau tumor suppressor protein (pVHL) suppresses tumor formation by binding the α subunits of hypoxia-inducible-factors responsible for stimulating tumor angiogenesis and glycolysis, and targeting them for ubiquitination and proteasomal destruction. Loss of pVHL leads to tumorigenesis and development of sporadic renal cell carcinomas and central nervous system hemangioblastomas. In the present study, we investigated whether engineered overexpression of pVHL in C6 glioma cells, which already express endogenous pVHL, would suppress the tumorigenicity of this particular tumor cell type. C6 cells overexpressing VHL displayed a reduced growth rate (70% inhibition) compared to the parental cell line when subcutaneously implanted in athymic (nu/nu) mice. Growth inhibition was associated with a 50% reduction in the number of tumor vessels and a 60% increase in tumor cell apoptosis, due in part to downregulation of HIF-1, VEGF, and the antiapoptotic factor Bcl-2, respectively. Gene transfer of VHL suppressed the growth of established C6 gliomas, and synergized with antisense HIF-1 to completely eradicate tumors. The data suggest that VHL gene therapy and/or agents that increase VHL expression could have utility in the treatment of gliomas, particularly when combined with agents that inhibit the expression or function of HIF-1.

title: Antisense HIF-1α prevents acquired tumor resistance to angiostatin gene therapy
abstract: Angiostatin is a naturally occurring inhibitor of angiogenesis that is being developed as a drug to fight cancer. In this study we reveal that EL-4 tumors established in mice rapidly develop resistance to angiostatin gene therapy by upregulating hypoxia-inducible pathways. Angiostatin initially delayed tumor growth for 6 days by reducing blood vessel density. However, tumors quickly responded by upregulating the production of hypoxia-inducible factor-1α (HIF-1α) and its effector vascular endothelial growth factor (VEGF) in response to increasing tumor hypoxia, leading to restored angiogenesis and rapid tumor growth. Theoretically, blockade of HIF-1 should prevent resistance to anti-angiogenic therapy by preventing a tumor from responding to induced hypoxia. Antisense HIF-1α inhibited the expression of HIF-1α and of the HIF-1 effectors VEGF, glucose transporter-1 and lactate dehydrogenase. As a monotherapy, it was effective in eradicating small 0.1 cm diameter tumors, but only delayed the growth of large 0.4 cm diameter tumors. In contrast, timed injection of a combination of angiostatin and antisense HIF-1α plasmids completely eradicated large EL-4 tumors within 2 weeks, and prevented upregulation of hypoxia-inducible pathways induced by angiostatin. The data indicate that blocking hypoxia-inducible pathways by antisense HIF-1α can circumvent hypoxia-induced drug resistance and thereby augment the efficacy of anti-angiogenic therapies.

title: Bovine milk fat enriched in conjugated linoleic and vaccenic acids attenuates allergic dermatitis in mice.
abstract: Orally administered milk fat enriched in conjugated linoleic acid (CLA) and trans-vaccenic acid (VA) ('enriched milk fat'), produced by supplementing the diet of pasture-fed cows with fish and sunflower oil, has been shown previously to suppress the development of allergic airway disease in mice.



To investigate whether topical or oral application of enriched milk fat and its two major fatty acids cis-9, trans-11 CLA (c9,t11-CLA) and VA inhibit allergic dermatitis in mice.



Allergic dermatitis was induced in C57BL/6 mice by epicutaneous sensitization of tape-stripped skin with ovalbumin (OVA). Enriched milk fat and its two major fatty acids were either topically applied to the OVA-sensitized skin, or orally fed to mice by supplementation of the diet. Blood and skin tissues were collected for analysis after the third skin sensitization.



Both topical and oral administration of enriched milk fat and its two major fatty acids led to significant suppression of allergic dermatitis as evidenced by reduced clinical and histological scores of affected skins, infiltration of inflammatory cells, and circulating allergen-specific IgE levels, compared with treatment with normal milk fat or the base control diet. C9,t11-CLA and VA individually inhibited multiple facets of allergic dermatitis when topically applied, and their combination produced a strong additive effect.



Enriched milk fat, and its two major fatty acids c9,t11-CLA and vaccenic acid attenuate allergic dermatitis in mice.

title: Intramuscular delivery of antiangiogenic genes suppresses secondary metastases after removal of primary tumors
abstract: The success of surgery to remove primary tumors can be compromised by the subsequent outgrowth of metastases. It is recognized that primary tumors secrete antiangiogenic factors that suppress the outgrowth of their daughter metastases. In accord we show here that surgical removal of primary EL-4 lymphomas led to a marked decrease in the levels of circulating angiostatin and endostatin, and promoted the growth of distant nodular tumors. Expression vectors encoding angiostatin and endostatin, formulated with poly-N-vinyl pyrrolidone (PVP), were injected into the tibialis and gastrocnemia muscles, leading to expression of angiostatin and endostatin in muscle fibers. High levels of biologically active exogenous proteins were secreted into the circulation. Intramuscular gene therapy with angiostatin and endostatin plasmids significantly inhibited tumor vascularity and induced tumor cell apoptosis, and thereby suppressed the growth of secondary subcutaneous and disseminated metastatic tumors in the lung and liver. Simultaneous intramuscular delivery of both angiostatin and endostatin plasmids significantly prolonged the survival of mice after removal of primary tumors. These results suggest that intramuscular gene transfer of angiostatin and endostatin might serve as a prophylactic cancer-prevention strategy to combat the recurrence of cancer after surgical resection of primary tumors.

title: Up-regulation of survivin by AKT and hypoxia-inducible factor 1α contributes to cisplatin resistance in gastric cancer.
abstract: This study investigated the contribution of survivin and its upstream regulators, AKT and hypoxia-inducible factor 1α (HIF-1α), to the resistance of gastric cancer cells to cisplatin (CDDP). We found that over-expression of survivin increased the resistance of SGC7901 and BGC823 gastric cancer cells to CDDP. Its over-expression abrogated CDDP-induced inhibition of cell proliferation and CDDP-induced cell apoptosis. In contrast, down-regulation of survivin expression using small hairpin RNA (shRNA) vectors and the small-molecule inhibitor YM155, or inhibition of survivin function using a recombinant cell-permeable dominant-negative survivin protein (dNSur9), promoted CDDP-induced apoptosis. CDDP-resistant sub-lines generated from the parental SGC7901 and BGC823 cells by exposure to increasing concentrations of CDDP expressed higher levels of HIF-1α and survivin in response to hypoxia, and higher levels of phosphorylated AKT (pAKT). Specific inhibition of AKT reduced the expression of HIF-1α and survivin, whereas specific inhibition or depletion of HIF-1α reduced survivin expression but had no effect on the expression of phosphorylated AKT. The expression levels of survivin affected the therapeutic efficacy of CDDP in treating gastric tumors in mice. Specific inhibition of survivin, AKT and HIF-1α enhanced the sensitivity of CDDP-resistant cells to CDDP. Specific inhibition of survivin, AKT and HIF-1α synergized with CDDP to suppress the growth of gastric tumors that had been engineered to overexpress survivin. In summary, the results provide evidence that up-regulation of survivin by AKT and HIF-1α contributes to CDDP resistance, indicating that inhibition of these pathways may be a potential strategy for overcoming CDDP resistance in the treatment of gastric cancer.

title: “Iron-saturated” bovine lactoferrin improves the chemotherapeutic effects of tamoxifen in the treatment of basal-like breast cancer in mice
abstract: BackgroundTamoxifen is used in hormone therapy for estrogen-receptor (ER)-positive breast cancer, but also has chemopreventative effects against ER-negative breast cancers. This study sought to investigate whether oral iron-saturated bovine lactoferrin (Fe-Lf), a natural product which enhances chemotherapy, could improve the chemotherapeutic effects of tamoxifen in the treatment of ER-negative breast cancers.MethodsIn a model of breast cancer prevention, female Balb/c mice treated with tamoxifen (5 mg/Kg) were fed an Fe-Lf supplemented diet (5 g/Kg diet) or the base diet. At week 2, 4T1 mammary carcinoma cells were injected into an inguinal mammary fat pad. In a model of breast cancer treatment, tamoxifen treatment was not started until two weeks following tumor cell injection. Tumor growth, metastasis, body weight, and levels of interleukin 18 (IL-18) and interferon γ (IFN-γ) were analyzed.ResultsTamoxifen weakly (IC50 ~ 8 μM) inhibited the proliferation of 4T1 cells at pharmacological concentrations in vitro. In the tumor prevention study, a Fe-Lf diet in combination with tamoxifen caused a 4 day delay in tumor formation, and significantly inhibited tumor growth and metastasis to the liver and lung by 48, 58, and 66% (all P < 0.001), respectively, compared to untreated controls. The combination therapy was significantly (all P < 0.05) more effective than the respective monotherapies. Oral Fe-Lf attenuated the loss of body weight caused by tamoxifen and cancer cachexia. It prevented tamoxifen-induced reductions in serum levels of IL-18 and IFN-γ, and intestinal cells expressing IL-18 and IFN-γ. It increased the levels of Lf in leukocytes residing in gut-associated lymphoid tissues. B, T and Natural killer (NK) cells containing high levels of Lf were identified in 4T1 tumors, suggesting they had migrated from the intestine. Similar effects of Fe-Lf and tamoxifen on tumor cell viability were seen in the treatment of established tumors.ConclusionsThe results indicate that Fe-Lf is a potent natural adjuvant capable of augmenting the chemotherapeutic activity of tamoxifen. It could have application in delaying relapse in tamoxifen-treated breast cancer patients who are at risk of developing ER-negative tumors.

title: Dumbbell-like PtPd-Fe₃O₄ nanoparticles for enhanced electrochemical detection of H₂O₂.
abstract: Dumbbell-like Pt(x)Pd(100-x)-Fe(3)O(4) nanoparticles (NPs) were synthesized and studied for electrocatalytic reduction and sensing of H(2)O(2). In 0.1 M phosphate buffered saline (PBS) solution, the 4-10 nm Pt(x)Pd(100-x)-Fe(3)O(4) NPs showed the Pt/Pd composition-dependent catalysis with Pt(48)Pd(52)-Fe(3)O(4) NPs having the best activity. The Pt(48)Pd(52)-Fe(3)O(4) NPs were tested for H(2)O(2) detection, and their H(2)O(2) detection limit reached 5 nM, which was suitable for monitoring H(2)O(2) generated from Raw 264.7 cells. These dumbbell-like PtPd-Fe(3)O(4) NPs are the most sensitive probe ever reported and can be used to achieve real-time quantitative detection of H(2)O(2) in biological environment for biological and biomedical applications.

title: Effect of burn injury on glucocorticoid receptor binding activity in rat muscle.
abstract: Burn injury is associated with increased muscle proteolysis and up-regulated gene expression in the proteolytic pathway. Glucocorticoids (GCs) are the most important mediator of burn injury induced muscle cachexia. However, the understanding of the mechanisms of GCs action in response to burn injury remains elusive. It is well known that GC acts by binding its own receptor. Therefore, in the present study, we examined the influence of burn injury on the hormone binding activity of the glucocorticoid receptor (GR) in skeletal muscle. Burn injury resulted in increased hormone binding activity in extensor digitorum longus (EDL) and soleus muscles. Scatchard plots revealed that the increased GR hormone binding activity reflected increased numbers of hormone binding sites, whereas receptor affinity for GCs was unchanged. Western blot analysis showed that dissociation of GR/heat shock protein 90 heterocomplex or increase in GR protein may account for the effect of burn injury. The GR antagonist RU 38486 blocked the burn injury-induced increase in GR hormone binding activity, implicating a positive regulatory effect of GCs on GR binding activity under the present experimental conditions.

title: Aspects of endocannabinoid signaling in periimplantation biology
abstract: Physiological roles of endocannabinoids, a group of endogenously produced cannabinoid-like lipid molecules that activate G protein-coupled cannabinoid receptors, are being increasingly appreciated in female reproduction. Adverse effects of cannabinoids on female fertility have been suspected for decades; however, underlying molecular and genetic bases by which they exert these effects were not clearly understood. The discovery of cannabinoid receptors (CB1 and CB2), endocannabinoid ligands (anandamide and 2-acylglycerol) as well as their key synthetic and hydrolytic pathways has helped to better understand the roles of cannabinoid/endocannabinoid signaling in preimplantation embryo development, oviductal embryo transport, embryo implantation and postimplantation embryonic growth. This review focuses on various aspects of the endocannabinoid system in female fertility based on studies that used knockout mouse models. The information generated from studies in mice is likely to shed deeper insight into fertility regulation in women.

title: Expression of uncoupling protein 3 is upregulated in skeletal muscle during sepsis.
abstract: Uncoupling protein 3 (UCP3) is a member of the mitochondrial transporter superfamily that is expressed primarily in skeletal muscle. UCP3 is upregulated in various conditions characterized by skeletal muscle atrophy, including hyperthyroidism, fasting, denervation, diabetes, cancer, lipopolysaccharide (LPS), and treatment with glucocorticoids (GCs). The influence of sepsis, another condition characterized by muscle cachexia, on UCP3 expression and activity is not known. We examined UCP3 gene and protein expression in skeletal muscles from rats after cecal ligation and puncture and from sham-operated control rats. Sepsis resulted in a two- to threefold increase in both mRNA and protein levels of UCP3 in skeletal muscle. Treatment of rats with the glucocorticoid receptor antagonist RU-38486 prevented the sepsis-induced increase in gene and protein expression of UCP3. The UCP3 mRNA and protein levels were increased 2.4- to 3.6-fold when incubated muscles from normal rats were treated with dexamethasone (DEX) and/or free fatty acids (FFA) ex vivo. In addition, UCP3 mRNA and protein levels were significantly increased in normal rat muscles in vivo with treatment of either DEX or FFA. The results suggest that sepsis upregulates the gene and protein expression of UCP3 in skeletal muscle, which may at least in part be mediated by GCs and FFA.

title: Effect of Aspirin on the Expression of Hepatocyte NF-κB and Serum TNF-α in Streptozotocin-Induced Type 2 Diabetic Rats
abstract: Aspirin is a kind of anti-inflammatory drug and may be used to reverse hyperglycemia, hyperinsulinemia, and dyslipidemia by improving insulin resistance. We hypothesized that aspirin improves insulin resistance in type 2 diabetes by inhibiting hepatic nuclear factor kappa-β (NF-κB) activation and serum tumor necrosis factor-α (TNF-α). Adult male Wistar rats were randomly divided into four groups: control, untreated diabetic, diabetic treated with metformin (100 mg/kg/day), and diabetic treated with aspirin (120 mg/kg/day). Diabetes was induced by high-fat feeding and a low dose of streptozotocin (30 mg/kg). After treatment, plasma glucose, insulin, lipids, free fatty acids (FFAs) concentrations and serum TNF-α were determined. The expression of NF-κB in hepatocytes was analyzed by immunohistochemistry and western blot. The results showed administration of aspirin caused no significant lowering in fasting glucose level but significant reduction of hepatic NF-κB expression and serum TNF-α level with improved insulin resistance compared to the diabetic group. The relevant analysis showed positive correlation between the expression of homeostasis model assessment-insulin resistance (HOMA-IR) and NF-κB (r = 0.799, P < 0.01); HOMA-IR and serum TNF-α (r = 0.790, P < 0.01). It is concluded that aspirin improves insulin resistance by inhibiting hepatic NF-κB activation and TNF-α level in streptozotocin-induced type 2 diabetic rats.