Transtinib, a potent tyrosine kinase inhibitor inhibits L858R/T790M mutant NSCLC cell lines and xenografts.

Non-small cell lung cancer (NSCLC) patients with activating epidermal growth factor receptor (EGFR) mutations initially respond well to the EGFR tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib. However, clinical efficacy is limited by the development of resistance. In most cases, this resistance is in the form of the T790M mutation. Here, we report the design, synthesis and biochemical evaluation of a novel series of irreversible EGFR tyrosine kinase inhibitors (EGFR-TKIs) that are derived from the anilinoquinazoline scaffold. Guided by molecular modeling, this series of analogs was evolved to target a cysteine residue in the ATP binding site via covalent bond formation and to achieve high levels of anti-tumor activity in cell cultures and in xenografts. The most promising compound 13c ((E) -N - (4 - (4 - (3-fluorobenzyloxy) -3- chlorophenylamino) -7-ethoxyquinazolin-6-yl) -3- ((S) -pyrrolidin-2-yl)acrylamide, which we named Transtinib) displayed strong anti-proliferative activity against the H1975 and A431 cell lines with IC50 values of 34 nM and 62 nM, respectively. In xenograft models, Transtinib significantly decreases tumor size for a prolonged period of time. These results suggest that Transtinib is a potential cancer therapeutic drug lead for the inhibition of mutant EGFR to overcome the development of resistance.

Identification of Vß7.1_H3F7 as a therapeutic gene encoding TCR specific to hepatocellular carcinoma.

The feasibility of T-Cell receptor (TCR) gene therapy using a MART-1-specific TCR has been previously demonstrated in melanoma patients. However, it remains a challenge without a defined tumor-specific antigen in the therapy of hepatocellular carcinoma (HCC). In this study, through the analysis of clonal expansion of TCR Vß subfamily and DNA sequencing, we identified TCR Vß7.1_H3F7 as a potential therapeutic gene specifically for the HCC patients. Peripheral blood monouclear cells (PBMC) transfected with TCRV ß7.1_H3F7 gene were specifically cytotoxic against HCC cells in vitro. Adoptive transfer of this transfected PBMC resulted in a marked suppression of HCC tumor development in the animal model. These results demonstrated the value of TCRV ß7.1_H3F7 as a therapeutic gene specifically for HCC. More importantly, it provides a novel strategy for screening tumor-specific TCR genes, which may pave the way for TCR gene therapy in cancer patients currently without the defined tumor-specific antigens.

Circulating tumor cells (CTCs) as a liquid biopsy material and drug target.

Circulating tumor cells (CTCs) have attracted interest as biomarkers of cancer metastases but only recently has a reliable method of CTC detection been developed. CTCs can be thought of as a liquid biopsy from the blood, and they can be used in pathological and molecular assays. CTCs may ideally replace metastatic tissue biopsies in the prediction and monitoring of therapeutic responses and tumor recurrence. CTCs can be used to guide therapeutic cancer management and serve as drug targets. For this reason, the potential of this technology and the limitations of currently available methods of CTC detection are addressed here. The clinical applications of CTCs include the prediction of cancer prognosis; selection and monitoring of therapeutic regimens; and drug target applications. The manner in which CTC molecular profiling can facilitate prognosis and the selection of cancer therapies.

Structure, function, and pathogenesis of SHP2 in developmental disorders and tumorigenesis.

Src homology 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2), encoded by the human PTPN11 gene, is a ubiquitously expressed protein tyrosine phosphatase (PTP) that consists of two tandem Src homology (SH2) domains (N-SH2 and C-SH2), a PTP catalytic domain, and a C-terminal tail with tyrosyl phosphorylation sites. It plays critical roles in numerous cellular processes through the regulation of various signaling pathways in PTP catalytic activity-dependent and -independent manners. Dysfunction of SHP2 resulting from pathogenic mutations and aberrant expression leads to the dysregulation of multiple signaling pathways, thus contributing to different human disorders. Germline and somatic mutations in PTPN11 are involved in Noonan syndrome (NS), LEOPARD syndrome (LS), and hematological malignancies, as well as several solid tumors. In this report, we provide an overview of the current knowledge of the structure and function of SHP2, and further discuss the molecular and pathogenic mechanism of SHP2 in human diseases, with a special focus on tumorigenesis. Furthermore, we summarize that SHP2 might itself represent a potential drug target for cancer prevention and treatment. Ongoing research and development of SHP2-specific inhibitors would enhance this potential.

Chronic resveratrol intake reverses pro-inflammatory cytokine profile and oxidative DNA damage in ageing hybrid mice.

Thymic involution and shrinkage of secondary lymphoid organs are leading causes of the deterioration of the T-cell compartment with age. Inflamm-aging, a sustained inflammatory status, has been associated with chronic diseases and shortened longevity. This is the first study to investigate the effect of treating aging hybrid mice with long-term, low-dose resveratrol (RSV) in drinking water by assessing multiple immunological markers and profiles in the immune system. We found that hybrid mice exhibited marked age-related changes in the CD3+CD4+, C3+CD8+, CD4+CD25+, CD4M and CD8M surface markers. RSV reversed surface phenotypes of old mice to that of young mice by maintaining the CD4+ and CD8+ population in splenocytes as well as reducing CD8+CD44+ (CD8M) cells in the aged. RSV also enhanced the CD4+CD25+ population in old mice. Interestingly, pro-inflammatory status in young mice was transiently elevated by RSV but it consequently mitigated the age-dependent increased pro-inflammatory cytokine profile while preserving the anti-inflammatory cytokine condition in the old mice. Age-dependent increase in 8OHdG, an oxidative DNA damage marker was ameliorated by RSV. Immunological-focused microarray gene expression analysis showed that only the CD72 gene was significantly downregulated in the 12-month RSV-treated mice compared to age-matched controls. Our study indicates that RSV even at low physiological relevant levels is able to affect the immune system without causing marked gene expression changes.

Elevation of oxidative-damage biomarkers during aging in F2 hybrid mice: protection by chronic oral intake of resveratrol.

Resveratrol (RSV), a naturally occurring phytoalexin that can be found in red wine, berries, and peanuts, has been shown to extend both mean and maximum life span in model organisms. RSV has also been reported to shift the physiology of middle-aged mice on a high-calorie diet toward that of mice on a standard diet. These beneficial effects of RSV have been suggested to resemble caloric restriction. Our study in F2 four-way cross-hybrid mice was the first to evaluate the effects of aging and long-term RSV treatment (14.09+/-3.4 mg/L in drinking water for 6 or 12 months) on biomarkers of oxidative damage to DNA, 8-hydroxy-2'-deoxyguanosine (8OHdG); lipid, 8-iso-prostaglandin(2 alpha) (8-iso-PGF(2 alpha)); and protein, protein carbonyl content (PCC). There was a significant age-dependent accumulation of oxidative damage to DNA, lipid, and protein as well as a clear increase in urine 8-iso-PGF(2 alpha) levels in the majority of mouse tissues. Rates of age-dependent increases in damage biomarkers varied between tissues. Chronic RSV treatment elevated total RSV plasma levels and reduced the observed age-dependent accumulation of (1) 8OHdG in liver and heart, (2) 8-iso-PGF(2 alpha) in heart and urine, and (3) PCC in liver and kidney. However, a 12-month RSV intake resulted in significant elevation of 8-iso-PGF(2 alpha) and PCC in kidney. Our studies demonstrate that RSV treatment consistently attenuated oxidative damage in tissues where age-related oxidative damage accumulation was prominent, but also suggested that chronic RSV treatment may induce nephrotoxicity.

Identifying stable reference genes for evaluation of antioxidative enzyme gene expression in auditory cortex and cochlea of young and old Fischer 344 rats.

CONCLUSION: This study suggests that the validation of reference genes is important for the accurate evaluation of differential gene expression in age-related hearing loss (AHL). OBJECTIVES: To identify the most stable reference gene in the auditory cortex and cochlea of young and old rats. Materials and methods. mRNA levels of some common reference genes such as beta-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin C, hypoxanthine phosphoribosyl-transferase and eukaryotic translation elongation factor (EF) were quantified in the auditory cortex and cochlea of young and old rats by quantitative RT-PCR, and then their stability was evaluated using GeNorm and Normfinder software. RESULTS: There were some variations in the expression of reference genes found in the auditory cortex and cochlea of rats, wherein EF and GAPDH were identified as the most stable reference genes in auditory cortex and cochlea, respectively. mRNA levels of Cu/Zn-superoxide dismutase (SOD), Mn-SOD, catalase and glutathione peroxidase in the auditory cortex and cochlea do not vary significantly in young and old rats, when they are normalized with EF or GAPDH, the valid reference genes, respectively. However, Cu/Zn-SOD and catalase levels differ significantly in the auditory cortex when they are normalized with beta-actin, an invalid reference gene, resulting in the misinterpretation of the target gene expression levels.

Identifying the changes in gene profiles regulating the amelioration of age-related oxidative damages in kidney tissue of rats by the intervention of adult-onset calorie restriction.

This study was initiated to investigate gene expression profiles that are involved in the molecular mechanisms regulating the amelioration of age-related oxidative damages in male Fischer-344 rats (12 months) through adult-onset calorie restriction (CR) intervention for 6 months. The adult-onset CR was initiated with 10 and 25% restriction for the first and second weeks, respectively, and then maintained at 40% throughout the experiment. The adult-onset CR significantly (p < 0.05) decreased urinary 8-isoprostane and protein carbonyl in kidney for the markers of lipid peroxidation and protein oxidation, respectively, in rats from the CR group when compared with control group. Based on Yu's and Melk's methods, the age-related renal pathological changes in the kidney of rats from CR group were retarded by adult-onset CR. Such changes could result from the decrease of plasminogen activation inhibition-1 and clusterin and the increase of kallikrein mRNA expressions significantly (p < 0.05) in the kidneys of rats from the CR group. They were further confirmed by quantitative RT-PCR. Moreover, inflammatory response pathway was down-regulated significantly (p < 0.05) in rats from the CR group, while fatty acid synthesis, mitochondrial fatty acid betaoxidation, glycolysis, and gluconeogenesis were considerably up-regulated in kidney tissue of rats. In conclusion, the adult-onset CR could retard the age-related oxidative damages and renal pathological changes due to variations in gene expressions and biological pathways.

Polyethylenimine-mediated cochlear gene transfer in guinea pigs.

OBJECTIVE: To demonstrate and compare polycationic-mediated cochlear gene transfer with linear polyethylenimine (PEI) via cochleostomy and osmotic pump infusion method. DESIGN: A dissociated cochlear culture was used to select the optimum nitrogen to phosphate ratio of PEI/DNA complexes to be used in vivo. The PEI-enhanced green fluorescent protein reporter gene DNA complex was introduced with single inoculation (cochleostomy) or with sustained delivery (osmotic pump method) into guinea pig cochleas and examined for transgene expression. SUBJECTS: Male Albino Hartley guinea pigs (250-350 g). RESULTS: The relatively low transfection efficiency of PEI limits its potential when compared with viral counterparts; however, sustained release of the vector solution was able to improve PEI's transfection efficiency. The PEI-infused cochleas maintained intact cellular and tissue architecture with absence of inflammation. Transfection confined to the perilymphatic space highlights the need to target the gene vector into the scala media if transfection is targeted at cells within the organ of Corti. CONCLUSION: These findings indicate that PEI is able to transfect the cochlea in vivo with sustained delivery and present an alternative for nonviral cochlear gene therapy.

Regulating the age-related oxidative damage, mitochondrial integrity, and antioxidative enzyme activity in Fischer 344 rats by supplementation of the antioxidant epigallocatechin-3-gallate.

In this study, epigallocatechin-3-gallate (EGCG) was examined for the first time for its anti-aging effect on middle-aged male Fischer 344 rats as a dietary supplement at 50 (low dose) and 500 (high dose) mg/kg/day over a 6-month period. Such levels of EGCG concentration were well-tolerated by rats without causing tissue damage or dysfunction in the liver and kidney, as evaluated by histopathological and biochemical observations. Compared to the rats in the low-dose and control groups, rats fed with high-dose EGCG showed a significant decline in the concentration of 8-hydroxy-2'-deoxyguanosine in the plasma while maintaining a better mitochondrial potential in the peripheral lymphocytes and preventing the deletion of ND4 region from mitochondrial DNA in the liver. The protective effects of high-dose EGCG against oxidative stress were comparable with the effects of caloric restriction, a well-established dietary intervention that retards aging. However, the supplementation of EGCG influenced merely the antioxidative enzyme activities and their gene expressions in rats, suggesting that EGCG may either function as an antioxidant itself or regulate other bioprocesses, including energy metabolism, biosynthesis, and stress response, as shown in the gene profiling analysis of microarray data. Thus, the present study provides preliminary information on the anti-aging property of EGCG in male Fischer 344 rats.

Bone-marrow-derived cells that home to acoustic deafened cochlea preserved their hematopoietic identity.

The high degree of bone marrow cell (BMC) plasticity has prompted us to test its restoration possibility in inner ear repair. Our aim was to determine the potential of these cells to transdifferentiate into specialized cochlea cell types after acoustic injury and BMC mobilization. Lethally irradiated mice were transplanted with BMCs from green fluorescent protein (GFP) transgenic mice and subjected to acoustic deafening 3 months later. In a separate experiment, stem cell factor and granulocyte colony-stimulating factor were administered to test the effect of BMC mobilization on bone marrow-derived cell (BMDC) transdifferentiation. All mice showed almost complete chimerism 3 months after bone marrow transplantation. Upon acoustic trauma, robust BMDC migration was observed in the deafened cochlea. GFP+ cell migration was most prominent during the first week after acoustic deafening, and these cells accumulated significantly at the spiral ligament, perilymphatic compartment walls, and limbus regions. Most of the BMDCs expressed CD45 and CD68 and were identified as macrophages. Upregulation of stromal-derived factor 1 (SDF-1) was also observed in the spiral ligament during the first week after acoustic deafening. Cytokine treatment resulted in increased BMC mobilization in the systemic circulation. However, the presence of any stem cell progenitors or the differentiation of BMDCs into any cell types expressing cochlea sensory, supporting, fibrocytic, or neuronal markers were not detected in the deafened cochlea. In conclusion, we have demonstrated the homing capability of BMDCs to the deafened cochlea, and these cells displayed mature hematopoietic properties without spontaneous transdifferentiation to any cochlea cell types after acoustic trauma or bone marrow mobilization.

Effects of epigallocatechin-3-gallate on mitochondrial integrity and antioxidative enzyme activity in the aging process of human fibroblast.

Mitochondrial integrity and antioxidative enzyme activity are two of the determinants of intracellular reactive oxygen species (ROS) accumulation probably underlying the aging mechanism. In this study, epigallocatechin-3-gallate (EGCG) was examined for its antiaging effect on human diploid fibroblasts (HDF). EGCG was evaluated for its cytotoxicity, and LC50 values were 78.0 and 84.4 microM for young and old HDF, respectively. HDF treated with EGCG at 25 and 50 microM for 24 h considerably increased catalase, superoxide dismutase (SOD)1, SOD2, and glutathione peroxidase gene expressions and their enzyme activities, thus protecting HDF against H2O2-induced oxidative damage, accompanied with decreased intracellular ROS accumulation and well-maintained mitochondrial potential. Moreover, HDF treated with EGCG at 12.5 microM for long term showed less intracellular ROS with higher mitochondrial potential, more intact mitochondrial DNA, much elevated antioxidative enzyme efficiency, and more juvenile cell status compared to those of the untreated group. Taken together, in this study we investigated the effects of EGCG in the regulation of mitochondrial integrity and antioxidative enzyme activity of HDF, suggesting that EGCG can be considered one of the possible antiaging reagents in the future.

Age-related changes in mitochondrial function and antioxidative enzyme activity in fischer 344 rats.

We have previously reported the changes of mitochondrial function and/or antioxidative enzyme efficiency in a few organs of rats as a result of aging. However, there is a further need to reach a conclusion about their interactions in biological functions based on other evaluation tips like the usage of advanced methods and the exploring of crucial biochemical parameters. Therefore, we investigated the mitochondrial inner membrane functional integrity by the analysis of respiration control ratio and membrane potential in the liver and brain of young (8 months) and old (26 months) Fischer 344 rats. The disintegration of mitochondrial membrane integrity was determined higher in the liver of old rats than that of young rats. This was well correlated with the decrease of total superoxide dismutase (SOD), Cu/Zn-SOD, Mn-SOD and glutathione peroxidase activities in most of the organs, except for the increase of catalase activity in heart of old rats. Similarly, the protein expressions of these enzymes were down regulated in the liver and kidney of old rats. Taken together, we suggest that the mitochondrial malfunction in old rats is associated with the decrease of antioxidative enzyme efficiency. And the data are also discussed with changes in the results from inter-laboratories.

Low-dose, long-term caroverine administration attenuates impulse noise-induced hearing loss in the rat.

CONCLUSION: Physiological and morphological assessments indicated that low-dose and long-term caroverine delivery might be a new approach to protect against impulse noise-induced hearing loss. BACKGROUND: Although the exact mechanisms by which impulse noise causes hearing loss are still unclear, there is accumulating evidence that increased reactive oxygen species (ROS) production and excessive glutamate released from the inner hair cells lead to hair cell loss and consequently hearing loss. Caroverine is an antagonist of two glutamate receptors, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors in the inner ear, as well as an antioxidant. MATERIALS AND METHODS: In this study, caroverine was delivered subcutaneously using an osmotic pump. This kind of delivery has the advantage, via continuous, long-term and low dose drug administration, of avoiding systemic side effects. RESULTS: It was shown that caroverine could significantly protect the cochlea against impulse noise trauma.

Relationship between levels of oxidative DNA damage, lipid peroxidation and mitochondrial membrane potential in young and old F344 rats.

The extent of in vivo oxidative damage has been known to be cumulative over the period of the life of mammals. Our hypothesis is that there should be a positive correlation between the levels of 8-hydroxy-2'-deoxyguanosine (8OHdG) and 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)) in major rat tissues. We also investigated whether increased level of oxidative stress causes a decrease in the mitochondrial membrane potential of peripheral lymphocytes of old rats using the MitoTracker Red fluorochrome. Our results show positive correlations between 8OHdG and 8-iso-PGF(2alpha) for liver, brain and kidney measured by HPLC-UV-ECD (electrochemical detector) and EIA methods, respectively. However, heart tissues show a negative correlation. The mitochondrial membrane potential of old rat lymphocytes records significant decrease compared with the young lymphocytes. Based on our results, we conclude that in ageing studies, specific tissues need to be examined in order to measure the localised DNA damage and lipid peroxidation as different tissues display different extent of oxidative damage. We believe this approach of using combined markers is useful to verify the true efficacy of health intervention studies in animals and humans. In addition, the isoprostane assay can be further developed looking at lipid peroxidation as a potential marker in ageing studies.

Identification of valid housekeeping genes and antioxidant enzyme gene expression change in the aging rat liver.

Valid housekeeping genes (HKG) are a prerequisite for accurate gene quantification. We performed real-time reverse transcription-polymerase chain reaction to investigate the gene expression of five commonly used HKGs (beta-actin, glyceraldehyde-3-phosphate dehydrogenase [GAPDH], ubiquitin C [UBC], hypoxanthine phosphoribosyl-transferase [HPRT], and cyclophilin A [CYPa]) and antioxidant enzymes in the liver of young and old male Fischer rats. A wide variation in HKG expression existed during the aging process, and HPRT was identified as the most stable HKG in rat liver aging. When Cu/Zn-superoxide dismutase gene expression was normalized to HPRT, there was no detectable difference between young and old rats; however, a significant difference was seen when it was normalized to UBC. The variation of UBC caused the misinterpretation of Cu/Zn-superoxide dismutase expression. Catalase expression was significantly decreased, whereas glutathione peroxidase expression was not altered with age. We demonstrated that HPRT was an appropriate HKG, validation of HKGs was vital for accurate quantification, and decreased catalase expression might be involved in the decline of antioxidant defenses during rat liver aging.

Analysis of differential gene expression in the cochlea and kidney of mouse by cDNA microarrays.

Microarray hybridization analysis of gene expression in the cochlea and kidney suggest a relationship between these tissues at the genomic level, indicating the common gene expression, likely serving a common function in both the organs primarily maintaining ion transport, and implied previously from morphological, pharmaco-kinetic and teratogenic studies. The cDNAs of more than 100 genes listed on the hereditary hearing loss homepage were amplified as targets by RT-PCR and were hybridized with probes prepared from total RNA of the cochlea and the kidney. Thirteen of the genes analyzed showed altered fluorescence ratios of more than two logs. Of these, the expressions of 11 genes were over expressed and two were under expressed in the cochlea than in the kidney. Our data is the first report to corroborate the genomic similarities between these two important organs and may help to explain the somewhat similar response of these organs to certain therapeutic drugs.

Protection of auditory function against noise trauma with local caroverine administration in guinea pigs.

Glutamate is the most likely neurotransmitter at the synapse between the inner hair cell and its afferent neuron in the peripheral auditory system. Intense noise exposure may result in excessive glutamate release, binding to the post-synaptic receptors and leading to neuronal degeneration and hearing impairment. The present study investigated the protective effect of caroverine, an antagonist of two glutamate receptors, N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, on noise-induced hearing loss. Two different doses of caroverine were applied onto the round window membrane with gelfoam, followed by one-third-octave band noise centered at 6.3 kHz (110 dB SPL) for 1 h. Auditory brainstem responses were measured at regular time intervals afterwards. Caroverine was found to offer significant protection of the cochlear function against noise-induced hearing loss.

Acute treatment of noise trauma with local caroverine application in the guinea pig.

Intense sound stimulation may result in excessive glutamate release from the inner hair cells, resulting in binding to the postsynaptic glutamate receptors and leading to neuronal degeneration and functional impairment. In this study we investigated the therapeutic effect and time window of caroverine, an N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist, on noise-induced hearing loss Guinea pigs were exposed to one-third octave band noise centered at 6.3 kHz (110 dB sound pressure limit) for 1 h. One or 24 h after noise exposure, caroverine was applied to the round window membrane. Auditory brainstem responses were recorded at regular time intervals. It was shown that caroverine could significantly decrease hearing impairment after noise trauma when applied 1 but not 24 h after noise exposure.