A novel HIF-1α/VMP1-autophagic pathway induces resistance to photodynamic therapy in colon cancer cells.

Colon cancer is the third most frequent cancer and the fourth most common cause of cancer-related mortality worldwide and the standard therapy is surgical resection plus adjuvant chemotherapy. Photodynamic therapy (PDT) has been proposed as an adjuvant therapy because it can prevent the tumor recurrence after surgical excision in colon cancer patients. Hypoxia is a common feature in solid tumors and leads to chemo/radioresistance. Recently, it has been shown that in response to hypoxia, cells can induce HIF-1α-mediated autophagy to survive in this hostile microenvironment. Moreover, hypoxia and autophagy have been implicated in the resistance to antitumor PDT. However, the molecular signals by which HIF-1α induces autophagy in the PDT context have not been studied yet. Here we evaluate the interplay between HIF-1α and autophagy as well as the underlying mechanism in the PDT resistance of colon cancer cells. Our study demonstrates that HIF-1α stabilization significantly increases VMP1-related autophagy through binding to hypoxia responsive elements in the VMP1 promoter. We show that HIF-1α-induced autophagy increases colon cancer cell survival as well as decreases cell death after PDT. Moreover, here we demonstrate that HIF-1α-induced autophagy is mediated by VMP1 expression, since the downregulation of VMP1 by the RNA interference strategy reduces HIF-1α-induced autophagy and cell survival after PDT. In conclusion, PDT induces autophagy as a survival mechanism and the induction of the novel HIF-1α/VMP1-autophagic pathway may explain, at least in part, the resistance of colon cancer cells to PDT. The knowledge of the molecular mechanisms involved in PDT resistance may lead to more accurate therapeutic strategies.

Nitric oxide and apoptosis induced in Peyer's patches by attenuated strains of Salmonella enterica serovar Enteritidis.

Nitric oxide (NO) is a toxic molecule of the immune system which contributes to the control of microbial pathogens. Additional functions of NO in innate and adaptive immunity have recently been described; these functions include the modulation of the cytokine response of lymphocytes and the regulation of immune cell apoptosis. In addition to direct microbicidal actions, NO has immunoregulatory effects relevant to the control of infections. In turn, infected macrophages and macrophage-regulating lymphocytes may undergo apoptosis during infection by Salmonella spp. In this work we investigated the ability of attenuated strains of Salmonella enterica serovar Enteritidis with different protective capacities to induce intestinal inducible nitric oxide synthase (iNOS) and apoptosis in Peyer's patches (PP) in mice. Results showed that the intestinal iNOS activity correlated with increased apoptosis in PP. Furthermore, the ability to induce intestinal NO production and apoptosis within the first few hours after immunization seemed to correlate with the protective capacity of mutant E/1/3 of S. enterica serovar Enteritidis. It was found that nonprotective mutant C/2/2, which was unable to induce intestinal NO production, also failed to induce apoptosis in PP. Moreover, aminoguanidine treatment at the time of immunization resulted in inhibition of the NO production and apoptosis induced by protective mutant E/1/3 and completely abolished protection against challenge. These results suggest that the induction of iNOS in the intestinal mucosa by attenuated mutant E/1/3 of S. enterica serovar Enteritidis at the time of immunization is necessary to generate a protective immune response.

Diminished percentage of antigen bearing cells in the lymph nodes of immune aged rats.

We have demonstrated previously that during experimental autoimmune prostatitis (EAP), aged rats show a diminished humoral autoimmune response. In the present paper we have studied the transport of the autoantigen from the site of injection toward lymphatic organs in rats of different ages with or without EAP. We used as autoantigen prostatic components (rat accessory glands (RAG)) conjugated with fluorescein isothiocyanate (FITC). Studies of flow cytometry, fluorescent microscopy and confocal microscopy show no differences in the percentage of RAG-FITC positive cells or in the localization of the cells in the popliteal lymph nodes of not-immunized young and aged rats. On the other hand, in 18-month-old rats immunized with either RAG or Ovalbumin there were lower levels of specific IgG antibodies and fewer antigen containing cells in the draining lymph nodes than those of 3- or 12-month-old rats. In all groups fluorescent cells were MHC class II positive and some were IgM positive. Our results demonstrate that in immunized 18-month-old rats there is a diminished percentage of cells bearing the antigen in the draining lymph nodes after antigen injection in the skin, related to the levels of specific antibodies able to form antigen-antibody complexes in the periphery.

Changes in the development of experimental autoimmune prostatitis (EAP) by castration in aged rats.

During Experimental Autoimmune Prostatitis (EAP), 12-month-old rats show a higher cellular autoimmune response and lower humoral autoimmune response against prostatic components than 3-month-old rats subjected to the same antigen stimulus. We analyzed if thymus recovery by orchidectomy could affect the development of EAP in 12-month-old rats. Thirty days after gonadectomy, 12-month-old rats showed an increment in the thymic mass and in the thymocytes absolute number, with percentages of the four main cell subpopulations (defined by CD4-CD8 molecules expression) similar to the 3-month-old rats. The DTH response of castrated 12-month-old with EAP were diminished in comparison with sham-castrated 12-month-old rats with EAP, resembling the values observed in 3-month-old rats with EAP. The prostates of castrated 12-month-old rats with EAP did not show inflammatory mononuclear cell infiltration, as did control 3- and 12-month-old rats with EAP. Castration seems to modulate negatively EAP in 12-month-old rats, possibly through the regeneration of thymus after testosterone deprivation.

Pancreatic acinar cells submitted to stress activate TNF-alpha gene expression.

To elucidate whether pancreatic acinar cell submitted to stress is able to express TNF-alpha, we studied TNF-alpha mRNA expression by Northern blot and in situ hybridization in healthy pancreas, in tissue from caerulein-induced pancreatitis and after lipopolysaccharide (LPS) treatment. In specimens from normal pancreas, TNF-alpha mRNA expression, as judged by both Northern blot and in situ hybridization, was negative, whereas a strong but transient expression was observed in acinar cells from caerulein pancreatitis and LPS treatment. TNF-alpha mRNA appeared as rapidly as 30 min after treatment, and was maximal 6 h after. At this time, there was mild infiltration consisting mostly of polymorphonuclear leukocytes (PMNL) and no signal of TNF-alpha transcript was found in their cytoplasm. Our results strongly indicate that pancreatic acinar cell is the source of TNF-alpha early in the course of acute pancreatitis and LPS treatment, and suggest that the expression of this cytokine is a part of a general response of the acinar cell to aggression.

Cloning and expression of the mouse PIP49 (Pancreatitis Induced Protein 49) mRNA which encodes a new putative transmembrane protein activated in the pancreas with acute pancreatitis.

We have used a microarray-based strategy to characterize, at the molecular level, the pancreatic emergency program set up by the pancreatic cells in response to pancreatitis. In this strategy, the phenotype of the pancreatitis-affected pancreas is established by characterization of a large number of its transcripts using a high-density mouse cDNA microarray. This method allows identification of transcripts differentially expressed during pancreatitis. We describe here the cloning, sequencing, and expression analysis of a new gene, named PIP49 (Pancreatitis Induced Protein 49). Its very strong expression is specific of acinar cells and occurs rapidly after initiation of the acute phase of pancreatitis. Analysis of its primary and secondary structures strongly suggests that PIP49 encodes a putative transmembrane protein.

Effect of aging on experimental autoimmune prostatitis: differential kinetics of development.

We have studied the influence of aging on the kinetics of autoimmune response in Experimental Autoimmune Prostatis (EAP). EAP was induced in 3- and 12-month-old Wistar rats by i.d. immunization with a saline extract of rat male sex accessory glands (RAG), chemically modified, and emulsioned in CFA. After immunization, 12-month-old rats developed a faster and stronger specific DTH response against RAG and mononuclear infiltration in the prostate. The levels of total IgM and IgG against RAG were lower in 12-month-old rats than in 3-month-old rats, with a prevalence of IgG2a, IgG2b, and IgG2c subclasses in both ages. Immunization stimulated slightly the appearance of specific IgG1 to RAG only in 3-month-old rats but in 12-month-old rats there was no specific IgG1 to RAG. On the other hand, normal 12-month-old rats showed higher levels of some natural antibodies and their thymocytes and peripheral lymphocytes had a diminished proliferative capacity compared to 3-month-old rats. These data demonstrated that 12-month-old rats show parameters of an aged immune system and present an exacerbated autoimmune prostatitis compared with 3-month-old rats.