Testosterone induces apoptosis in vascular smooth muscle cells via extrinsic apoptotic pathway with mitochondria-generated reactive oxygen species involvement.

Testosterone exerts both beneficial and harmful effects on the cardiovascular system. Considering that testosterone induces reactive oxygen species (ROS) generation and ROS activate cell death signaling pathways, we tested the hypothesis that testosterone induces apoptosis in vascular smooth muscle cells (VSMCs) via mitochondria-dependent ROS generation. Potential mechanisms were addressed. Cultured VSMCs were stimulated with testosterone (10(-7) mol/l) or vehicle (2-12 h) in the presence of flutamide (10(-5) mol/l), CCCP (10(-6) mol/l), mimetic manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP; 3 × 10(-5) mol/l), Z-Ile-Glu(O-ME)-Thr-Asp(O-Me) fluoromethyl ketone (Z-IETD-FMK; 10(-5) mol/l), or vehicle. ROS were determined with lucigenin and dichlorodihydrofluorescein; apoptosis, with annexin V and calcein; O2 consumption, with a Clark-type electrode, and procaspases, caspases, cytochrome c, Bax, and Bcl-2 levels by immunoblotting. Testosterone induced ROS generation (relative light units/mg protein, 2 h; 162.6 ± 16 vs. 100) and procaspase-3 activation [arbitrary units, (AU), 6 h; 166.2 ± 19 vs. 100]. CCCP, MnTMPyP, and flutamide abolished these effects. Testosterone increased annexin-V fluorescence (AU, 197.6 ± 21.5 vs. 100) and decreased calcein fluorescence (AU, 34.4 ± 6.4 vs. 100), and O2 consumption (nmol O2/min, 18.6 ± 2.0 vs. 34.4 ± 3.9). Testosterone also reduced Bax-to-Bcl-2 ratio but not cytochrome-c release from mitochondria. Moreover, testosterone (6 h) induced cleavage of procaspase 8 (AU, 161.1 ± 13.5 vs. 100) and increased gene expression of Fas ligand (2(ΔΔCt), 3.6 ± 1.2 vs. 0.7 ± 0.5), and TNF-α (1.7 ± 0.4 vs. 0.3 ± 0.1). CCCP, MnTMPyP, and flutamide abolished these effects. These data indicate that testosterone induces apoptosis in VSMCs via the extrinsic apoptotic pathway with the involvement of androgen receptor activation and mitochondria-generated ROS.

Restoring adiponectin via rosiglitazone ameliorates tissue wasting in mice with lung cancer.

AIM: To investigate systemic regulators of the cancer-associated cachexia syndrome (CACS) in a pre-clinical model for lung cancer with the goal to identify therapeutic targets for tissue wasting. METHODS: Using the Kras/Lkb1 (KL) mouse model, we found that CACS is associated with white adipose tissue (WAT) dysfunction that directly affects skeletal muscle homeostasis. WAT transcriptomes showed evidence of reduced adipogenesis, and, in agreement, we found low levels of circulating adiponectin. To preserve adipogenesis and restore adiponectin levels, we treated mice with the PPAR-γ agonist, rosiglitazone. RESULTS: Rosiglitazone treatment increased serum adiponectin levels, delayed weight loss, and preserved skeletal muscle and adipose tissue mass, as compared to vehicle-treated mice. The preservation of muscle mass with rosiglitazone was associated with increases in AMPK and AKT activity. Similarly, activation of the adiponectin receptors in muscle cells increased AMPK activity, anabolic signaling, and protein synthesis. CONCLUSION: Our data suggest that PPAR-γ agonists may be a useful adjuvant therapy to preserve tissue mass in lung cancer.

Restoring adiponectin via rosiglitazone ameliorates tissue wasting in mice with lung cancer.

The cancer associated cachexia syndrome (CACS) is a systemic metabolic disorder resulting in loss of body weight due to skeletal muscle and adipose tissues atrophy. CACS is particularly prominent in lung cancer patients, where it contributes to poor quality of life and excess mortality. Using the Kras/Lkb1 (KL) mouse model, we found that CACS is associated with white adipose tissue (WAT) dysfunction that directly affects skeletal muscle homeostasis. WAT transcriptomes showed evidence of reduced adipogenesis, and, in agreement, we found low levels of circulating adiponectin. To preserve adipogenesis and restore adiponectin levels, we treated mice with the PPAR-γ agonist, rosiglitazone. Rosiglitazone treatment increased serum adiponectin levels, delayed weight loss, and preserved skeletal muscle and adipose tissue mass, as compared to vehicle-treated mice. The preservation of muscle mass with rosiglitazone was associated with increases in AMPK and AKT activity. Similarly, activation of the adiponectin receptors in muscle cells increased AMPK activity, anabolic signaling, and protein synthesis. Our data suggest that PPAR-γ agonists may be a useful adjuvant therapy to preserve tissue mass in lung cancer.

Blocking ActRIIB and restoring appetite reverses cachexia and improves survival in mice with lung cancer.

Cancer cachexia is a common, debilitating condition with limited therapeutic options. Using an established mouse model of lung cancer, we find that cachexia is characterized by reduced food intake, spontaneous activity, and energy expenditure accompanied by muscle metabolic dysfunction and atrophy. We identify Activin A as a purported driver of cachexia and treat with ActRIIB-Fc, a decoy ligand for TGF-ß/activin family members, together with anamorelin (Ana), a ghrelin receptor agonist, to reverse muscle dysfunction and anorexia, respectively. Ana effectively increases food intake but only the combination of drugs increases lean mass, restores spontaneous activity, and improves overall survival. These beneficial effects are limited to female mice and are dependent on ovarian function. In agreement, high expression of Activin A in human lung adenocarcinoma correlates with unfavorable prognosis only in female patients, despite similar expression levels in both sexes. This study suggests that multimodal, sex-specific, therapies are needed to reverse cachexia.

The deubiquitinase JOSD2 is a positive regulator of glucose metabolism.

Cancer cells undergo complex metabolic alterations. The mechanisms underlying the tuning of cancer metabolism are under active investigation. Here, we identify the uncharacterized deubiquitinase JOSD2 as a positive regulator of cancer cell proliferation by displaying comprehensive effects on glucose catabolism. We found that JOSD2 directly controls a metabolic enzyme complex that includes Aldolase A, Phosphofructokinase-1 and Phosphoglycerate dehydrogenase, in vitro and in vivo. Further, JOSD2 expression, but not a catalytically inactive mutant, deubiquitinates and stabilizes the enzyme complex, thereby enhancing their activities and the glycolytic rate. This represents a selective JOSD2 feature that is not shared among other Machado-Joseph disease DUBs or observed in nontransformed cells. JOSD2 deficiency displays cytostatic effects and reduces glycolysis in a broad spectrum of tumor cells of distinct origin and its expression correlates with poor prognosis in non-small cell lung cancer. Overall, our study provides evidence for a previously unknown biological mechanism in which JOSD2 integrates glucose and serine metabolism with potential therapeutic implications.

SET protein accumulation prevents cell death in head and neck squamous cell carcinoma through regulation of redox state and autophagy.

Molecular alterations in cell death pathways and imbalances in regulators of up- or downstream signaling pathways can lead to resistance to cell death, which is one of the hallmarks of cancer. These signaling modifications are strategies that tumor cells use to resist chemotherapy and that contribute to the high recurrence rate of head and neck squamous cell carcinoma (HNSCC). The SET oncoprotein is a PP2A inhibitor that accumulates in HNSCC and represents a promising therapeutic target. Here we report the role that SET protein plays in resistance to death of two HNSCC cell lines: Cal 27 and HN13. SET protein regulated intracellular redox balance by controlling cellular localization of APE 1 - an endonuclease that is part of the SET complex and regulates antioxidant gene transcription. SET protein knockdown (siSET) associated with tert-butyl hydroperoxide-induced oxidative stress sensitized Cal 27 and HN13 cells to apoptosis via the extrinsic and intrinsic pathways, respectively. SET protein upregulated autophagy in HNSCC cells in a PP2A-dependent manner and apparently regulated ULK1 expression. The fact that siSET lowered Bcl-2 phosphorylation levels indicated that SET protein interfered with an alternative pathway that modulated autophagy in HNSCC cells. Overall, SET protein regulated intracellular redox state and sustained autophagy in HNSCC cells, which may explain resistance to death of HNSCC cells. Altogether, the findings reported herein support SET protein as therapeutic target for HNSCC.

Resistant to Targeted Therapy - Aim for Metabolic Liabilities.

The advent of targeted therapies generated much optimism when discovered. Targeted therapies, are however associated with rapid acquisition of resistance. In a recent study by Dong et al. (Theranostics 2018; 8(7):1808-1823. doi:10.7150/thno.23177) it was shown that lung tumors resistant to the EGFR-inhibitor (Erlotinib), reprogram their metabolism and acquire a pro-survival dependency on Phosphoglycerate Dehydrogenase (PHGDH) that can be targeted to eliminate resistant tumors.

The AMPK-related kinase SNARK regulates muscle mass and myocyte survival.

The maintenance of skeletal muscle mass is critical for sustaining health; however, the mechanisms responsible for muscle loss with aging and chronic diseases, such as diabetes and obesity, are poorly understood. We found that expression of a member of the AMPK-related kinase family, the SNF1-AMPK-related kinase (SNARK, also known as NUAK2), increased with muscle cell differentiation. SNARK expression increased in skeletal muscles from young mice exposed to metabolic stress and in muscles from healthy older human subjects. The regulation of SNARK expression in muscle with differentiation and physiological stress suggests that SNARK may function in the maintenance of muscle mass. Consistent with this hypothesis, decreased endogenous SNARK expression (using siRNA) in cultured muscle cells resulted in increased apoptosis and decreased cell survival under conditions of metabolic stress. Likewise, muscle-specific transgenic animals expressing a SNARK dominant-negative inactive mutant (SDN) had increased myonuclear apoptosis and activation of apoptotic mediators in muscle. Moreover, animals expressing SDN had severe, age-accelerated muscle atrophy and increased adiposity, consistent with sarcopenic obesity. Reduced SNARK activity, in vivo and in vitro, caused downregulation of the Rho kinase signaling pathway, a key mediator of cell survival. These findings reveal a critical role for SNARK in myocyte survival and the maintenance of muscle mass with age.

HELICOBACTER PYLORI INFECTION IN PATIENTS WITH DIFFERENT GASTROINTESTINAL DISEASES FROM NORTHERN BRAZIL.

BACKGROUND: The mechanisms whereby Helicobacter pylori produces different pathological manifestations in the stomach and duodenum are not fully understood. Considering the geographic diversity in the prevalence of virulence factors of this microorganism and their association with the development of different diseases, the search for pathogenicity markers such as CagA and VacA alleles by molecular techniques has intensified. OBJECTIVES: To investigate the presence of H. pylori infection and the frequency of different genotypes of this bacterium in patients with gastrointestinal diseases from Northern Brazil, and to establish their association with the histopathological findings. METHODS: In a prospective study, samples were collected from 554 patients with different gastrointestinal diseases (gastritis, duodenal ulcer, gastric ulcer, and gastric cancer) seen at a referral hospital attending the entire State of Pará, located in the metropolitan region of Belém. Data such as gender and age obtained with an epidemiological questionnaire were analyzed. The presence of H. pylori and the bacterial genotype were investigated by PCR. Gastric biopsies were assessed histologically. RESULTS: The prevalence of H. pylori infection was 91%. Infection was more frequent among patients with gastric ulcer and gastric cancer. In these groups, there was a predominance of men and older patients when compared to the other two groups studied. The predominant bacterial genotype was s1m1cagA+, which was more frequent among patients with gastric ulcer, duodenal ulcer and gastric cancer. A significant association was observed between s1m1cagA+ strains and a higher degree of inflammation, neutrophil activity and development of intestinal metaplasia. CONCLUSION: The present study demonstrates a high incidence of H. pylori infection in the patients analyzed, especially among those with gastric ulcer and gastric cancer. Virulent s1m1cagA+ strains predominated and were associated with more severe lesions.

HELICOBACTER PYLORI INFECTION IN PATIENTS WITH DIFFERENT GASTROINTESTINAL DISEASES FROM NORTHERN BRAZIL

Background - The mechanisms whereby Helicobacter pylori produces different pathological manifestations in the stomach and duodenum are not fully understood. Considering the geographic diversity in the prevalence of virulence factors of this microorganism and their association with the development of different diseases, the search for pathogenicity markers such as CagA and VacA alleles by molecular techniques has intensified. Objectives - To investigate the presence of H. pylori infection and the frequency of different genotypes of this bacterium in patients with gastrointestinal diseases from Northern Brazil, and to establish their association with the histopathological findings. Methods - In a prospective study, samples were collected from 554 patients with different gastrointestinal diseases (gastritis, duodenal ulcer, gastric ulcer, and gastric cancer) seen at a referral hospital attending the entire State of Pará, located in the metropolitan region of Belém. Data such as gender and age obtained with an epidemiological questionnaire were analyzed. The presence of H. pylori and the bacterial genotype were investigated by PCR. Gastric biopsies were assessed histologically. Results - The prevalence of H. pylori infection was 91%. Infection was more frequent among patients with gastric ulcer and gastric cancer. In these groups, there was a predominance of men and older patients when compared to the other two groups studied. The predominant bacterial genotype was s1m1cagA+, which was more frequent among patients with gastric ulcer, duodenal ulcer and gastric cancer. A significant association was observed between s1m1cagA+ strains and a higher degree of inflammation, neutrophil activity and development of intestinal metaplasia. Conclusion - The present study demonstrates a high incidence of H. pylori infection in the patients analyzed, especially among those with gastric ulcer and gastric cancer. Virulent s1m1cagA+ strains predominated and were associated with more severe lesions.

Contexto - Os mecanismos pelos quais o H. pylori produz diferentes quadros patológicos no estômago e no duodeno não são totalmente conhecidos. Considerando a diversidade geográfica relacionada à prevalência dos fatores de virulência desse microrganismo e sua associação com o desenvolvimento de diferentes doenças, vem se intensificando a pesquisa de marcadores de patogenicidade, como o CagA e os alelos do VacA por técnicas moleculares. Objetivos - O objetivo desse estudo foi investigar a presença da infecção por H. pylori, e a frequência dos diferentes genótipos dessa bactéria em pacientes com doenças gastrointestinais da nossa região, procurando estabelecer sua associação com os achados histopatológicos. Métodos - Em estudo prospectivo, foram coletadas amostras de 554 pacientes com diferentes doenças gastrointestinais (gastrite, úlcera duodenal, úlcera gástrica e câncer gástrico), atendidos em hospital de referência para todo o Estado do Pará, localizado na região metropolitana de Belém. Foram analisados dados obtidos através de questionário epidemiológico, relacionados ao sexo e faixa etária desses pacientes. A presença do H. pylori e do genótipo bacteriano foi detectada utilizando a PCR. As biopsias gástricas foram avaliadas histologicamente. Resultados - Observou-se uma prevalência de 91% da infecção pelo H. pylori, sendo mais frequente nos portadores de úlcera gástrica e câncer gástrico, nos quais houve predomínio do sexo masculino e a idade foi maior que a dos outros dois grupos estudados. O genótipo bacteriano predominante foi o s1m1cagA positivo, sendo mais frequentes entre os pacientes com úlcera gástrica, úlcera duodenal e câncer gástrico. Houve associação significante das cepas com o genótipo s1m1cagA positivo com maior grau de inflamação, atividade neutrofílica e desenvolvimento de metaplasia intestinal. Conclusão - Nosso estudo demonstra a alta incidência da infecção pelo H. pylori nos pacientes analisados em nosso meio, especialmente em portadores de úlcera e câncer gástricos. As cepas virulentas s1m1cagA+ foram predominantes e estavam associadas a lesões mais graves.