LQFM030 reduced Ehrlich ascites tumor cell proliferation and VEGF levels.

AIMS: This study reports the biological properties of LQFM030 in vivo, a molecular simplification of the compound nutlin-1. MAIN METHODS: Ehrlich ascites tumor (EAT)-bearing mice were treated intraperitoneally with LQFM030 (50, 75 or 150mg/kg) for 10days to determine changes in ascites tumor volume, body weight, cytotoxicity and angiogenesis. Moreover, flow cytometric expression of p53 and p21 proteins and caspase-3/7, -8 and -9 activation were investigated in EAT cells from mice treated. Acute oral systemic toxicity potential of LQFM030 in mice was also investigated using an alternative method. KEY FINDINGS: Treatment of EAT-bearing mice with LQFM030 resulted in a marked decline in tumor cell proliferation and the vascular endothelial growth factor (VEGF) levels along with enhanced survival of the mice. Apoptotic tumor cell death was detected through p53 and p21 modulation and increase of caspase-3/7, -8 and -9 activity. LQFM030 also showed orally well tolerated, being classified in the UN GHS category 5 (LD50>2000-5000mg/Kg). SIGNIFICANCE: LQFM030 seems to be a promising antitumor candidate for combinatory therapy with typical cytotoxic compounds, reducing the toxicity burden while allowing a superior anticancer activity. Moreover, these data also open new perspectives for LQFM030 as an antiangiogenic agent for treatment of diseases involving VEGF overexpression.

Cooperation of p53 mutations with other oncogenic alterations in cancer.

Following the initial findings suggesting a pro-oncogenic role for p53 point mutants, more than 30 years of research have unveiled the critical role exerted by these mutants in human cancer. A growing body of evidence, including mouse models and clinical data, has clearly demonstrated a connection between mutant p53 and the development of aggressive and metastatic tumors. Even if the molecular mechanisms underlying mutant p53 activities are still the object of intense scrutiny, it seems evident that full activation of its oncogenic role requires the functional interaction with other oncogenic alterations. p53 point mutants, with their pleiotropic effects, simultaneously activating several mechanisms of aggressiveness, are engaged in multiple cross-talk with a variety of other cancer-related processes, thus depicting a complex molecular landscape for the mutant p53 network. In this chapter revealing evidence illustrating different ways through which this cooperation may be achieved will be discussed. Considering the proposed role for mutant p53 as a driver of cancer aggressiveness, disarming mutant p53 function by uncoupling the cooperation with other oncogenic alterations, stands out as an exciting possibility for the development of novel anti-cancer therapies.

Fibroblast growth factor 2 restrains Ras-driven proliferation of malignant cells by triggering RhoA-mediated senescence.

Fibroblast growth factor 2 (FGF2) is considered to be a bona fide oncogenic factor, although results from our group and others call this into question. Here, we report that exogenous recombinant FGF2 irreversibly inhibits proliferation by inducing senescence in Ras-dependent malignant mouse cells, but not in immortalized nontumorigenic cell lines. We report the following findings in K-Ras-dependent malignant Y1 adrenocortical cells and H-Ras V12-transformed BALB-3T3 fibroblasts: (a) FGF2 inhibits clonal growth and tumor onset in nude and immunocompetent BALB/c mice, (b) FGF2 irreversibly blocks the cell cycle, and (c) FGF2 induces the senescence-associated beta-galactosidase with no accompanying signs of apoptosis or necrosis. The tyrosine kinase inhibitor PD173074 completely protected malignant cells from FGF2. In Y1 adrenal cells, reducing the constitutively high levels of K-Ras-GTP using the dominant-negative RasN17 mutant made cells resistant to FGF2 cytotoxicity. In addition, transfection of the dominant-negative RhoA-N19 into either Y1 or 3T3-B61 malignant cell lines yielded stable clonal transfectants that were unable to activate RhoA and were resistant to the FGF2 stress response. We conclude that in Ras-dependent malignant cells, FGF2 interacts with its cognate receptors to trigger a senescence-like process involving RhoA-GTP. Surprisingly, attempts to select FGF2-resistant cells from the Y1 and 3T3-B61 cell lines yielded only rare clones that (a) had lost the overexpressed ras oncogene, (b) were dependent on FGF2 for proliferation, and (c) were poorly tumorigenic. Thus, FGF2 exerted a strong negative selection that Ras-dependent malignant cells could rarely overcome.

Preliminary results from a phase I/II study of perillyl alcohol intranasal administration in adults with recurrent malignant gliomas.

BACKGROUND: Activation of the p21-ras signaling pathway from aberrantly expressed receptors promotes the growth of malignant human astrocytomas. Perillyl alcohol has shown to have both chemopreventive and chemotherapeutic activities in preclinical studies. The underlying action mechanism(s) of POH has yet to be delineated but may involve effects on the TGF-beta and/or the Ras signaling pathways. The intranasal delivery allows drugs that do not cross the BBB to enter the CNS; moreover, it eliminates the need for systemic delivery, thereby reducing unwanted systemic side effects. METHODS: We are conducting a phase I/II study to evaluate the antitumoral activity of POH intranasal delivery in a 4x daily schedule in patients with recurrent MG. The objective was to determine PFS at 6 months and the safety for POH in adult patients who failed conventional treatment. Assessments were performed every 27 days. Thirty-seven patients with progressive disease after prior surgery, radiotherapy, and at least temozolomide-based chemotherapy were enrolled, 29 of whom had GBM, 5 who had anaplastic astrocytoma, and 3 had AO. RESULTS: One patient (3.4%) with GBM and 1 patient (33.3%) with AO achieved partial response; 13 patients (44.8%) with GBM, 3 patients (60%) with AA, and 1 (33.3%) with AO achieved stable disease; 15 (51.7%) patients with GBM, 2 (40%) patients with AA, and 1 (33.3%) with AO showed progressive disease. Progression-free survival (partial response and stable disease) was 48.2% for patients with GBM, 60% for patients with AA, and 66.6% for patients with AO. CONCLUSIONS: There were no toxicity events. Perillyl alcohol is well tolerated and regression of tumor size in some patients is suggestive of antitumor activity. This work discusses POH intranasal delivery as a potential adjuvant therapeutic strategy for patients with malignant gliomas.

H-RAS gene expression in human multinodular goiter.

The RAS protooncogene has an important, although not yet established role in thyroid neoplasia. In this study, we evaluated the H-RAS mRNA and protein levels in human samples of nontoxic and toxic multinodular goiter samples, according to serum TSH levels. The mean of H-RAS mRNA levels in nodules of nontoxic nodular goiter were significantly increased compared to nonnodular tissue (1.49+/-1.21 vs. 0.94+/-0.81 AU, P=0.016). Nine of the 18 specimens (50%) of nontoxic multinodular goiter exhibited increased levels of H-RAS mRNA. The increased H-RAS mRNA levels were paralleled by inRAcreased H-Ras protein levels in about 90% of the cases. Interestingly, no differences were observed in H-RAS expression between nodules and adjacent nonnodular tissue in toxic nodular goiters (0.58+/-0.27 vs. 0.58+/-0.20 AU, P=0.88). None of the 10 samples from toxic multinodular goiters exhibited overexpression of H-RAS. The H-RAS expression was positively correlated with thyroglobulin expression (r2=0.51; P=0.04). In conclusion, we demonstrated increased levels of H-RAS mRNA and protein in samples of nontoxic multinodular goiter, indicating that it might be involved in goiter pathogenesis. In contrast, H-RAS overexpression was not detected in any of the samples of toxic multinodular goiter, suggesting different mechanisms for cell proliferation in nodular goiter according to thyroid status.

Mutational analysis of K-ras and Ras protein expression in larynx squamous cell carcinoma.

The ras gene family (H, K and N-ras) encodes the Ras protein, a GTPase-activating protein that regulates several signal transduction pathways including cellular proliferation and differentiation. Mutations in codons 12, 13 and 61 of the ras genes constitute one of the most frequent alterations in human cancer. In the Western Hemisphere, a low frequency of mutations in these genes has been observed in head and neck carcinomas; a higher frequency has been found in countries such as India and Taiwan. Increased protein expression is a relatively frequent event in larynx carcinomas. This study was aimed to evaluate the participation of the k-ras gene and Ras expression in 20 Mexican patients with larynx squamous carcinoma, 2 with dysplasia and 4 with normal mucosa. Samples (of 26 patients) were embedded in paraffin and immunohistochemical analysis was performed for the Ras protein, as well as amplification of the k-ras gene exon 1 (108 bp) by laser capture microdissection. Then, DNA extraction, PCR and sequencing were performed looking for possible mutation in codons 12 and 13. All patients with larynx carcinoma were men, median age 62 years. Eighty-five percent of the patients had risk factors such as smoking and/or alcohol consumption, 25% were in clinical stages I and II, and 75% in stages III and IV; 45% of the patients presented tumor recurrence or persistence. In this study, no mutations were found in codons 12 or 13 of the k-ras gene; however, protein expression was observed in 95% of the samples and a higher expression of the protein was associated with tumor recurrence or persistence, although this was not statistically significant. Unexpectedly, well-differentiated carcinomas and dysplasias presented an increase in protein expression. These results suggest that ras may be involved in early stages of larynx carcinogenesis and may be activated by other mechanisms different from mutations, such as epigenetic events.

Ha-Ras sensitizes transformed mouse skin cells to Anisomycin-induced apoptosis.

Efforts have been made to develop a chemoprevention that selectively triggers apoptosis in malignant cancer cells. Here, we demonstrated that a mutated Ha-Ras activity is required in Anisomycin-induced apoptosis in transformed keratinocytes. Anisomycin stimulates JNK activity and apoptosis in oncogenic Ha-Ras positive cells, but not in normal keratinocytes. This effect was demonstrated in stably transfected cells with dominant negative Ha-Ras, that protected transformed cells, and oncogenic Ha-Ras that sensitized non-transformed cells to Anisomycin-induced apoptosis. Lastly, the treatment of cells with inhibitors of the JNK displayed resistance to Anisomycin induced apoptosis. These data suggests that the oncogenic Ha-Ras is important for Anisomycin-induced JNK activation and apoptosis in transformed keratinocytes.

Regresión de cáncer de mama después del tratamiento hormonal en un modelo murino / Mammary cancer regression after hormonal treatment in a murine model

Carcinomas mamarios murinos, originalmente inducidos por acetato de medroxiprogesterona, con crecimiento autónomo y receptores de estrógenos y progesterona regresionan con estradiol (E2) o con antiprogestágenos. Con el objeto de analizar los mecanismos de la regresión tumoral, estudiamos las características morfológicas y la participación de los reguladores del ciclo celular tales como p21, p27, p53 y MDM2 mediante inmunohistoquímica utilizando dos tumores sensibles al E2 o a los antiprogestágenos y uno sensible al E2 y antiprogestágenos resistente. La regresión se asocia a disminución del parénquima tumoral con aumento del estroma, a un efecto antiproliferativo y proapoptótico y a la inducción de proteínas inhibitorias del ciclo celular tales como p21 y p27. Sin embargo el patrón de expresión de los reguladores del ciclo celular varió. En los tumores sensibles a ambos tratamientos aumentó p21 y p27, los valores basales de p53 fueron altos y los de MDM2 bajos. En el tumor sensible sólo a E2 aumentó únicamente p27 y p21 permaneció en valores bajos acompañados por altos niveles basales de p53 y MDM2. Estos hallazgos sugieren que p21 podría ser esencial para la acción de los antiprogestágenos y que alteraciones en la vía p21/p53 podrían participar en la resistencia al tratamiento hormonal.

Regresión de cáncer de mama después del tratamiento hormonal en un modelo murino / Mammary cancer regression after hormonal treatment in a murine model

Carcinomas mamarios murinos, originalmente inducidos por acetato de medroxiprogesterona, con crecimiento autónomo y receptores de estrógenos y progesterona regresionan con estradiol (E2) o con antiprogestágenos. Con el objeto de analizar los mecanismos de la regresión tumoral, estudiamos las características morfológicas y la participación de los reguladores del ciclo celular tales como p21, p27, p53 y MDM2 mediante inmunohistoquímica utilizando dos tumores sensibles al E2 o a los antiprogestágenos y uno sensible al E2 y antiprogestágenos resistente. La regresión se asocia a disminución del parénquima tumoral con aumento del estroma, a un efecto antiproliferativo y proapoptótico y a la inducción de proteínas inhibitorias del ciclo celular tales como p21 y p27. Sin embargo el patrón de expresión de los reguladores del ciclo celular varió. En los tumores sensibles a ambos tratamientos aumentó p21 y p27, los valores basales de p53 fueron altos y los de MDM2 bajos. En el tumor sensible sólo a E2 aumentó únicamente p27 y p21 permaneció en valores bajos acompañados por altos niveles basales de p53 y MDM2. Estos hallazgos sugieren que p21 podría ser esencial para la acción de los antiprogestágenos y que alteraciones en la vía p21/p53 podrían participar en la resistencia al tratamiento hormonal. (AU)

Relevância clínica dos carcinomas de grande células pulmonares com relação às características neuroendócrinas, biomoleculares e estromais / Neuroendocrine and biologic features of primary tumors and tissue in pulmonary large cell carcinomas

Avaliou-se p21 e outros marcadores em espécimes cirúrgicos de 61 pacientes com carcinoma de grande células de pulmão. Utilizamos imunohistoquímica para avaliar a proteína p21 e a densidade de microvasos. A análise através do modelo multivariado de Cox mostrou que após o tratamento cirúrgico, o subtipo histológico foi significante com relação a sobrevida (p=0.02), mas a quantificação do tumor para o p21 e a densidade de microvasos adicionaram importante informação ao estudo do prognóstico e foram fatores mais fortemente relacionados com a sobrevida do que o subtipo histológico (P=0.00).We examined p21waf1/cip1 and other markers in tissue from 61 patients with surgically excised large cell carcinoma. We used immunohistochemistry and morphometry to evaluate the amount of tumor staining for p21waf1/cip1 and microvessel density. The study outcome was survival time until death from recurrent lung cancer. Multivariate Cox model analysis demonstrated that after surgical excision control, histologic subtypes were significantly related to survival time (P=0.02), but quantitative staining of the tumor for p21waf1/cip1 and microvessel density added prognostic information and were more strongly prognostic than histologic subtype (P=0.00)...

Importância de fatores celulares em associaçäo ao índice de prognóstico internacional na resposta ao tratamento inicial e na evoluçäo clínica de pacientes com linfoma difuso de grandes células B / Importance of cellular factors associated with international prognostic index on treatment response and clinical outcome in difuse large B-cell non-Hodgkin's lymphoma

Objetivos: O presente estudo teve os seguintes objetivos: 1) avaliar a aplicabilidade do índice de Prognóstico Internacional em uma população de pacientes com LDGCB; 2) correlacionar a expressão das proteínas P53, P21 e Mdm2 com a resposta ao tratamento inicial e evolução clínica de pacientes com LDGCB; 3) determinar a importância da expressão das proteínas Bcl-2 e Bcl-6 na resposta ao tratamento inicial e evolução clínica de pacientes com LDGCB; 4) detectar a presença de mutação nos exons 5 a 9 do gene p53 e correlacioná-la à resposta ao tratamento inicial e evolução clínica de pacientes com LDGCB; 5) detectar a presença do rearranjo bcl-2/IgH e correlacioná-la à resposta ao tratamento inicial e evolução clínica de pacientes com LDGCB. Métodos: Foram analisados, retrospectivamente, os 51 pacientes tratados no ambulatório de LNH do Hospital São Paulo (UNIFESP/EPM) com diagnóstico de LDGCB, no período de janeiro de 1990 a janeiro de 2002, e que apresentavam informações clínicas e blocos de parafina disponíveis para análise imunohistoquímica e molecular. A análise da expressão das proteínas P53, P21, Mdm2, Bcl-2 e Bcl-6 foi realizada por técnicas de imunohistoquímica. A pesquisa de mutação no gene p53 foi avaliada pelo método de PCR-SSCP, seguido de seqüenciamento automatizado das amostras contendo desvio de mobilidade (shift). A pesquisa do rearranjo bcl-2/IgH foi realizada pelo método de PCR para a detecção do rearranjo na região MBR do gene bcl-2. Resultados: O IPI pôde ser aplicado a esta população de pacientes com LDGCB, confimando seu valor como marcador prognóstico na sobrevida geral (p = 0,029) e como fator preditivo de resposta à terapia inicial (p= 0,003). A expressão das proteínas P53 e P21 se correlacionou com a sobrevida geral, havendo melhor evolução para o grupo de pacientes com expressão de P53 >=10 por cento e P21 >=10 por cento e pior evolução para P53 >=10 por cento e P21 <10 por cento (p= 0,031). A expressão de Mdm2 apresentou tendência à associação com a expressão de P53...(au)

RalA mediates v-Src, v-Ras, and v-Raf regulation of CD44 and fibronectin expression in NIH3T3 fibroblasts.

Oncogenic transformation of fibroblasts by v-Src and v-Ras is often associated with downregulation of fibronectin (FN) and increased expression of CD44, a receptor for hyaluronan. Both v-Src and v-Ras as well as v-Raf activate phospholipase D through the small GTPase, RalA, an important mediator of transformation and tumorigenesis in vivo. We have therefore investigated whether RalA is involved in the downregulation of FN and overproduction of CD44 upon oncogenic transformation. We report here that compared to untransfected cells NIH3T3 cells transformed by v-Src, v-Ras, or v-Raf have reduced levels of FN and increased levels of CD44. Moreover, the ability to form extracellular FN fibrils was significantly reduced in the oncogene-transformed cells compared to parental controls. Coexpression of the dominant negative S28N-RalA mutant restored the levels of CD44 and FN and the capacity of v-Src-, v-Ras-, and v-Raf-expressing cells to form extracellular FN fibrils, to those observed in NIH3T3 cells. The data presented here show a novel regulatory role for RalA, which is required for tumor formation in transformed NIH3T3 cells, in mediating the signal transduction pathway activated by v-Src, v-Ras, and v-Raf, that leads to FN downregulation and CD44 overexpression.

Inhibition of ras oncogene: a novel approach to antineoplastic therapy.

The most frequently detected oncogene alterations, both in animal and human cancers, are the mutations in the ras oncogene family. These oncogenes are mutated or overexpressed in many human tumors, with a high incidence in tumors of the pancreas, thyroid, colon, lung and certain types of leukemia. Ras is a small guanine nucleotide binding protein that transduces biological information from the cell surface to cytoplasmic components within cells. The signal is transduced to the cell nucleus through second messengers, and it ultimately induces cell division. Oncogenic forms of p21(ras) lead to unregulated, sustained signaling through downstream effectors. The ras family of oncogenes is involved in the development of both primary tumors and metastases making it a good therapeutic target. Several therapeutic approaches to cancer have been developed pointing to reducing the altered gene product or to eliminating its biological function: (1) gene therapy with ribozymes, which are able to break down specific RNA sequences, or with antisense oligonucleotides, (2) immunotherapy through passive or active immunization protocols, and (3) inhibition of p21(ras) farnesylation either by inhibition of farnesyl transferase or synthesis inhibition of farnesyl moieties.

Carcinoma de la vesícula biliar: expresión de productos de los oncogenes C-MYC y RAS-P-21 / Carcinoma of gallblader: expression of products of C-MYC and RAS-P-21 oncogene

The expression of c-myc and p-ras-21 oncogenes was studied using an immunohistochemical method with monoclonal antibodies in 126 samples of gallbladder carcinoma (103 primary tumors and 23 metastatic lesions). Twenty five gallbladder samples without tumor evidence were used as controls. C-myc oncoprotein was positive in one control sample and p-ras-21 oncoprotein was negative in all. Among primary carcinomas c-myc was positive in 9 (9 per cent) and 4 (4 per cent); among metastatic carcinomas c-myc was positive in 6 (26 per cent, p=0.03 vs primary carcinoma) and p-ras-21 in 11 (48 per cent, p <0.001 vs primary carcinoma). There was a non significant association between c-myc and p-ras-21 expression and degree of histological differentiation and levelñ of tumoral infiltration. It is concliuded that c-myc and p-ras-21 oncoprotein expression is observed in less than 10 per cent of primary gallbladder carcinomas and that this expression is significantly higher in metastatic cells

Image analysis of Feulgen-stained transformed NIH 3T3 cells differing in p21 expression and ras-induced metastatic ability.

Nuclear phenotypes of clonal and polyclonal T24 H-ras-transformed NIH 3T3 cells differing in p21 expression and experimental metastatic ability were studied in Feulgen-stained preparations by image analysis. The objective was to determine if these cells varied in their degree of chromatin condensation, as previously reported with cell transformation, or in any other chromatin texture property highlighted by image analysis parameters. The majority of nuclei in all of these ras-transformed cells exhibited increased levels of chromatin condensation, independent of ras levels or metastatic properties. This chromatin texture characteristic was assumed to be related to the transformed phenotype. No significant changes in chromatin supraorganization that could be correlated directly with ras levels or metastatic ability were found, with the exception of an increased frequency of a relatively rare phenotype in highly metastatic cells. This phenotype was characterized by an extreme contrast in packing state between condensed and noncondensed chromatin. It is suggested that ras transformation results in alterations in chromatin structure but that there is not a simple relationship between ras p21 expression levels or ras-induced metastatic ability and the degree of these changes.

Free L-phosphotyrosine activates human platelets: molecular evidence for a new signal transducer.

Human platelets, either purified or plasma-enriched, are activated when exposed to free L-phosphotyrosine. Physical aggregation is similar to that induced by collagen, although with distinctive biochemical features. Among these, a mobility shift of a GTP-binding protein specifically recognized by an anti-Ki-v-ras monoclonal antibody and an altered pattern of low molecular weight phosphorylated polypeptides are the most outstanding features. Since free phosphotyrosine is detected in platelets extracts, its role as a new signal transducer as well as its putative modulating action over protein phosphorylation are discussed.

Glucocorticoid dexamethasone reversibly complements EJ-RAS oncogene to transform mouse embryo BALB-3T3 cells.

EJ-A is a Balb-3T3 transfectant cell line that bears a small number of EJ-ras oncogene copies/cell, has low EJ-ras expression, and resembles the parental cell line in displaying a non-transformed phenotype. The glucocorticoid hormone dexamethasone reversibly induces transformation traits in EJ-A cells, namely: 1) morphological transformation; 2) increased growth rate and saturation density; 3) reduced G1 length; and 4) independence of the FGF requirement to initiate DNA synthesis. Western blot analysis revealed that dexamethasone does not increase the p21ras protein intracellular level. beta-IFN, added to the culture medium, does not suppress the dexamethasone-induced growth stimulation and morphological transformation. Therefore, glucocorticoid hormones can complement low EJ-ras expression to transform Balb-3T3 cells, by a mechanism that is likely to be independent of p21ras increase and beta-IFN decrease.