E-learning environments, such as Moodle, provide a technology that fosters the improvement of the... more E-learning environments, such as Moodle, provide a technology that fosters the improvement of the educational system in developed countries, where education is traditionally performed with relatively high standards of quality. A large number of case studies and research have been conducted to demonstrate how e-learning technologies can be applied to improve both training and learning processes. However, these technologies have not been proved efficient when applied to developing countries. The challenges that must be addressed in developing countries, both technological and societal, are much more complex and the possible solution margins are more constrained than those existing in the context where these technologies have been created. In this paper we show how Moodle can be used to improve the quality of education in developing countries and, even more important, how can be used to turn the educational system more sustainable and effective in the long-term. We describe our experience in implementing a programming course in Moodle for the Higher School of Informatics at the Université Polytechnique de Bobo-Dioulasso, in Burkina Faso (West Africa), joining efforts with local professors in designing and implementing the learning system. The case example has been designed having in mind a number of contextual problems: lack of lecturers, excessive teaching hours per lecturer, massive classes, and curricula organization and stability, among others. We finally discuss how the teaching effort is reduced, the students' knowledge and capacity improves, and the institutional academic model can be guaranteed with the proposal. For this reason, we claim that information technologies in developing countries are a cost-effective way to guarantee the objectives originally defined in the academic curricula and, therefore, deal with the problem of the education.
Bacground Evaluation of renal replacement therapy with haemodialysis is essential for its improve... more Bacground Evaluation of renal replacement therapy with haemodialysis is essential for its improvement. However, the methods used have important epistemological limitations. The present study aimed to determine the opinions and preferences of stakeholders (patients, clinicians, and managers) and establish their relative importance to facilitating an adequate evaluation. Methods Successive working groups (WGs) were established using a multicriteria methodology. WG1 created a draft of criteria and sub-criteria, WG2 agreed, and WG3 was composed of three face-to-face subgroups (WG3-A, WG3-B, and WG3-C) that weighted them using two methodologies: weighting sum (WS) and analytic hierarchy process (AHP). Subsequently, they determined a preference for the WS or AHP results. Finally, via the Internet, WG4 weighted the criteria and sub-criteria by the method preferred by WG3, and WG5 analysed the results. Results WG1 and WG2 identified and agreed on the following evaluation criteria: evidence-...
Mitochondrion is an accepted molecular target in cancer treatment since it exhibits a higher tran... more Mitochondrion is an accepted molecular target in cancer treatment since it exhibits a higher transmembrane potential in cancer cells, making it susceptible to be targeted by lipophilic-delocalized cations of triphenylphosphonium (TPP(+)). Thus, we evaluated five TPP(+)-linked decyl polyhydroxybenzoates as potential cytotoxic agents in several human breast cancer cell lines that differ in estrogen receptor and HER2/neu expression, and in metabolic profile. Results showed that all cell lines tested were sensitive to the cytotoxic action of these compounds. The mechanism underlying the cytotoxicity would be triggered by their weak uncoupling effect on the oxidative phosphorylation system, while having a wider and safer therapeutic range than other uncouplers and a significant lowering in transmembrane potential. Noteworthy, while the TPP(+)-derivatives alone led to almost negligible losses of ATP, when these were added in the presence of an AMP-activated protein kinase inhibitor, the levels of ATP fell greatly. Overall, data presented suggest that decyl polyhydroxybenzoates-TPP(+) and its derivatives warrant future investigation as potential anti-tumor agents.
Environmental chemicals may be involved in the etiology of breast cancer. There is substantial ev... more Environmental chemicals may be involved in the etiology of breast cancer. There is substantial evidence that breast cancer risk is associated with prolonged exposure to female hormones. Among these hormonal influences a leading role is attributed to the ovarian hormone estradiol, since breast cancer does not develop in the absence of ovaries. The rat mammary gland has special characteristics that make it an ideal organ for studying development, cell proliferation and transformation. In vivo and in vitro model systems for cell proliferation and mammary carcinogenesis have allowed morphological and biochemical analysis under different experimental conditions. The aim of this study was to examine the effect of eserine, an acetylcholinesterase inhibitor, as are the organophosphorous compounds malathion and parathion, and 17ß estradiol on cell proliferation and tumor formation that takes place in the rat mammary gland after in vivo and in vitro treatment. These studies showed that eserine and 17ß estradiol were capable of inducing carcinogenesis in the epithelium of rat mammary glands. It was found that there was a significant increase in the number of cells per duct of the 44-day-old rat mammary gland after the 10-day eserine treatment, compared to the control. A higher increase was observed in the animals treated for 10 days with eserine followed by 30-daily injections of estrogen in comparison to control animals. In 12 animals, two mammary tumors were directly developed in response to 17ß estradiol injected at 39 days of age with a latency period of 180 and 245 days, respectively. Such tumors were metastatic to the lung. These results suggest that terminal end buds are major targets related to rat mammary carcinogenesis and 17ß estradiol can be an initiator and promoter in this process.
The role of Rel and activation protein-1 (AP-1) in IL-2 promoter activity in B7-1- and leukocyte ... more The role of Rel and activation protein-1 (AP-1) in IL-2 promoter activity in B7-1- and leukocyte function-associated Ag-3 (LFA-3)-costimulated T cells has been evaluated. We demonstrate that overexpression of c-Jun but not c-Fos increases IL-2 promoter activity in both B7-1- and LFA-3-costimulated Jurkat T cells. Cotransfection of both c-Jun and c-Fos substitutes for B7-1 costimulation in driving an activation protein-1 response element but not for the IL-2 promoter. Overexpression of Rel proteins demonstrated that p65-expressing Jurkat cells transcribed equally well a nuclear factor κβ reporter construct when costimulated with B7-1 or LFA-3, but transcription of IL-2 promoter or CD28 response element (CD28RE)-driven reporters was superior in B7-1-costimulated cells. Combined expression of c-Jun and p65 induced vigorous transcription of IL-2 promoter- and CD28RE-driven reporter constructs in both LFA-3- and B7-1-costimulated Jurkat cells. Mutating the CD28RE but not the upstream nuc...
We examined receptor binding and metabolic effects of the platelet-derived growth factor (PDGF) i... more We examined receptor binding and metabolic effects of the platelet-derived growth factor (PDGF) isoforms AA, AB, and BB in cultures of osteoblastic cells from fetal rat calvaria. Saturation binding experiments demonstrated the presence of 6,000 binding sites for PDGF-AA, 42,000 for PDGF-AB, and 60,000 for PDGF-BB. Binding competition experiments were compatible with the recently postulated model of three PDGF receptor subtypes with differential affinity for the PDGF isoforms. The effects of the PDGF isoforms on DNA synthesis, collagen synthesis, and alkaline phosphatase activity in osteoblasts strictly correlated with the number of available binding sites. Accordingly, PDGF-BB was the most potent isoform, PDGF-AB was slightly less potent, and PDGF-AA was the least potent. In contrast, we found that PDGF-BB was less potent than PDGF-AB in increasing plasminogen activator activity in the osteoblast-conditioned medium. Our data strongly suggest that the PDGF receptor subtypes in fetal rat osteoblasts not only selectively bind one or more PDGF isoforms, but are also capable of responding differently to these isoforms.
Wortmannin is an important regulator of Phosphoinositide 3-kinase (PI(3)K) signaling pathway. Cha... more Wortmannin is an important regulator of Phosphoinositide 3-kinase (PI(3)K) signaling pathway. Changes in expression and activity of PI3-kinase and PDGF are major positive and negative regulators, respectively, of the PI3-kinase pathway, which regulates growth, survival, and proliferation. Here we have shown that cells dosed with platelet-derived-growth-factor (PDGF) and /or wortmannin, an inhibitor of PI3 kinase, proliferated at expected rates with respect to cells deprived of any additions. Cells with added platelet-derived-growth-factor (PDGF) multiplied substantially faster than naturally growing cells-some thirty percent. As anticipated, cells given only wortmannin divided over forty percent slower than cells without any dosage. Additionally, cells transfected with a luciferase reporter carrying a consensus sequences of the nuclear factor NF-κB binding site and treated with wortmannin inhibited the activation of luciferase in T98G cells. However, this inhibition was not affected by the treatment of PDFG. Our data indicate that Wortmannin and PDGF play different role in the control of expression of Phosphoinositide 3-kinase in glioma T98G cell line.
We previously demonstrated that alkyl gallates coupled to triphenylphosphine have a selective and... more We previously demonstrated that alkyl gallates coupled to triphenylphosphine have a selective and efficient antiproliferative effect by inducing mitochondrial uncoupling in vitro due to the increased mitochondrial transmembrane potential of tumor cells. Therefore, in this work, the in vivo antitumor activities of alkyl gallate triphenylphosphonium derivatives (TPP(+)C8, TPP(+)C10 and TPP(+)C12) were evaluated in a syngeneic murine model of breast cancer. We found that TPP(+)C10 increased the cytosolic ADP/ATP ratio and significantly increased the AMP levels in a concentration-dependent manner in TA3/Ha murine mammary adenocarcinoma cells. Interestingly, TPP(+)C10 induced a decrease in the levels of cellular proliferation markers and promoted caspase-3 activation in tumor-bearing mice. Additionally, TPP(+)C10 inhibited tumor growth in the syngeneic mouse model. Importantly, 30days of intraperitoneal (i.p.) administration of the combination of TPP(+)C10 (10mg/kg/48h) and the antibioti...
We have characterized the regulation of nuclear factors involved in transcriptional control of th... more We have characterized the regulation of nuclear factors involved in transcriptional control of the interleukin-2 (IL-2) promoter-enhancer activity in Jurkat T cells stimulated with superantigen presented on HLA-DR transfectants combined with the ligands LFA-3 (CD58) and B7-1 (CD80). Gel shift analyses showed that NF-AT was strongly induced in LFA-3-costimulated Jurkat T cells, suggesting that NF-AT is a key target nuclear factor for the CD2-LFA-3 pathway. Studies using HLA-DR-B7-1-LFA-3 triple transfectants showed that the LFA-3-induced NF-AT DNA binding activity was negatively regulated by B7-1 costimulation. In contrast, induction of a CD28 response complex containing only c-Rel proteins was seen after B7-1 costimulation. Both LFA-3 costimulation and B7-1 costimulation induced the AP-1 and NF-kappaB nuclear factors. Distinct compositions of the NF-AT complexes were seen in B7-1- and LFA-3-costimulated cells. LFA-3 induced primarily Jun-D, Fra-1, and Fra-2, while B7-1 induced June-...
The Tax protein of human T cell leukemia virus type 1 plays a major role in the pathogenesis of a... more The Tax protein of human T cell leukemia virus type 1 plays a major role in the pathogenesis of adult T cell leukemia (ATL), an aggressive neoplasia of CD4+ T cells. In the present study, we investigated whether the EGR-1 pathway is involved in the regulation of Tax-induced JNK expression in human Jurkat T cells transfected to express the Tax protein in the presence or absence of PMA or ionomycin. Overexpression of EGR-1 in Jurkat cells transfected to express Tax, promoted the activation of several genes, with the most potent being those that contained AP-1 (Jun/c-Fos), whereas knockdown of endogenous EGR-1 by small interfering RNA (siRNA) somewhat reduced Tax-mediated JNK-1 transcription. Additionally, luciferase-based AP-1 and NF-κB reporter gene assays demonstrated that inhibition of EGR-1 expression by an siRNA did not affect the transcriptional activity of a consensus sequence of either AP-1 or NF-κB. On the other hand, the apoptosis assay, using all-trans retinoic acid (ATRA) ...
Previous studies suggest that the effects of Egr-1 on tumorigenesis are critically dependent on t... more Previous studies suggest that the effects of Egr-1 on tumorigenesis are critically dependent on the levels of Egr-1 and the cellular context. For this reason, we examined the effects of blocking the Egr-1 activity by a short interfering RNA (siRNA) against Egr-1 on the expression of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) signaling in the PC-3 and LNCaP prostate carcinoma cell lines. We observed that the reduction in expression of Egr-1 in PC-3 and LNCaP cells by effects of the siRNA vector resulted in lower cell viability and increased apoptosis at 24 and 120 h after transfection. This reduced cell viability correlated well with a reduced activity of the NF-κB and AP-1 factors. We analyzed the expression and activity of these factors and found that p65 and IκB but not p50 were reduced in the siRNA-treated cells. Similarly, we found that c-Fos but not c-Jun was reduced in the siRNA treated cells. These effects were translated to reduced transcriptional activity of N...
Breast cancer mortality has been increasing in Arica Chile where it has surpassed the national ra... more Breast cancer mortality has been increasing in Arica Chile where it has surpassed the national rates 11 times between 1990 and 2010. The city of Arica was sprayed with the organophosphrous pesticide malathion in order to control the Mediterranean fly 33 years ago. Moreover we have demonstrated that a malathion treatment induces the formation of breast carcinomas in Sprague Dowley female rats. The objective of this work was to find a relationship between malathion aerial spraying and the increased mortality rate due to breast cancer that has been observed in Arica in recent years. We extracted city data bases with all breast cancer cases diagnosed from 1995 to 2005 from the Dr. Juan Noe Crevani Hospital of Arica city and Ernesto Torres Hospital of Iquique. The number of patients was 100 in Arica and 58 in Iquique, nearby city that has never been sprayed with malathion which had a similar population than Arica in those years. The statistical analysis of the characteristics of the sample related to breast cancer risk showed that there is no significant difference between women from Arica and from Iquique. Nevertheless the patients with more times of exposure to malathion were 5.7-times more likely to be diagnosed with breast cancer (OR= 5.7; p<0.02). In addition, metastases were found in 30.5% of the malathion-exposed group and only in 16% in the group never exposed (p<0.05). This study suggests that the increase in the mortality rate due to breast cancer occurring in Arica has a significant correlation with the exposure to malathion sprayed over the city more than 30 years ago.
To address elements that might uniquely characterize EGR-1 mediated signaling, the expression of ... more To address elements that might uniquely characterize EGR-1 mediated signaling, the expression of two transcription factors, namely, nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1) were studied. PC-3 and LNCaP prostate carcinoma cell lines were transiently transfected with wildtype Egr-1 expression plasmid (pCMV-Egr-1) and treated with cisplatin and TPA. Overexpression of EGR-1 was found to induce nuclear expression of both, NF-κB and AP1. However, the intensity of the induced AP-1 and NF-κB was diminished after cisplatin treatment, but not after TPA. Our findings confirm that the overexpression of wild-type Egr-1 caused a marked increase in cell proliferation in PC-3 and LNCaP proliferation in a 14-day soft agar colony forming assay. In addition, luciferase reporter gene assay showed that the transcriptional activity of AP-1 and NF-κB in PC-3 and LNCaP prostate carcinoma cell lines was also modulated by the overexpression of EGR-1 in these cells using tandem repeated Luc-AP-1 and Luc-NF-κB. The activation of both NF-κB and AP-1 are key steps in the cascade of events following the activation of the EGR-1 gene. It was revealed that overexpression of EGR-1 selectively increased AP-1 and NF-κB activation, and that the activation of these nuclear factors appears to be essential for the induction of proliferation and anchorage independence in activated PC-3 and LNCaP cells. However, the mechanism underlying the modulation of AP-1 and NF-κB by the overexpression of EGR-1 is still unknown.
The present study examined the effect of the overexpression of early growth response gene (Egr-1)... more The present study examined the effect of the overexpression of early growth response gene (Egr-1) on transforming growth factor β-1 (TGF-β1) and p14ARF levels, in PC-3 and LNCaP prostate carcinoma cell lines. Amplification of EGR-1, TGF-β1 and p14ARF were observed in the two cell lines treated with different stimuli and resulted in a corresponding mRNA and protein expression. The downregulation of TGF-β1 and the attenuation of p14ARF expression by siRNA against Egr-1 predominantly suggested that TGF-β1 and p14ARF may be regulated by the transcription factor EGR-1. A marginal attenuation of cell growth in PC-3 and LNCaP prostate carcinoma cell lines overexpressing p14ARF was observed. Cells transfected with Egr-1 wild-type were able to grow and avoid cell cycle arrest and apoptosis in the presence or absence of p14ARF. In addition, EGR-1 stimulated the expression of TGF β-l as well as the accumulation of the p14ARF proteins. The results suggested that TGF-β1 and p14ARF activities in ...
Inhibition of basal Jun kinase (JNK) activity by small interfering RNAs (siRNAs) enhances cisplat... more Inhibition of basal Jun kinase (JNK) activity by small interfering RNAs (siRNAs) enhances cisplatin sensitivity and decreases DNA repair in T98G glioblastoma cells. Although the JNK pathway has been extensively studied in recent years, little is known concerning the signaling pathways that control their expression in glioma cells. The aim of the present study was to assess the role of c-Jun-NH2-terminal kinases (JNKs) in the regulation of T98G glioblastoma cells treated with cisplatin in the presence or absence of siRNAs against JNK1 and JNK2. Addition of either small interfering JNK1-siRNA or JNK2-siRNA induced decreased DNA repair and sensitized the T98G glioblastoma cells to the DNA damaging drug cisplatin (cis-diamminedichloroplatinum). This effect was associated with reduced cell survival and loss of anchorage‑independent colony formation. The results indicate that effective inhibition of the JNK pathway significantly sensitizes glioblastoma cells to cisplatin, a compound of pr...
Changes in mitochondrial ATP synthesis can affect the function of tumor cells due to the dependen... more Changes in mitochondrial ATP synthesis can affect the function of tumor cells due to the dependence of the first step of glycolysis on mitochondrial ATP. The oxidative phosphorylation (OXPHOS) system is responsible for the synthesis of approximately 90% of the ATP in normal cells and up to 50% in most glycolytic cancers; therefore, inhibition of the electron transport chain (ETC) emerges as an attractive therapeutic target. We studied the effect of a lipophilic isoprenylated catechol, 3-hydroxybakuchiol (3-OHbk), a putative ETC inhibitor isolated from Psoralea glandulosa. 3-OHbk exerted cytotoxic and anti-proliferative effects on the TA3/Ha mouse mammary adenocarcinoma cell line and induced a decrease in the mitochondrial transmembrane potential, the activation of caspase-3, the opening of the mitochondrial permeability transport pore (MPTP) and nuclear DNA fragmentation. Additionally, 3-OHbk inhibited oxygen consumption, an effect that was completely reversed by succinate (an electron donor for Complex II) and duroquinol (electron donor for Complex III), suggesting that 3-OHbk disrupted the electron flow at the level of Complex I. The inhibition of OXPHOS did not increase the level of reactive oxygen species (ROS) but caused a large decrease in the intracellular ATP level. ETC inhibitors have been shown to induce cell death through necrosis and apoptosis by increasing ROS generation. Nevertheless, we demonstrated that 3-OHbk inhibited the ETC and induced apoptosis through an interaction with Complex I. By delivering electrons directly to Complex III with duroquinol, cell death was almost completely abrogated. These results suggest that 3-OHbk has antitumor activity resulting from interactions with the ETC, a system that is already deficient in cancer cells.
Tumor cells present a known metabolic reprogramming, which makes them more susceptible for a sele... more Tumor cells present a known metabolic reprogramming, which makes them more susceptible for a selective cellular death by modifying its mitochondrial bioenergetics. Anticancer action of the antioxidant 9,10dihydroxy-4,4-dimethyl-5,8-dihydroanthracen-1(4H)-one (HQ) on mouse mammary adenocarcinoma TA3, and its multiresistant variant TA3-MTXR, were evaluated. HQ decreased the viability of both tumor cells, affecting slightly mammary epithelial cells. This hydroquinone blocked the electron flow through the NADH dehydrogenase (Complex I), leading to ADP-stimulated oxygen consumption inhibition, transmembrane potential dissipation and cellular ATP level decrease, without increasing ROS production. Duroquinol, an electron donor at CoQ level, reversed the decrease of cell viability induced by HQ. Additionally, HQ selectively induced G 2 /M-phase arrest. Taken together, our results suggest that the bioenergetic dysfunction provoked by HQ is implicated in its anticancer action.
E-learning environments, such as Moodle, provide a technology that fosters the improvement of the... more E-learning environments, such as Moodle, provide a technology that fosters the improvement of the educational system in developed countries, where education is traditionally performed with relatively high standards of quality. A large number of case studies and research have been conducted to demonstrate how e-learning technologies can be applied to improve both training and learning processes. However, these technologies have not been proved efficient when applied to developing countries. The challenges that must be addressed in developing countries, both technological and societal, are much more complex and the possible solution margins are more constrained than those existing in the context where these technologies have been created. In this paper we show how Moodle can be used to improve the quality of education in developing countries and, even more important, how can be used to turn the educational system more sustainable and effective in the long-term. We describe our experience in implementing a programming course in Moodle for the Higher School of Informatics at the Université Polytechnique de Bobo-Dioulasso, in Burkina Faso (West Africa), joining efforts with local professors in designing and implementing the learning system. The case example has been designed having in mind a number of contextual problems: lack of lecturers, excessive teaching hours per lecturer, massive classes, and curricula organization and stability, among others. We finally discuss how the teaching effort is reduced, the students' knowledge and capacity improves, and the institutional academic model can be guaranteed with the proposal. For this reason, we claim that information technologies in developing countries are a cost-effective way to guarantee the objectives originally defined in the academic curricula and, therefore, deal with the problem of the education.
Bacground Evaluation of renal replacement therapy with haemodialysis is essential for its improve... more Bacground Evaluation of renal replacement therapy with haemodialysis is essential for its improvement. However, the methods used have important epistemological limitations. The present study aimed to determine the opinions and preferences of stakeholders (patients, clinicians, and managers) and establish their relative importance to facilitating an adequate evaluation. Methods Successive working groups (WGs) were established using a multicriteria methodology. WG1 created a draft of criteria and sub-criteria, WG2 agreed, and WG3 was composed of three face-to-face subgroups (WG3-A, WG3-B, and WG3-C) that weighted them using two methodologies: weighting sum (WS) and analytic hierarchy process (AHP). Subsequently, they determined a preference for the WS or AHP results. Finally, via the Internet, WG4 weighted the criteria and sub-criteria by the method preferred by WG3, and WG5 analysed the results. Results WG1 and WG2 identified and agreed on the following evaluation criteria: evidence-...
Mitochondrion is an accepted molecular target in cancer treatment since it exhibits a higher tran... more Mitochondrion is an accepted molecular target in cancer treatment since it exhibits a higher transmembrane potential in cancer cells, making it susceptible to be targeted by lipophilic-delocalized cations of triphenylphosphonium (TPP(+)). Thus, we evaluated five TPP(+)-linked decyl polyhydroxybenzoates as potential cytotoxic agents in several human breast cancer cell lines that differ in estrogen receptor and HER2/neu expression, and in metabolic profile. Results showed that all cell lines tested were sensitive to the cytotoxic action of these compounds. The mechanism underlying the cytotoxicity would be triggered by their weak uncoupling effect on the oxidative phosphorylation system, while having a wider and safer therapeutic range than other uncouplers and a significant lowering in transmembrane potential. Noteworthy, while the TPP(+)-derivatives alone led to almost negligible losses of ATP, when these were added in the presence of an AMP-activated protein kinase inhibitor, the levels of ATP fell greatly. Overall, data presented suggest that decyl polyhydroxybenzoates-TPP(+) and its derivatives warrant future investigation as potential anti-tumor agents.
Environmental chemicals may be involved in the etiology of breast cancer. There is substantial ev... more Environmental chemicals may be involved in the etiology of breast cancer. There is substantial evidence that breast cancer risk is associated with prolonged exposure to female hormones. Among these hormonal influences a leading role is attributed to the ovarian hormone estradiol, since breast cancer does not develop in the absence of ovaries. The rat mammary gland has special characteristics that make it an ideal organ for studying development, cell proliferation and transformation. In vivo and in vitro model systems for cell proliferation and mammary carcinogenesis have allowed morphological and biochemical analysis under different experimental conditions. The aim of this study was to examine the effect of eserine, an acetylcholinesterase inhibitor, as are the organophosphorous compounds malathion and parathion, and 17ß estradiol on cell proliferation and tumor formation that takes place in the rat mammary gland after in vivo and in vitro treatment. These studies showed that eserine and 17ß estradiol were capable of inducing carcinogenesis in the epithelium of rat mammary glands. It was found that there was a significant increase in the number of cells per duct of the 44-day-old rat mammary gland after the 10-day eserine treatment, compared to the control. A higher increase was observed in the animals treated for 10 days with eserine followed by 30-daily injections of estrogen in comparison to control animals. In 12 animals, two mammary tumors were directly developed in response to 17ß estradiol injected at 39 days of age with a latency period of 180 and 245 days, respectively. Such tumors were metastatic to the lung. These results suggest that terminal end buds are major targets related to rat mammary carcinogenesis and 17ß estradiol can be an initiator and promoter in this process.
The role of Rel and activation protein-1 (AP-1) in IL-2 promoter activity in B7-1- and leukocyte ... more The role of Rel and activation protein-1 (AP-1) in IL-2 promoter activity in B7-1- and leukocyte function-associated Ag-3 (LFA-3)-costimulated T cells has been evaluated. We demonstrate that overexpression of c-Jun but not c-Fos increases IL-2 promoter activity in both B7-1- and LFA-3-costimulated Jurkat T cells. Cotransfection of both c-Jun and c-Fos substitutes for B7-1 costimulation in driving an activation protein-1 response element but not for the IL-2 promoter. Overexpression of Rel proteins demonstrated that p65-expressing Jurkat cells transcribed equally well a nuclear factor κβ reporter construct when costimulated with B7-1 or LFA-3, but transcription of IL-2 promoter or CD28 response element (CD28RE)-driven reporters was superior in B7-1-costimulated cells. Combined expression of c-Jun and p65 induced vigorous transcription of IL-2 promoter- and CD28RE-driven reporter constructs in both LFA-3- and B7-1-costimulated Jurkat cells. Mutating the CD28RE but not the upstream nuc...
We examined receptor binding and metabolic effects of the platelet-derived growth factor (PDGF) i... more We examined receptor binding and metabolic effects of the platelet-derived growth factor (PDGF) isoforms AA, AB, and BB in cultures of osteoblastic cells from fetal rat calvaria. Saturation binding experiments demonstrated the presence of 6,000 binding sites for PDGF-AA, 42,000 for PDGF-AB, and 60,000 for PDGF-BB. Binding competition experiments were compatible with the recently postulated model of three PDGF receptor subtypes with differential affinity for the PDGF isoforms. The effects of the PDGF isoforms on DNA synthesis, collagen synthesis, and alkaline phosphatase activity in osteoblasts strictly correlated with the number of available binding sites. Accordingly, PDGF-BB was the most potent isoform, PDGF-AB was slightly less potent, and PDGF-AA was the least potent. In contrast, we found that PDGF-BB was less potent than PDGF-AB in increasing plasminogen activator activity in the osteoblast-conditioned medium. Our data strongly suggest that the PDGF receptor subtypes in fetal rat osteoblasts not only selectively bind one or more PDGF isoforms, but are also capable of responding differently to these isoforms.
Wortmannin is an important regulator of Phosphoinositide 3-kinase (PI(3)K) signaling pathway. Cha... more Wortmannin is an important regulator of Phosphoinositide 3-kinase (PI(3)K) signaling pathway. Changes in expression and activity of PI3-kinase and PDGF are major positive and negative regulators, respectively, of the PI3-kinase pathway, which regulates growth, survival, and proliferation. Here we have shown that cells dosed with platelet-derived-growth-factor (PDGF) and /or wortmannin, an inhibitor of PI3 kinase, proliferated at expected rates with respect to cells deprived of any additions. Cells with added platelet-derived-growth-factor (PDGF) multiplied substantially faster than naturally growing cells-some thirty percent. As anticipated, cells given only wortmannin divided over forty percent slower than cells without any dosage. Additionally, cells transfected with a luciferase reporter carrying a consensus sequences of the nuclear factor NF-κB binding site and treated with wortmannin inhibited the activation of luciferase in T98G cells. However, this inhibition was not affected by the treatment of PDFG. Our data indicate that Wortmannin and PDGF play different role in the control of expression of Phosphoinositide 3-kinase in glioma T98G cell line.
We previously demonstrated that alkyl gallates coupled to triphenylphosphine have a selective and... more We previously demonstrated that alkyl gallates coupled to triphenylphosphine have a selective and efficient antiproliferative effect by inducing mitochondrial uncoupling in vitro due to the increased mitochondrial transmembrane potential of tumor cells. Therefore, in this work, the in vivo antitumor activities of alkyl gallate triphenylphosphonium derivatives (TPP(+)C8, TPP(+)C10 and TPP(+)C12) were evaluated in a syngeneic murine model of breast cancer. We found that TPP(+)C10 increased the cytosolic ADP/ATP ratio and significantly increased the AMP levels in a concentration-dependent manner in TA3/Ha murine mammary adenocarcinoma cells. Interestingly, TPP(+)C10 induced a decrease in the levels of cellular proliferation markers and promoted caspase-3 activation in tumor-bearing mice. Additionally, TPP(+)C10 inhibited tumor growth in the syngeneic mouse model. Importantly, 30days of intraperitoneal (i.p.) administration of the combination of TPP(+)C10 (10mg/kg/48h) and the antibioti...
We have characterized the regulation of nuclear factors involved in transcriptional control of th... more We have characterized the regulation of nuclear factors involved in transcriptional control of the interleukin-2 (IL-2) promoter-enhancer activity in Jurkat T cells stimulated with superantigen presented on HLA-DR transfectants combined with the ligands LFA-3 (CD58) and B7-1 (CD80). Gel shift analyses showed that NF-AT was strongly induced in LFA-3-costimulated Jurkat T cells, suggesting that NF-AT is a key target nuclear factor for the CD2-LFA-3 pathway. Studies using HLA-DR-B7-1-LFA-3 triple transfectants showed that the LFA-3-induced NF-AT DNA binding activity was negatively regulated by B7-1 costimulation. In contrast, induction of a CD28 response complex containing only c-Rel proteins was seen after B7-1 costimulation. Both LFA-3 costimulation and B7-1 costimulation induced the AP-1 and NF-kappaB nuclear factors. Distinct compositions of the NF-AT complexes were seen in B7-1- and LFA-3-costimulated cells. LFA-3 induced primarily Jun-D, Fra-1, and Fra-2, while B7-1 induced June-...
The Tax protein of human T cell leukemia virus type 1 plays a major role in the pathogenesis of a... more The Tax protein of human T cell leukemia virus type 1 plays a major role in the pathogenesis of adult T cell leukemia (ATL), an aggressive neoplasia of CD4+ T cells. In the present study, we investigated whether the EGR-1 pathway is involved in the regulation of Tax-induced JNK expression in human Jurkat T cells transfected to express the Tax protein in the presence or absence of PMA or ionomycin. Overexpression of EGR-1 in Jurkat cells transfected to express Tax, promoted the activation of several genes, with the most potent being those that contained AP-1 (Jun/c-Fos), whereas knockdown of endogenous EGR-1 by small interfering RNA (siRNA) somewhat reduced Tax-mediated JNK-1 transcription. Additionally, luciferase-based AP-1 and NF-κB reporter gene assays demonstrated that inhibition of EGR-1 expression by an siRNA did not affect the transcriptional activity of a consensus sequence of either AP-1 or NF-κB. On the other hand, the apoptosis assay, using all-trans retinoic acid (ATRA) ...
Previous studies suggest that the effects of Egr-1 on tumorigenesis are critically dependent on t... more Previous studies suggest that the effects of Egr-1 on tumorigenesis are critically dependent on the levels of Egr-1 and the cellular context. For this reason, we examined the effects of blocking the Egr-1 activity by a short interfering RNA (siRNA) against Egr-1 on the expression of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) signaling in the PC-3 and LNCaP prostate carcinoma cell lines. We observed that the reduction in expression of Egr-1 in PC-3 and LNCaP cells by effects of the siRNA vector resulted in lower cell viability and increased apoptosis at 24 and 120 h after transfection. This reduced cell viability correlated well with a reduced activity of the NF-κB and AP-1 factors. We analyzed the expression and activity of these factors and found that p65 and IκB but not p50 were reduced in the siRNA-treated cells. Similarly, we found that c-Fos but not c-Jun was reduced in the siRNA treated cells. These effects were translated to reduced transcriptional activity of N...
Breast cancer mortality has been increasing in Arica Chile where it has surpassed the national ra... more Breast cancer mortality has been increasing in Arica Chile where it has surpassed the national rates 11 times between 1990 and 2010. The city of Arica was sprayed with the organophosphrous pesticide malathion in order to control the Mediterranean fly 33 years ago. Moreover we have demonstrated that a malathion treatment induces the formation of breast carcinomas in Sprague Dowley female rats. The objective of this work was to find a relationship between malathion aerial spraying and the increased mortality rate due to breast cancer that has been observed in Arica in recent years. We extracted city data bases with all breast cancer cases diagnosed from 1995 to 2005 from the Dr. Juan Noe Crevani Hospital of Arica city and Ernesto Torres Hospital of Iquique. The number of patients was 100 in Arica and 58 in Iquique, nearby city that has never been sprayed with malathion which had a similar population than Arica in those years. The statistical analysis of the characteristics of the sample related to breast cancer risk showed that there is no significant difference between women from Arica and from Iquique. Nevertheless the patients with more times of exposure to malathion were 5.7-times more likely to be diagnosed with breast cancer (OR= 5.7; p<0.02). In addition, metastases were found in 30.5% of the malathion-exposed group and only in 16% in the group never exposed (p<0.05). This study suggests that the increase in the mortality rate due to breast cancer occurring in Arica has a significant correlation with the exposure to malathion sprayed over the city more than 30 years ago.
To address elements that might uniquely characterize EGR-1 mediated signaling, the expression of ... more To address elements that might uniquely characterize EGR-1 mediated signaling, the expression of two transcription factors, namely, nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1) were studied. PC-3 and LNCaP prostate carcinoma cell lines were transiently transfected with wildtype Egr-1 expression plasmid (pCMV-Egr-1) and treated with cisplatin and TPA. Overexpression of EGR-1 was found to induce nuclear expression of both, NF-κB and AP1. However, the intensity of the induced AP-1 and NF-κB was diminished after cisplatin treatment, but not after TPA. Our findings confirm that the overexpression of wild-type Egr-1 caused a marked increase in cell proliferation in PC-3 and LNCaP proliferation in a 14-day soft agar colony forming assay. In addition, luciferase reporter gene assay showed that the transcriptional activity of AP-1 and NF-κB in PC-3 and LNCaP prostate carcinoma cell lines was also modulated by the overexpression of EGR-1 in these cells using tandem repeated Luc-AP-1 and Luc-NF-κB. The activation of both NF-κB and AP-1 are key steps in the cascade of events following the activation of the EGR-1 gene. It was revealed that overexpression of EGR-1 selectively increased AP-1 and NF-κB activation, and that the activation of these nuclear factors appears to be essential for the induction of proliferation and anchorage independence in activated PC-3 and LNCaP cells. However, the mechanism underlying the modulation of AP-1 and NF-κB by the overexpression of EGR-1 is still unknown.
The present study examined the effect of the overexpression of early growth response gene (Egr-1)... more The present study examined the effect of the overexpression of early growth response gene (Egr-1) on transforming growth factor β-1 (TGF-β1) and p14ARF levels, in PC-3 and LNCaP prostate carcinoma cell lines. Amplification of EGR-1, TGF-β1 and p14ARF were observed in the two cell lines treated with different stimuli and resulted in a corresponding mRNA and protein expression. The downregulation of TGF-β1 and the attenuation of p14ARF expression by siRNA against Egr-1 predominantly suggested that TGF-β1 and p14ARF may be regulated by the transcription factor EGR-1. A marginal attenuation of cell growth in PC-3 and LNCaP prostate carcinoma cell lines overexpressing p14ARF was observed. Cells transfected with Egr-1 wild-type were able to grow and avoid cell cycle arrest and apoptosis in the presence or absence of p14ARF. In addition, EGR-1 stimulated the expression of TGF β-l as well as the accumulation of the p14ARF proteins. The results suggested that TGF-β1 and p14ARF activities in ...
Inhibition of basal Jun kinase (JNK) activity by small interfering RNAs (siRNAs) enhances cisplat... more Inhibition of basal Jun kinase (JNK) activity by small interfering RNAs (siRNAs) enhances cisplatin sensitivity and decreases DNA repair in T98G glioblastoma cells. Although the JNK pathway has been extensively studied in recent years, little is known concerning the signaling pathways that control their expression in glioma cells. The aim of the present study was to assess the role of c-Jun-NH2-terminal kinases (JNKs) in the regulation of T98G glioblastoma cells treated with cisplatin in the presence or absence of siRNAs against JNK1 and JNK2. Addition of either small interfering JNK1-siRNA or JNK2-siRNA induced decreased DNA repair and sensitized the T98G glioblastoma cells to the DNA damaging drug cisplatin (cis-diamminedichloroplatinum). This effect was associated with reduced cell survival and loss of anchorage‑independent colony formation. The results indicate that effective inhibition of the JNK pathway significantly sensitizes glioblastoma cells to cisplatin, a compound of pr...
Changes in mitochondrial ATP synthesis can affect the function of tumor cells due to the dependen... more Changes in mitochondrial ATP synthesis can affect the function of tumor cells due to the dependence of the first step of glycolysis on mitochondrial ATP. The oxidative phosphorylation (OXPHOS) system is responsible for the synthesis of approximately 90% of the ATP in normal cells and up to 50% in most glycolytic cancers; therefore, inhibition of the electron transport chain (ETC) emerges as an attractive therapeutic target. We studied the effect of a lipophilic isoprenylated catechol, 3-hydroxybakuchiol (3-OHbk), a putative ETC inhibitor isolated from Psoralea glandulosa. 3-OHbk exerted cytotoxic and anti-proliferative effects on the TA3/Ha mouse mammary adenocarcinoma cell line and induced a decrease in the mitochondrial transmembrane potential, the activation of caspase-3, the opening of the mitochondrial permeability transport pore (MPTP) and nuclear DNA fragmentation. Additionally, 3-OHbk inhibited oxygen consumption, an effect that was completely reversed by succinate (an electron donor for Complex II) and duroquinol (electron donor for Complex III), suggesting that 3-OHbk disrupted the electron flow at the level of Complex I. The inhibition of OXPHOS did not increase the level of reactive oxygen species (ROS) but caused a large decrease in the intracellular ATP level. ETC inhibitors have been shown to induce cell death through necrosis and apoptosis by increasing ROS generation. Nevertheless, we demonstrated that 3-OHbk inhibited the ETC and induced apoptosis through an interaction with Complex I. By delivering electrons directly to Complex III with duroquinol, cell death was almost completely abrogated. These results suggest that 3-OHbk has antitumor activity resulting from interactions with the ETC, a system that is already deficient in cancer cells.
Tumor cells present a known metabolic reprogramming, which makes them more susceptible for a sele... more Tumor cells present a known metabolic reprogramming, which makes them more susceptible for a selective cellular death by modifying its mitochondrial bioenergetics. Anticancer action of the antioxidant 9,10dihydroxy-4,4-dimethyl-5,8-dihydroanthracen-1(4H)-one (HQ) on mouse mammary adenocarcinoma TA3, and its multiresistant variant TA3-MTXR, were evaluated. HQ decreased the viability of both tumor cells, affecting slightly mammary epithelial cells. This hydroquinone blocked the electron flow through the NADH dehydrogenase (Complex I), leading to ADP-stimulated oxygen consumption inhibition, transmembrane potential dissipation and cellular ATP level decrease, without increasing ROS production. Duroquinol, an electron donor at CoQ level, reversed the decrease of cell viability induced by HQ. Additionally, HQ selectively induced G 2 /M-phase arrest. Taken together, our results suggest that the bioenergetic dysfunction provoked by HQ is implicated in its anticancer action.
Uploads
Papers by Eduardo Parra