RESUMEN

The aim of this work was to systematically obtain quantitative imaging parameters with static and dynamic contrast-enhanced (CE) X-ray imaging techniques and to evaluate their correlation with histological biomarkers of angiogenesis in a subcutaneous C6 glioma model. Enhancement (E), iodine concentration (CI), and relative blood volume (rBV) were quantified from single- and dual-energy (SE and DE, respectively) micro-computed tomography (micro-CT) images, while rBV and volume transfer constant (Ktrans) were quantified from dynamic contrast-enhanced (DCE) planar images. CI and rBV allowed a better discernment of tumor regions from muscle than E in SE and DE images, while no significant differences were found for rBV and Ktrans in DCE images. An agreement was found in rBV for muscle quantified with the different imaging protocols, and in CI and E quantified with SE and DE protocols. Significant strong correlations (Pearson r > 0.7, p < 0.05) were found between a set of imaging parameters in SE images and histological biomarkers: E and CI in tumor periphery were associated with microvessel density (MVD) and necrosis, E and CI in the complete tumor with MVD, and rBV in the tumor periphery with MVD. In conclusion, quantitative imaging parameters obtained in SE micro-CT images could be used to characterize angiogenesis and necrosis in the subcutaneous C6 glioma model.

RESUMEN

CD8+ T cell-mediated immune response plays a major role in the clearance of virus-infected cells, including human papillomavirus (HPV). The effective treatment of women with normal cytology but persistent high risk-HPV infection or with low-grade intraepithelial lesions could take advantage of novel strategies based on vaccination with viral immunological targets with a wide spectrum of cross-protection. The helicase E1, expressed early during viral replication in HPV infection, is among the most conserved papillomavirus proteins, which makes it a good vaccine candidate. In the present study, we examined E1-specific CD8+ T cell and NK immune responses in a mouse model with α-galactosylceramide (α-GalCer) as an adjuvant. We found that mice immunized with E1 combined with α-GalCer elicited an E1-specific CD8+ T and NK cell cytotoxic responses, which correlated with growth inhibition of grafted melanoma B16-F0 cells expressing E1, both in prophylactic and therapeutic protocols.

Asunto(s)

Vacunas contra el Cáncer/inmunología , Citotoxicidad Inmunológica , Galactosilceramidas/administración & dosificación , Proteínas Oncogénicas Virales/inmunología , Linfocitos T Citotóxicos/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Femenino , Galactosilceramidas/inmunología , Papillomavirus Humano 18 , Humanos , Células Asesinas Naturales/inmunología , Melanoma Experimental/prevención & control , Melanoma Experimental/terapia , Melanoma Experimental/virología , Ratones , Ratones Endogámicos C57BL , Proteínas Oncogénicas Virales/administración & dosificación , Infecciones por Papillomavirus/inmunología , Infecciones por Papillomavirus/prevención & control , Infecciones por Papillomavirus/terapia , Trasplantes , Células Tumorales Cultivadas/inmunología , Vacunación

RESUMEN

INTRODUCTION: Metastasis involves the accumulation of genetic and epigenetic alterations leading to activation of prometastatic genes and inactivation of antimetastatic genes. Among epigenetic alterations, DNA hypermethylation and histone hypoacetylation are the focus of intense translational research because their pharmacological inhibition has been shown to produce antineoplastic activity in a variety of experimental models. AIMS: This study aimed to evaluate the antimetastatic effect of the DNA-methylation inhibitor, hydralazine, and the histone deacetylase inhibitor, valproic acid. METHODS: NIH 3T3-Ras murine cells were treated with hydralazine and valproic acid to evaluate their effects upon cell proliferation, cell motility, chemotaxis, gelatinase activity, and gene expression. Lung metastases were developed by intravenous injection of NIH 3T3-Ras cells in BALB/c nu/nu mice and then treated with the drug combination. RESULTS: Treatment induced a growth-inhibitory effect on NIH 3T3-Ras cells, showed a trend toward increased gelatinase activity of MMP2 and MMP9, and inhibited chemotaxis and cell motility. The combination led to a strong antimetastatic effect in lungs of nude mice. CONCLUSION: Hydralazine and valproic acid, two repositioned drugs as epigenetic agents, exhibit antimetastatic effects in vitro and in vivo and hold potential for cancer treatment.

RESUMEN

INTRODUCTION: Cancer cells have increased glycolysis and glutaminolysis. Their third feature is increased de novo lipogenesis. As such, fatty acid (FA) synthesis enzymes are over-expressed in cancer and their depletion causes antitumor effects. As fatty acid synthase (FASN) plays a pivotal role in this process, it is an attractive target for cancer therapy. AREAS COVERED: This is a review of the lipogenic phenotype of cancer and how this phenomenon can be exploited for cancer therapy using inhibitors of FASN, with particular emphasis on orlistat as a repurposing drug. EXPERT OPINION: Disease stabilization only has been observed with a highly selective FASN inhibitor used as a single agent in clinical trials. It is too early to say whether the absence of tumor responses other than stabilization results because even full inhibition of FASN is not enough to elicit antitumor responses. The FASN inhibitor orlistat is a 'dirty' drug with target-off actions upon at least seven targets with a proven role in tumor biology. The development of orlistat formulations suited for its intravenous administration is a step ahead to shed light on the concept that drug promiscuity can or not be a virtue.

Asunto(s)

Acido Graso Sintasa Tipo I/antagonistas & inhibidores , Lactonas/uso terapéutico , Neoplasias/tratamiento farmacológico , Administración Intravenosa , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Diseño de Fármacos , Reposicionamiento de Medicamentos , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Humanos , Lactonas/administración & dosificación , Lactonas/farmacología , Terapia Molecular Dirigida , Neoplasias/enzimología , Neoplasias/patología , Orlistat

RESUMEN

The aim of the present study was to investigate in vivo the feasibility and efficacy of the combination of lonidamine (LND), 6-diazo-5-oxo-L-norleucine (DON) and orlistat to simultaneously target glycolysis, glutaminolysis and de novo synthesis of fatty acids, respectively. The doses of LND and DON used in humans were translated to mouse doses (77.7 mg/kg and 145.5 mg/kg, respectively) and orlistat was used at 240 mg/kg. Three schedules of LND, DON and orlistat at different doses were administered by intraperitoneal injection to BALB/c mice in a 21-day cycle (schedule 1: LND, 0.5 mg/day; DON, 0.25 mg/day 1, 5 and 9; orlistat, 240 mg/kg/day; schedule 2: LND, 0.1 mg/day; DON, 0.5 mg/day 1, 5 and 9; orlistat, 240 mg/kg/day; schedule 3: LND, 0.5 mg/day; DON, 0.08 mg/day 1, 5 and 9; orlistat, 360 mg/kg/day) to assess tolerability. To determine the antitumor efficacy, a syngeneic tumor model in BALB/c mice was created using colon cancer CT26.WT cells, and a xenogeneic tumor model was created in nude mice using the human colon cancer SW480 cell line. Mice were treated with schedule 1. Animals were weighed, clinically inspected during the experiment and the tumor volume was measured at day 21. The 3 schedules assessed in the tolerability experiments were well tolerated, as mice maintained their weight and no evident clinical signs of toxicity were observed. Combination treatment with schedule 1 significantly decreased tumor growth in each mouse model. No evident signs of toxicity were observed and mice maintained their weight during treatment. The triple metabolic blockade of the malignant phenotype appears feasible and promising for cancer therapy.

RESUMEN

INTRODUCTION: Natural Killer cells (NK) are key for the innate immune response against tumors and viral infections. Several viral proteins evade host immune response and target the NK cell receptor NKG2D and its ligands. Areas covered: This review aimed to describe the viruses and their proteins that interfere with the NKG2D receptor and their ligands, and how these interactions lead to immune evasion, host protection, and tissue damage from acute and chronic viral infections. Expert opinion: The study of viral proteins has already impacted the field of oncology. A prime example is the HBV vaccine and the development of antiviral drugs for HIV, Hepatitis C, and the family of Herpesviridae viruses. The NKG2D system seems to be a rational therapeutic target. Nevertheless, an effective cytotoxic response by NK cells is mediated by a network of activating and inhibitory receptors, the integration of which determines if the NK cell becomes cytotoxic or permissive. Immunotherapeutic agents that increase the antitumor lytic activity of NK cells through modulating activation and inhibitory signaling of NK cells are being developed. Nevertheless, more research is needed to dissect the integrative mechanism of NK cells function to fully exploit their antitumor and antiviral effector mechanisms.

Asunto(s)

Inmunoterapia/métodos , Células Asesinas Naturales/metabolismo , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Antivirales/farmacología , Humanos , Inmunidad Innata/inmunología , Células Asesinas Naturales/inmunología , Ligandos , Subfamilia K de Receptores Similares a Lectina de Células NK/inmunología , Neoplasias/inmunología , Neoplasias/terapia , Neoplasias/virología , Transducción de Señal/inmunología , Virosis/complicaciones , Virosis/inmunología , Virosis/terapia

RESUMEN

Background Cell-free DNA circulates in cancer patients and induces in vivo cell transformation and cancer progression in susceptible cells. Based on this, we hypothesized that depletion of circulating DNA with DNAse I and a protease mix could have antitumor effects. Study design The study aimed to demonstrate that DNAse I and a protease mix can degrade in vitro DNA and proteins from the serum of healthy individuals and cancer patients, and in vivo in serum of Wistar rats,. Moreover, the antitumor effect of the systemically administered enzyme mix treatmentwas evaluated in nude mice subcutaneously grafted with the human colon cancer cell line SW480. Results The serum DNA of cancer patients or healthy individuals was almost completely degraded in vitro by the enzymatic treatment, but no degradation was found with the enzymes given separately. The intravenous administration of the enzymes led to significant decreases in DNA and proteins from rat serum. No antitumor effect was observed in immunodeficient mice treated with the enzymes given separately. In contrast, the animals that received both enzymes exhibited a marked growth inhibition of tumors, 40% of them having pathological complete response. Conclusion This study demonstrated that systemic treatment with DNAse I and a protease mix in rats decreases DNA and proteins from serum and that this treatment has antitumor effects. Our results support the hypothesis that circulating DNA could have a role in tumor progression, which can be offset by depleting it. Further studies are needed to prove this concept.

Asunto(s)

Desoxirribonucleasa I/farmacología , Péptido Hidrolasas/farmacología , Adulto , Animales , Neoplasias de la Mama/sangre , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Neoplasias del Colon/sangre , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , ADN/sangre , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Proteínas/metabolismo , Ratas , Ratas Wistar

RESUMEN

The aim of the present study was to demonstrate that ribavirin, a known inhibitor of eIF4E and inosine 5'-phosphate dehydrogenase (IMPDH), also inhibits histone methyltransferase zeste homolog 2 (EZH2). A computational searching revealed that ribavirin has a high structural similarity to 3-deazaneplanocin A (DZNep). The growth inhibitory effects of ribavirin as well as its effects upon epigenetic enzymes were evaluated in various cancer cell lines. siRNA assays were used to downregulate eIF4E, EZH2 and IMPDH to determine the contribution of these targets to the growth inhibitory effects of ribavirin. Ribavirin decreased EZH2 expression, inhibited histone methyltransferase activity and decreased H3K27 trimethylation. Ribavirin induced variable growth inhibition in a number of cell lines and downregulation of the targets, EZH2, eIF4E and IMPDH1 and 2 by siRNA led to comparable growth inhibition while no significant further reduction in viability was observed when siRNA transfected cells were treated with ribavirin. The results showed that ribavirin inhibits these cancer targets and should thus be studied for cancer therapy.

Asunto(s)

Antineoplásicos/farmacología , Antivirales/farmacología , Proliferación Celular/efectos de los fármacos , Factor 4E Eucariótico de Iniciación/efectos de los fármacos , IMP Deshidrogenasa/efectos de los fármacos , Neoplasias/genética , Complejo Represivo Polycomb 2/efectos de los fármacos , Ribavirina/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Simulación por Computador , Reposicionamiento de Medicamentos , Proteína Potenciadora del Homólogo Zeste 2 , Factor 4E Eucariótico de Iniciación/genética , Células HeLa , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/efectos de los fármacos , Humanos , IMP Deshidrogenasa/genética , Células MCF-7 , Neoplasias/metabolismo , Complejo Represivo Polycomb 2/genética , ARN Interferente Pequeño

RESUMEN

BACKGROUND: Down regulation of genes coding for nucleoside transporters and drug metabolism responsible for uptake and metabolic activation of the nucleoside gemcitabine is related with acquired tumor resistance against this agent. Hydralazine has been shown to reverse doxorubicin resistance in a model of breast cancer. Here we wanted to investigate whether epigenetic mechanisms are responsible for acquiring resistance to gemcitabine and if hydralazine could restore gemcitabine sensitivity in cervical cancer cells. METHODOLOGY/PRINCIPAL FINDINGS: The cervical cancer cell line CaLo cell line was cultured in the presence of increasing concentrations of gemcitabine. Down-regulation of hENT1 & dCK genes was observed in the resistant cells (CaLoGR) which was not associated with promoter methylation. Treatment with hydralazine reversed gemcitabine resistance and led to hENT1 and dCK gene reactivation in a DNA promoter methylation-independent manner. No changes in HDAC total activity nor in H3 and H4 acetylation at these promoters were observed. ChIP analysis showed H3K9m2 at hENT1 and dCK gene promoters which correlated with hyper-expression of G9A histone methyltransferase at RNA and protein level in the resistant cells. Hydralazine inhibited G9A methyltransferase activity in vitro and depletion of the G9A gene by iRNA restored gemcitabine sensitivity. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that acquired gemcitabine resistance is associated with DNA promoter methylation-independent hENT1 and dCK gene down-regulation and hyper-expression of G9A methyltransferase. Hydralazine reverts gemcitabine resistance in cervical cancer cells via inhibition of G9A histone methyltransferase.

Asunto(s)

Antimetabolitos Antineoplásicos/metabolismo , Desoxicitidina/análogos & derivados , Resistencia a Antineoplásicos/efectos de los fármacos , Epigénesis Genética/fisiología , Regulación Neoplásica de la Expresión Génica/fisiología , Hidralazina/farmacología , Neoplasias del Cuello Uterino/tratamiento farmacológico , Antimetabolitos Antineoplásicos/uso terapéutico , Western Blotting , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Cartilla de ADN/genética , Desoxicitidina/metabolismo , Desoxicitidina/uso terapéutico , Tranportador Equilibrativo 1 de Nucleósido/metabolismo , Femenino , Antígenos de Histocompatibilidad , Histona Desacetilasas/metabolismo , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Gemcitabina

RESUMEN

It is known that cancer progresses by vertical gene transfer, but this paradigm ignores that DNA circulates in higher organisms and that it is biologically active upon its uptake by recipient cells. Here we confirm previous observations on the ability of cell-free DNA to induce in vitro cell transformation and tumorigenesis by treating NIH3T3 recipient murine cells with serum of colon cancer patients and supernatant of SW480 human cancer cells. Cell transformation and tumorigenesis of recipient cells did not occur if serum and supernatants were depleted of DNA. It is also demonstrated that horizontal cancer progression mediated by circulating DNA occurs via its uptake by recipient cells in an in vivo model where immunocompetent rats subjected to colon carcinogenesis with 1,2-dimethylhydrazine had increased rate of colonic tumors when injected in the dorsum with human SW480 colon carcinoma cells as a source of circulating oncogenic DNA, which could be offset by treating these animals with DNAse I and proteases. Though the contribution of biologically active molecules other than DNA for this phenomenon to occur cannot be ruled out, our results support the fact that cancer cells emit into the circulation biologically active DNA to foster tumor progression. Further exploration of the horizontal tumor progression phenomenon mediated by circulating DNA is clearly needed to determine whether its manipulation could have a role in cancer therapy.

Asunto(s)

Transformación Celular Neoplásica/genética , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Transferencia de Gen Horizontal , Animales , Secuencia de Bases , Línea Celular Tumoral , Medios de Cultivo Condicionados/química , Modelos Animales de Enfermedad , Femenino , Dosificación de Gen , Genes ras , Humanos , Ratones , Células 3T3 NIH , Ratas , Proteínas de Unión al GTP rab/química , Proteínas de Unión al GTP rab/genética

RESUMEN

The reversing of epigenetic aberrations using the inhibitors of DNA methylation and histone deacetylases may have therapeutic value in cervical cancer. This is a randomized phase III, placebo-controlled study of hydralazine and valproate (HV) added to cisplatin topotecan in advanced cervical cancer. Patients received hydralazine at 182 mg for rapid, or 83 mg for slow acetylators, and valproate at 30 mg/kg, beginning a week before chemotherapy and continued until disease progression. Response, toxicity, and PFS were evaluated, and 36 patients (17 CT + HV and 19 CT + PLA) were included. The median number of cycles was 6. There were four PRs to CT + HV and one in CT + PLA. Stable disease in five (29%) and six (32%) patients, respectively, whereas eight (47%) and 12 (63%) showed progression (P = 0.27). At a median follow-up time of 7 months (1-22), the median PFS is 6 months for CT + PLA and 10 months for CT + HV (P = 0.0384, two tailed). Although preliminary, this study represents the first randomized clinical trial to demonstrate a significant advantage in progression-free survival for epigenetic therapy over one of the current standard combination chemotherapy in cervical cancer. Molecular correlates with response and survival from this trial are pending to analyze.

Asunto(s)

Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Epigénesis Genética , Terapia Genética/métodos , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/terapia , Adulto , Anciano , Terapia Combinada/métodos , Método Doble Ciego , Femenino , Estudios de Seguimiento , Humanos , Hidralazina/administración & dosificación , Persona de Mediana Edad , Tasa de Supervivencia , Neoplasias del Cuello Uterino/mortalidad , Ácido Valproico/administración & dosificación

RESUMEN

Decitabine and azacitidine, two DNA methyltransferase (DNMT) inhibitors, are the current standard of treatment for myelodysplastic syndrome (MDS). Histone deacetylase (HDAC) inhibitors are also being tested against MDS. Both drug classes synergize in their gene reactivating and anticancer activities. The combination of hydralazine and valproate (Transkrip®), a DNMT and HDAC inhibitor, respectively), has been developed as epigenetic therapy under the drug repositioning concept. To evaluate the clinical efficacy and safety of hydralazine and valproate against MDS, an open phase-II study for previously treated patients with MDS was conducted. The hydralazine dose was given according with the acetylator phenotype, and valproate was dosed at 30 mg/kg/day. Response was graded with International Working Group criteria. Toxicity was evaluated by the Common Toxemia Criteria-National Cancer Institute version 3 scale. From November 2007 to January 2010, 12 patients were included. Median age±SD was 53±19.78 years (range, 23-79 years); median time from diagnosis to inclusion in the study was 7.9 months (range 2.6-36.1 months). Median of previous treatment was 2 (range, 1-6). Refractory cytopenia with multilineage dysplasia was diagnosed in ten cases, and refractory anemia with excess of blasts in two. Overall response was documented in six (50%) of 12 cases, including one CR, one PR, and four hematological improvements of the erythroid series. Two patients (16.6%) progressed to acute myeloid leukemia. Hemoglobin increased from 7.4 to 10.3 g/dL (in 13 weeks), neutrophils, from 1.1 to 2.0 (in 3 weeks), and platelets, from 66×10(9) to 72×10(9)/L (in 2 weeks). Transfusional requirements decreased from 2.3 to 0 U bi-monthly for red blood cells and from 0.5 to 0 U bi-monthly for platelets in responding patients. Main toxicities were mild, including somnolence and nausea. Preliminary results of this phase-II study suggest that the combination of hydralazine and valproate is a promising non-toxic and effective therapy for MDS.

Asunto(s)

Epigenómica , Hidralazina/uso terapéutico , Síndromes Mielodisplásicos/tratamiento farmacológico , Síndromes Mielodisplásicos/genética , Ácido Valproico/uso terapéutico , Adulto , Anciano , Azacitidina/análogos & derivados , Azacitidina/uso terapéutico , Análisis Citogenético , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , Metilasas de Modificación del ADN/antagonistas & inhibidores , Decitabina , Femenino , Inhibidores de Histona Desacetilasas/sangre , Inhibidores de Histona Desacetilasas/uso terapéutico , Humanos , Hidralazina/sangre , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/sangre , Resultado del Tratamiento , Ácido Valproico/sangre , Adulto Joven

RESUMEN

Aberrant DNA methylation and histone deacetylation participate in cancer development and progression; hence, their reversal by inhibitors of DNA methylation and histone deacetylases is a promising cancer therapy. Experimental data demonstrate that these inhibitors in combination do not only show synergy in antitumor effects but also in whole genome global expression. Ten pairs of pre- and post-treatment cervical tumor samples were analyzed by microarray analysis. Treatment for seven days with hydralazine and valproate (HV) in patients up-regulated 964 genes. The two pathways possessing the highest number of up-regulated genes comprised the ribosome protein and the oxidative phosphorylation pathways, followed by MAPK signaling, tight junction, adherens junction, actin cytoskeleton, cell cycle, focal adhesion, apoptosis, proteasome, Wnt signaling, and antigen processing and presentation pathways. Up-regulated genes by HV, clustered with down-regulated genes in untreated primary cervical carcinomas and were more alike as compared with up-regulated genes from untreated patients in terms of gene ontology. Increased acetylated p53 was also observed. Epigenetic therapy with HV leads to gene reactivation in primary tumors of cervical cancer patients as well as protein acetylation. A number of these reactivated genes have a definitive role as a tumor suppressors. The global expression pattern induced by HV suggests this therapy has an impact on pathways related to energy production which may promote apoptosis.

Asunto(s)

Carcinoma/genética , Epigénesis Genética/efectos de los fármacos , Hidralazina/farmacología , Transcripción Genética/efectos de los fármacos , Neoplasias del Cuello Uterino/genética , Ácido Valproico/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma/tratamiento farmacológico , Carcinoma/patología , Ensayos Clínicos como Asunto , Metilación de ADN/efectos de los fármacos , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas/administración & dosificación , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Hidralazina/administración & dosificación , Análisis por Micromatrices , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/patología , Ácido Valproico/administración & dosificación

RESUMEN

BACKGROUND: Germ-line mutations of the TP53 gene are known to cause Li-Fraumeni syndrome, an autosomal, dominantly inherited, high-penetrance cancer-predisposition syndrome characterized by the occurrence of a variety of cancers, mainly soft tissue sarcomas, adrenocortical carcinoma, leukemia, breast cancer, and brain tumors. METHODS: Mutation analysis was based on Denaturing high performance liquid chromatography (DHPLC) screening of exons 2-11 of the TP53 gene, sequencing, and cloning of DNA obtained from peripheral blood lymphocytes. RESULTS: We report herein on Li Fraumeni syndrome in a family whose members are carriers of a novel TP53 gene mutation at exon 4. The mutation comprises an insertion/duplication of seven nucleotides affecting codon 110 and generating a new nucleotide sequence and a premature stop codon at position 150. With this mutation, the p53 protein that should be translated lacks the majority of the DNA binding domain. CONCLUSION: To our knowledge, this specific alteration has not been reported previously, but we believe it is the cause of the Li-Fraumeni syndrome in this family.

Asunto(s)

Mutación de Línea Germinal/genética , Síndrome de Li-Fraumeni/genética , Proteína p53 Supresora de Tumor/genética , Adulto , Anciano , Cartilla de ADN/química , Cartilla de ADN/genética , Femenino , Humanos , Masculino , México , Persona de Mediana Edad , Linaje , Adulto Joven

RESUMEN

BACKGROUND: We explore the use of a clinical orthovoltage X-ray treatment unit as a small-animal radiation therapy system in a tumoral model of cervical cancer. METHODS: Nude mice were subcutaneously inoculated with 5 x 106 HeLa cells in both lower limbs. When tumor volume approximated 200 mm3 treatment was initiated. Animals received four 2 mg/kg intraperitoneal cycles (1/week) of cisplatin and/or 6.25 mg/kg of gemcitabine, concomitant with radiotherapy. Tumors were exposed to 2.5 Gy/day nominal surface doses (20 days) of 150 kV X-rays. Lead collimators with circular apertures (0.5 to 1.5 cm diameter) were manufactured and mounted on the applicator cone to restrict the X-ray beam onto tumors. X-ray penetration and conformality were evaluated by measuring dose at the surface and behind the tumor lobe by using HS GafChromic film. Relative changes in tumor volume (RTV) and a clonogenic assay were used to evaluate the therapeutic response of the tumor, and relative weight loss was used to assess toxicity of the treatments. RESULTS: No measurable dose was delivered outside of the collimator apertures. The analysis suggests that dose inhomogeneities in the tumor reach up to +/- 11.5% around the mean tumor dose value, which was estimated as 2.2 Gy/day. Evaluation of the RTV showed a significant reduction of the tumor volume as consequence of the chemoradiotherapy treatment; results also show that toxicity was well tolerated by the animals. CONCLUSION: Results and procedures described in the present work have shown the usefulness and convenience of the orthovoltage X-ray system for animal model radiotherapy protocols.

Asunto(s)

Neoplasias del Cuello Uterino/radioterapia , Animales , Modelos Animales de Enfermedad , Femenino , Células HeLa , Humanos , Ratones , Ratones Desnudos , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Among many anticancer drugs collectively named "targeted or molecular therapies" epigenetic drugs are clearly promising. Differently from other agents targeting a single gene product, epigenetic drugs have chromatin as their target through inhibition of histone deacetylases (HDACs) and DNA methyltransferases (DNMTs) therefore, yet unspecific, they may act upon most or all tumor types, as deregulation of the methylation and deacetylation machinery are a common hallmark of neoplasia. In the last years, valproic acid (VPA) as emerged as a promising drug for cancer treatment. VPA has shown potent antitumor effects in a variety of in vitro and in vivo systems, and encouraging results in early clinical trials either alone or in combination with demethylating and/or cytotoxic agents. In addition, whole genome expression by microarray analysis from the primary tumors of patients treated with VPA show significant up-regulation of hundred of genes belonging to multiple pathways including ribosomal proteins, oxidative phosphorylation, MAPK signaling; focal adhesion, cell cycle, antigen processing and presentation, proteasome, apoptosis, PI3K, Wnt signaling, calcium signaling, TGF-beta signaling, and ubiquitin-mediated proteolysis among others. Despite in general, industry is not particularly interested in funding the clinical development of VPA, -at least in comparison to novel HDAC inhibitors-, existing preclinical and preliminary clinical data strongly suggest that VPA could be a drug that eventually will be used in combination therapies, either with classical cytotoxics, other molecular-targeted drugs or radiation in a number of solid tumors.

Asunto(s)

Antineoplásicos/uso terapéutico , Epigénesis Genética , Neoplasias/tratamiento farmacológico , Ácido Valproico/uso terapéutico , Línea Celular Tumoral , Ensayos Clínicos como Asunto , Humanos , Procesamiento Proteico-Postraduccional

RESUMEN

BACKGROUND: The methylation status at the human papilloma virus (HPV) genome found in pre-invasive and invasive cervical lesions suggests that neoplastic transformation can be suppressed by gene hypermethylation, whereas hypomethylation accompanies or causes cancer progression; hence, epigenetic therapy aimed at reactivating cellular suppressor-gene expression has the potential to act as a tumor promoter by enhancing HPV oncoprotein expression in HPV-related malignancies. The objective of this study was to determine the influence of hydralazine and valproate on HPV oncogene expression in cervical cancer cell lines and the primary tumors of patients undergoing treatment with hydralazine and valproate. RESULTS: Overall, hydralazine and valproate either alone or combined exerted a growth inhibitory effect on cervical cancer cell lines. A cell line-specific up-regulating effect was observed on E6/E7 gene expression, which in general correlated with DNA hypomethylation and histone acetylation at the long control region (LCR). Nonetheless, E6/E7 expression was unchanged or decreased in the majority of patients with cervical cancer treated with hydralazine, valproate, or both. In some cervical cancer cell lines, these drugs led to increased transcription of p53, and increased its stabilization due to acetylation at lysines 273 and 282, which allowed a higher bax-protein transactivating effect. CONCLUSION: The results of this study demonstrate that hydralazine and valproate can be safely administered to HPV-related malignancies such as cervical cancer because they do not increase viral oncoprotein expression. Most importantly, the antitumor effect of hydralazine and valproate in cervical cancer may at least partially depend on an up-regulating effect on p53 gene and on the valproate-induced hyperacetylation of p53 protein, protecting it from degradation by E6.

Asunto(s)

Alphapapillomavirus/genética , Regulación Viral de la Expresión Génica/efectos de los fármacos , Hidralazina/farmacología , Proteínas Oncogénicas Virales/genética , Neoplasias del Cuello Uterino/virología , Ácido Valproico/farmacología , Acetilación , Alphapapillomavirus/efectos de los fármacos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Proliferación Celular , Metilación de ADN , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Femenino , Expresión Génica , Genes p53 , Inhibidores de Histona Desacetilasas , Histonas/metabolismo , Humanos , Hidralazina/uso terapéutico , Proteínas Oncogénicas Virales/biosíntesis , ARN/análisis , ARN/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias del Cuello Uterino/tratamiento farmacológico , Ácido Valproico/uso terapéutico

RESUMEN

BACKGROUND: Aberrant DNA methylation and histone deacetylation participate in cancer development and progression; hence, their reversal by inhibitors of DNA methylation and histone deacetylases (HDACs) is at present undergoing clinical testing in cancer therapy. As epigenetic alterations are common to breast cancer, in this proof-of-concept study demethylating hydralazine, plus the HDAC inhibitor magnesium valproate, were added to neoadjuvant doxorubicin and cyclophosphamide in locally advanced breast cancer to assess their safety and biological efficacy. METHODOLOGY: This was a single-arm interventional trial on breast cancer patients (ClinicalTrials.gov Identifier: NCT00395655). After signing informed consent, patients were typed for acetylator phenotype and then treated with hydralazine at 182 mg for rapid-, or 83 mg for slow-acetylators, and magnesium valproate at 30 mg/kg, starting from day -7 until chemotherapy ended, the latter consisting of four cycles of doxorubicin 60 mg/m2 and cyclophosphamide 600 mg/m2 every 21 days. Core-needle biopsies were taken from primary breast tumors at diagnosis and at day 8 of treatment with hydralazine and valproate. MAIN FINDINGS: 16 patients were included and received treatment as planned. All were evaluated for clinical response and toxicity and 15 for pathological response. Treatment was well-tolerated. The most common toxicity was drowsiness grades 1-2. Five (31%) patients had clinical CR and eight (50%) PR for an ORR of 81%. No patient progressed. One of 15 operated patients (6.6%) had pathological CR and 70% had residual disease <3 cm. There was a statistically significant decrease in global 5mC content and HDAC activity. Hydralazine and magnesium valproate up- and down-regulated at least 3-fold, 1,091 and 89 genes, respectively. CONCLUSIONS: Hydralazine and magnesium valproate produce DNA demethylation, HDAC inhibition, and gene reactivation in primary tumors. Doxorubicin and cyclophosphamide treatment is safe, well-tolerated, and appears to increase the efficacy of chemotherapy. A randomized phase III study is ongoing to support the efficacy of so-called epigenetic or transcriptional cancer therapy.

Asunto(s)

Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Ciclofosfamida/administración & dosificación , Doxorrubicina/administración & dosificación , Epigénesis Genética/efectos de los fármacos , Adulto , Anciano , Neoplasias de la Mama/patología , Metilación de ADN/efectos de los fármacos , Femenino , Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas/administración & dosificación , Humanos , Hidralazina/administración & dosificación , Persona de Mediana Edad , Terapia Neoadyuvante , Ácido Valproico/administración & dosificación

RESUMEN

BACKGROUND: The development of resistance to cytotoxic chemotherapy continues to be a major obstacle for successful anticancer therapy. It has been shown that cells exposed to toxic concentrations of commonly used cancer chemotherapy agents develop DNA hypermethylation. Hence, demethylating agents could play a role in overcoming drug resistance. METHODS: MCF-7 cells were rendered adriamycin-resistant by weekly treatment with adriamycin. Wild-type and the resulting MCF-7/Adr cells were analyzed for global DNA methylation. DNA methyltransferase activity and DNA methyltransferase (dnmt) gene expression were also determined. MCF-7/Adr cells were then subjected to antisense targeting of dnmt1, -3a, and -b genes and to treatment with the DNA methylation inhibitor hydralazine to investigate whether DNA demethylation restores sensitivity to adriamycin. RESULTS: MCF-7/Adr cells exhibited the multi-drug resistant phenotype as demonstrated by adriamycin resistance, mdr1 gene over-expression, decreased intracellular accumulation of adriamycin, and cross-resistance to paclitaxel. The mdr phenotype was accompanied by global DNA hypermethylation, over-expression of dnmt genes, and increased DNA methyltransferase activity as compared with wild-type MCF-7 cells. DNA demethylation through antisense targeting of dnmts or hydralazine restored adriamycin sensitivity of MCF-7/Adr cells to a greater extent than verapamil, a known inhibitor of mdr protein, suggesting that DNA demethylation interferes with the epigenetic reprogramming that participates in the drug-resistant phenotype. CONCLUSION: We provide evidence that DNA hypermethylation is at least partly responsible for development of the multidrug-resistant phenotype in the MCF-7/Adr model and that hydralazine, a known DNA demethylating agent, can revert the resistant phenotype.

RESUMEN

Hydralazine was one of the first orally active antihypertensive drugs developed. Currently, it is used principally to treat pregnancy-associated hypertension. Hydralazine causes two types of side effects. The first type is an extension of the pharmacologic effect of the drug and includes headache, nausea, flushing, hypotension, palpitation, tachycardia, dizziness, and salt retention. The second type of side effects is caused by immunologic reactions, of which the drug-induced lupus-like syndrome is the most common, and provides clues to underscoring hydralazine's DNA demethylating property in connection with studies demonstrating the participation of DNA methylation disorders in immune diseases. Abnormalities in DNA methylation have long been associated with cancer. Despite the fact that malignant tumors show global DNA hypomethylation, regional hypermethylation as a means to silence tumor suppressor gene expression has attracted the greatest attention. Reversibility of methylation-induced gene silencing by pharmacologic means, which in turns leads to antitumor effects in experimental and clinical scenarios, has directed efforts toward developing clinically useful demethylating agents. Among these, the most widely used comprise the nucleosides 5-azacytidine and 2'deoxy-5-azacytidine; however, these agents, like current cytotoxic chemotherapy, causes myelosuppression among other side effects that could limit exploitation of their demethylating properties. Among non-nucleoside DNA demethylating drugs currently under development, the oral drug hydralazine possess the ability to reactivate tumor suppressor gene expression, which is silenced by promoter hypermethylation in vitro and in vivo. Decades of extensive hydralazine use for hypertensive disorders that demonstrated hydralazine's clinical safety and tolerability supported its testing in a phase I trial in patients with cancer, confirming its DNA demethylating activity. Hydralazine is currently being evaluated, along with histone deacetylase inhibitors either alone or as adjuncts to chemotherapy and radiation, for hematologic and solid tumors in phase II studies.