Anti-metastatic action of an N 4-aryl substituted thiosemicarbazone on advanced triple negative breast cancer.

PURPOSE: Advanced triple negative breast cancer (ATNBC) is defined by a lack of expression of hormones receptors as well as HER2/neu and its high probability of visceral metastasis. This pathology is associated with a poor prognosis. Previously, we found that T2, an N 4-arylsubstituted thiosemicarbazone (N 4-TSC), had cytotoxic effect on human breast cancer cells lines. Hence, in this study, we investigated the anti-metastasic action of T2 on ATNBC. METHODS: In order to deepen T2 action mode on ATNBC, we first confirmed T2 cytotoxicity on a panel of TNBC cells and then continued studying T2 effects in vitro an in vivo on the syngeneic 4T1 mouse model. RESULTS: We found that T2 had a cytotoxic effect comparable to chemotherapeutics used in present treatment schemes for ATNBC. T2 treatment not only induced apoptosis, but it also down-modulated 4T1 invasive and metastatic-associated capacities, such as clonogenicity, migration and metallo-proteases activity. Moreover, this agent reduced the number of 4T1 cancer stem cells. Finally, T2 treatment induced a more differentiated cell phenotype and the overexpression of the metastasis suppressor gene NDRG-1. In vivo assays showed that T2 reduced tumor burden, down modulated local tumor invasion and significantly reduced the number of lung metastases in the 4T1 advanced TNBC murine model, while the compound did not exhibit intolerable toxicity. CONCLUSION: This study provided evidence that T2 not only exerted an anti-tumor activity but it also showed anti-invasive and anti-metastatic actions on ATNBC in vivo and in vitro, suggesting that T2 could be considered as a promising therapy that deserves further analysis.

The oxidative stress induced in vivo by Shiga toxin-2 contributes to the pathogenicity of haemolytic uraemic syndrome.

Typical haemolytic uraemic syndrome (HUS) is caused by Shiga toxin (Stx)-producing Escherichia coli infections and is characterized by thrombotic microangiopathy that leads to haemolytic anaemia, thrombocytopenia and acute renal failure. Renal or neurological sequelae are consequences of irreversible tissue damage during the acute phase. Stx toxicity and the acute inflammatory response raised by the host determine the development of HUS. At present there is no specific therapy to control Stx damage. The pathogenic role of reactive oxygen species (ROS) on endothelial injury has been largely documented. In this study, we investigated the in-vivo effects of Stx on the oxidative balance and its contribution to the development of HUS in mice. In addition, we analysed the effect of anti-oxidant agents as therapeutic tools to counteract Stx toxicity. We demonstrated that Stx induced an oxidative imbalance, evidenced by renal glutathione depletion and increased lipid membrane peroxidation. The increased ROS production by neutrophils may be one of the major sources of oxidative stress during Stx intoxication. All these parameters were ameliorated by anti-oxidants reducing platelet activation, renal damage and increasing survival. To conclude, Stx generates a pro-oxidative state that contributes to kidney failure, and exogenous anti-oxidants could be beneficial to counteract this pathogenic pathway.

Endogenous glucocorticoids attenuate Shiga toxin-2-induced toxicity in a mouse model of haemolytic uraemic syndrome.

The concept that during an immune challenge the release of glucocorticoids (GC) provides feedback inhibition on evolving immune responses has been drawn primarily from studies of autoimmune and/or inflammatory processes in animal models. The epidemic form of haemolytic uraemic syndrome (HUS) occurs secondary to infection with Gram-negative bacteria that produce Shiga toxin (Stx). Although Stx binding to the specific receptors present on renal tissue is the primary pathogenic mechanism, inflammatory or immune interactions are necessary for the development of the complete form of HUS. The aim of this study was to investigate the influence of endogenous GC on Stx-toxicity in a mouse model. Stx2 was injected into GC-deprived mice and survival rate, renal damage and serum urea levels were evaluated. Plasma corticosterone and cytosolic GC receptor (GR) concentration were also determined at multiple intervals post-Stx2 treatment. Higher sensitivity to Stx2 was observed in mice lacking endogenous GC, evidenced by an increase in mortality rates, circulating urea levels and renal histological damage. Moreover, Stx2 injection was associated with a transient but significant rise in corticosterone secretion. Interestingly, 24 h after Stx inoculation significant increases in total GR were detected in circulating neutrophils. These results indicate that interactions between the neuroendocrine and immune systems can modulate the level of damage significantly during a bacterial infection.

Molecular analysis of p53 tumor-suppressor gene and microsatellites in preneoplastic and neoplastic lesions of the colon and esophagus.

Mutations in exons 4-8 of the p53 gene by the PCR-SSCP analysis in preneoplastic and neoplastic lesions of the colon (n=11) and esophagus (n=18) were screened. p53 overexpression by immunohistochemistry in 11 colonic lesions and 13 microsatellites, in all the patients (n=29), were also studied. A positive result concordancy between the three techniques was found in 1 adenoma and 2 adenocarcinomas of the colon, each with loss of heterozygocity of microsatellites. Metaplastic lesions of esophagus showed biallelic mutations and low frequency of microsatellite alterations. The relationship between genetic alterations in p53, microsatellites and type of colon and esophageal lesions is discussed.

Impairment of mammary lobular development induced by expression of TGFbeta1 under the control of WAP promoter does not suppress tumorigenesis in MMTV-infected transgenic mice.

It has previously been shown that transgenic female mice expressing TGFbeta1 under control of regulatory elements of the whey-acidic protein (WAP) gene were unable to lactate. This was due to the increased apoptosis of the cells committed to the lobular-lactogenic phenotype. Our goal was to determine whether the expression of WAP-TGFbeta1 transgene could inhibit MMTV (mouse mammary tumor virus) tumorigenic activity in the mammary gland. It is well known that the infection with this virus produces focal hyperplastic secretory nodules (HANs) and, some variants can also induce ductal pregnancy-dependent lesions (plaques). In either case, MMTV infection leads ultimately to the appearance of malignant mammary tumors. The results shown herein demonstrate that TGFbeta1 expression in the secretory mammary epithelium does not suppress mammary tumorigenesis in MMTV infected mice. Although MMTV infected WAP-TGFbeta1 transgenic females displayed a strong impairment of lobule-alveolar development, carcinogenesis induced by any of the four MMTV variants used herein proceeded unabated. WAP-TGFbeta1 tumors that showed a strong expression at the WAP promoter, appeared later and grew more slowly than their wild-type counterparts. Transgenic females also had a lower incidence of HANs and plaques. Our study suggests that the epithelial target cells for tumorigenic mutations are probably progenitor cells that are not susceptible to the apoptotic effect of TGFbeta1. Alternatively, their daughters cells that display the secretory phenotype and could be more involved in the formation of premalignant lesions continue to die due to the expression of the transgene.

Effects of antiprogestins RU486 and ZK98299 on the expression of cell cycle proteins of a medroxyprogesterone acetate (MPA)-induced murine mammary tumor.

This study links the tumor inhibitory effect of anti-progestins RU486 and ZK98299 to the expression of cell cycle proteins. Medroxyprogesterone acetate-induced ductal mammary adenocarcinoma-bearing female BALB/c mice were treated daily either with RU486 or ZK98299, observing tumor growth retardation. p21 increased after 24 h treatment, peaked at day 4 and returned to control levels at day 7. Cyclin D1, cyclin E, CDK2 and CDK4, did not change during treatment. These results suggest that p21 might play a role in early inhibitory stages of tumor growth induced by RU486 and ZK98299.

Terapia fotodinámica en el tratamiento de tumores de piel inducidos quimicamente / Photodynamic therapy in the skin tumors tratment chemical induced

La terapia fotodinámica (TFD) es un tratamiento para tumores que utiliza luz y compuestos fotosensibles como porfirinas endógenas sintetizadas a partir del precursor ácido 5-Aminolevúlico (ALA). En el presente trabajo se estudió la acumulación de porfirinas luego de la aplicación de ALA tópico en diferentes vehículos y el grado de efectividad del tratamiento fotodinámico en tumores cutáneos inducidos químicamente en ratones SENCAR. Cada tipo de tumor (papilomas, queratoacantomas y carcinomas epidermoides) presentó una respuesta distinta al ALA-TFD. Sobre los carcinomas in situ e invasores, la acción de la TFD fue más evidente, observándose degeneración y desestructuración de los estratos más superficiales, hasta 1,5 mm, con cambios destructivos asociados a coagulación. Sobre la vascularización de la dermis se vieron fenómenos de congestión y hemorragia. Los efectos más encontrados fueron vacuolización y condensación de citoplasma, picnosis y desintegración nuclear. La acción sobre los papilomas en cambio, fue más limitada, debido a la escasa penetración del ALA y o la luz a través de la extensa capa de queratina. Sólo se encontraron respuestas talkes como concurrencia de mastocitos, infiltrados de leucocitos y esclerohialinizacion. Tambien se estudió la acumulación de porfirinas en los papilomas luego de la topicación de ALS en distintos vehículos sobre la superficie tumoral. La aplicación del ALA en loción fue la que mayor síntesis de porfirinas indujo (loción salina=2,31 mas menos 0,52 mi g/g. Con la aplicaciuón de una emulsión de aceite en agua, la acumulación de porfirinas fue de 1,30 mas menos 0,23 mi g/g y con una emulsión de agua en aceite, la concentración fue de 0,62 mas menos 0,12 mi g/g. Se concluye, que la TFD a partir de ALA es indicada para lesiones cutáneas con escasa queratinización, en particular para lesiones superficiales. El ALA topicado en loción sola o con etanol son los vejículos más efectivos para tratar estas lesiones

Terapia fotodinámica en el tratamiento de tumores de piel inducidos quimicamente / Photodynamic therapy in the skin tumors tratment chemical induced

La terapia fotodinámica (TFD) es un tratamiento para tumores que utiliza luz y compuestos fotosensibles como porfirinas endógenas sintetizadas a partir del precursor ácido 5-Aminolevúlico (ALA). En el presente trabajo se estudió la acumulación de porfirinas luego de la aplicación de ALA tópico en diferentes vehículos y el grado de efectividad del tratamiento fotodinámico en tumores cutáneos inducidos químicamente en ratones SENCAR. Cada tipo de tumor (papilomas, queratoacantomas y carcinomas epidermoides) presentó una respuesta distinta al ALA-TFD. Sobre los carcinomas in situ e invasores, la acción de la TFD fue más evidente, observándose degeneración y desestructuración de los estratos más superficiales, hasta 1,5 mm, con cambios destructivos asociados a coagulación. Sobre la vascularización de la dermis se vieron fenómenos de congestión y hemorragia. Los efectos más encontrados fueron vacuolización y condensación de citoplasma, picnosis y desintegración nuclear. La acción sobre los papilomas en cambio, fue más limitada, debido a la escasa penetración del ALA y o la luz a través de la extensa capa de queratina. Sólo se encontraron respuestas talkes como concurrencia de mastocitos, infiltrados de leucocitos y esclerohialinizacion. Tambien se estudió la acumulación de porfirinas en los papilomas luego de la topicación de ALS en distintos vehículos sobre la superficie tumoral. La aplicación del ALA en loción fue la que mayor síntesis de porfirinas indujo (loción salina=2,31 mas menos 0,52 mi g/g. Con la aplicaciuón de una emulsión de aceite en agua, la acumulación de porfirinas fue de 1,30 mas menos 0,23 mi g/g y con una emulsión de agua en aceite, la concentración fue de 0,62 mas menos 0,12 mi g/g. Se concluye, que la TFD a partir de ALA es indicada para lesiones cutáneas con escasa queratinización, en particular para lesiones superficiales. El ALA topicado en loción sola o con etanol son los vejículos más efectivos para tratar estas lesiones(AU)

Two different types of concomitant resistance induced by murine tumors: morphological aspects and intrinsic mechanisms.

Concomitant resistance (CR) is the phenomenon according to which a tumor-bearing host inhibits the growth of a secondary implant of the same tumor at a distant site. Confirming and extending previous results of our laboratory, histological studies have revealed that two temporally separate peaks of CR can be detected throughout tumor evolution. The first peak induced by immunogenic small tumors, in euthymic but not in nude mice, is associated with extensive necrosis of the secondary tumor implant and a profuse infiltration of polymorphonuclear granulocytes and mononuclear cells resulting in its final destruction; these features correspond to a typical immunological rejection. The second peak of CR induced by both immunogenic and non-immunogenic large tumors, in euthymic as well as in nude mice, is characterized by a dormant tumor stage with scarce or null mononuclear infiltration, associated with a significant reduction of tumor mitotic index and of the number of PCNA+ cells along with an increase in apoptosis and an arrest in S phase. In previous reports we suggested that a 1000 D serum fraction from mice bearing large tumors could be responsible for the induction of this dormant tumor stage. In this study tumor cells incubated in vitro with that serum factor mimicked the inhibition and cellular alterations observed in vivo in the secondary tumor inhibited by the second peak of CR. Moreover, the passive transfer of this factor by the intra-peritoneal (i.p.) route induced an in vivo inhibition of an i.p. tumor reproducing the image characteristic of the second peak of CR. This represents a direct proof that this serum factor can restrain tumor growth in vivo and that it is, most probably, the effector of the second peak of CR.

Inhibition of metastases by a serum factor associated to concomitant resistance induced by unrelated murine tumors.

Murine lung metatases growing undisturbed by the primary tumor were significantly inhibited by the concomitant resistance induced by a secondary subcutaneous implant of two unrelated tumors. Such inhibition was T-independent since it was also observed in nude mice; its full expression was dependent on the presence of the secondary tumor implant and it was exerted on both macroscopic and microscopic established metastases and not on the process of tumor cell dissemination from the primary tumor. Direct and indirect mechanisms seemed to be involved, the former affecting the metastatic cells per se by causing a decrease in proliferation and an increase in apoptosis while the latter affected neo-vascularization. These antitumor and antiangiogenic effects could be attributed to a serum factor induced by the unrelated tumors generating concomitant resistance. This factor proved to be heat, acid and alkaline resistant and dialysable; it was recovered in an HPLC column with maximum absorption at 215 and 266 nm; it was anionic at neutral pH, exhibiting free carboxil groups and one or more molecules of tyrosine, with a molecular weight between 870 and 1300 Dalton. Intravenous administration of this factor significantly inhibited lung metastases, decreasing mitosis and increasing apoptosis similar to that observed in the presence of the unrelated tumors.