Non-clinical studies for oncology drug development

Non-clinical studies are necessary at each stage of the development of oncology drugs. Many experimental cancer models have been developed to investigate carcinogenesis, cancer progression, metastasis, and other aspects in cancer biology and these models turned out to be useful in the efficacy evaluation and the safety prediction of oncology drugs. While the diversity and the degree of engagement in genetic changes in the initiation of cancer cell growth and progression are widely accepted, it has become increasingly clear that the roles of host cells, tissue microenvironment, and the immune system also play important roles in cancer. Therefore, the methods used to develop oncology drugs should continuously be revised based on the advances in our understanding of cancer. In this review, we extensively summarize the effective use of those models, their advantages and disadvantages, ranges to be evaluated and limitations of the models currently used for the development and for the evaluation of oncology drugs

G Protein Coupling and Second Messenger Generation Are Indispensable for Metalloprotease-dependent, Heparin-binding Epidermal Growth Factor Shedding Through Angiotensin II Type-1 Receptor

A G protein-coupled receptor agonist, angiotensin II (AngII), induces epidermal growth factor (EGF) receptor (EGFR) transactivation possibly through metalloprotease- dependent, heparin-binding EGF (HB-EGF) shedding. Here, we have investigated signal transduction of this process by using COS7 cells expressing an AngII receptor, AT1. In these cells AngII-induced EGFR transactivation was completely inhibited by pretreatment with a selective HB-EGF inhibitor, or with a metalloprotease inhibitor. We also developed a COS7 cell line permanently expressing a HB-EGF construct tagged with alkaline phosphatase, which enabled us to measure HB-EGF shedding quantitatively. In the COS7 cell line AngII stimulated release of HB-EGF. This effect was mimicked by treatment either with a phospholipase C activator, a Ca2 ionophore, a metalloprotease activator, or H2O2. Conversely, pretreatment with an intracellular Ca2 antagonist or an antioxidant blocked AngII-induced HB-EGF shedding. Moreover, infection of an adenovirus encoding an inhibitor of Gq markedly reduced EGFR transactivation and HB-EGF shedding through AT1. In this regard, AngII-stimulated HB-EGF shedding was abolished in an AT1 mutant that lacks Gq protein coupling. However, in cells expressing AT1 mutants that retain Gq protein coupling, AngII is still able to induce HB-EGF shedding. Finally, the AngII-induced EGFR transactivation was attenuated in COS7 cells overexpressing a catalytically inactive mutant of ADAM17. From these data we conclude that AngII stimulates a metalloprotease ADAM17-dependent HB-EGF shedding through AT1/Gq/phospholipase C-mediated elevation of intracellular Ca2 and reactive oxygen species production, representing a key mechanism indispensable for EGFR transactivation

Chronic heat stress induces renal fibrosis and mitochondrial dysfunction in laying hens

Abstract Background Heat stress in laying hens negatively affects egg production and shell quality by disrupting the homeostasis of plasma calcium and phosphorus levels. Although the kidney plays an important role in calcium and phosphorus homeostasis, evidence regarding the effect of heat stress on renal injury in laying hens is yet to be elucidated. Therefore, the aim of this study was to evaluate the effects of chronic heat stress on renal damage in hens during laying periods. Methods A total of 16 white-leghorn laying hens (32 weeks old) were randomly assigned to two groups (n = 8). One group was exposed to chronic heat stress (33 °C for 4 weeks), whereas the other group was maintained at 24 °C. Results Chronic heat exposure significantly increased plasma creatinine and decreased plasma albumin levels (P < 0.05). Heat exposure also increased renal fibrosis and the transcription levels of fibrosis-related genes (COLA1A1, αSMA, and TGF-β) in the kidney. These results suggest that renal failure and fibrosis were induced by chronic heat exposure in laying hens. In addition, chronic heat exposure decreased ATP levels and mitochondrial DNA copy number (mtDNA-CN) in renal tissue, suggesting that renal mitochondrial dysfunction occurs under conditions of heat stress. Damaged mitochondria leak mtDNAs into the cytosol and mtDNA leakage may activate the cyclic GMP-AMP synthase (cGAS) stimulator of interferon genes (STING) signaling pathway. Our results showed that chronic heat exposure activated the cGAS-STING pathway as indicated by increased expression of MDA5, STING, IRF7, MAVS, and NF-κB levels. Furthermore, the expression of pro-inflammatory cytokines (IL-12) and chemokines (CCL4 and CCL20) was upregulated in heat-stressed hens. Conclusions These results suggest that chronic heat exposure induces renal fibrosis and mitochondrial damage in laying hens. Mitochondrial damage by heat stress may activate the mtDNA-cGAS-STING signaling and cause subsequent inflammation, which contributes to the progression of renal fibrosis and dysfunction

Heat Stress Modulates Cytokine Gene Expression in the Spleen of Broiler Chickens

Heat stress has negative effects on biological defense mechanisms such as the immune response in chickens, and organs of the immune system, such as the spleen, are atrophied by heat stress in broiler chickens. In order to assess heat stress-induced functional changes of immune mechanisms in the spleen of broiler chickens, expression of genes encoding splenic cytokines (Th1 type, Th2 type, and pro-inflammatory cytokines) was analyzed. Heat exposure at 34°C for 15 days significantly induced the spleen involution, increased interleukin (IL)-4 and IL-12 expression and decreased interferon (IFN)-γ. However, expression of IL-6, 10, 13, and 18 was not affected. Heat stress reduced feed intake, which may affect the spleen weight and cytokine expression. Therefore, a pair-fed group at 24°C (24PF) was included in which chickens received the same amount of feed as those in the group at 34°C. Spleen weight was not affected by the reduction in feed intake. IL-4 expression in the 24PF group was higher than that in the control group. Further, IFN-γ expression increased and IL-12 expression was not affected by the reduction of feed intake, suggesting that the feed intake reduction induced by heat stress does not modulate splenic cytokine expression in broiler chickens. These data suggest that heat stress induces spleen involution and affects the expression of splenic cytokines such as IL-12 and IFN-γ in broiler chickens, but not through the feed intake reduction

Effects of Dietary Brown Rice on the Growth Performance, Systemic Oxidative Status, and Splenic Inflammatory Responses of Broiler Chickens under Chronic Heat Stress

The aim of this study was to evaluate the effects of dietary brown rice on the growth performance, systemic oxidative status, and splenic inflammatory responses of broiler chickens under both thermo-neutral and chronic heat stress conditions. Forty 12-day-old male broiler chickens (ROSS 308) were randomly assigned to two groups and fed either a control diet (corn-based) or a brown rice-based diet. After seven days (19 days old), both groups were randomly divided into two sub-groups (n=10), one of which was exposed to heat stress (33°C for 14 days), while the other was maintained at 24°C. Heat exposure reduced the body weight gain and feed intake (p&lt;0.01) of both groups. In terms of oxidative plasma states, heat exposure reduced the glutathione peroxidase activity and increased the ceruloplasmin content, while the 2-thiobarbituric acid reactive substance and reduced glutathione levels were not affected adversely. Heat exposure activated the immune responses, as evidenced by increased plasma immunoglobin levels, and altered splenic immune-related gene expressions including heat shock proteins, toll-like receptor 4, and interleukin-12. Under both thermo-neutral and heat stress conditions, dietary brown rice improved the growth performance, decreased the immunoglobulin levels, and down-regulated the expression of splenic immune-related genes of broilers, although their systemic oxidative status was not affected. Dietary brown rice should be considered as a valuable component of broiler chicken feeds subjected to both thermo-neutral and heat stress conditions. The positive effects of brown rice on bird performance may be associated with the modulation of the immune responses, as reflected by the decreased production of immunoglobulins and altered splenic immune-related gene expression