Evaluación de nuevas estrategias para el desarrollo de terapias anticancerosas selectivas

Texto completo descargado en TeseoEvaluación de nuevas estrategias para el desarrollo de terapias anticancerosas selectivas El cáncer es una enfermedad muy común y difícil de tratar. En países desarrollados, se estima que una de cada tres personas desarrolla esta enfermedad a lo largo de sus vidas y que únicamente la mitad de los afectados logra sobrevivir más de 5 años1. A pesar de la implementación de campañas de diagnóstico precoz, un gran número de cánceres se diagnostican cuando células de un tumor primario ya han invadido tejidos cercanos y se han diseminado a través de la sangre o la linfa a otros tejidos del organismo. En esta fase de la enfermedad, denominada metástasis, las células del tumor ya no están localizadas y, por tanto, ya no pueden eliminarse mediante la cirugía o la radioterapia. La principal forma de tratamiento en esta fase de la enfermedad es la quimioterapia, que consiste en administrar fármacos de forma sistémica con el objetivo de que alcancen y maten a las células tumorales. Pero la gran mayoría de estos fármacos son tóxicos tanto para las células tumorales como para las células sanas, lo que causa una alta toxicidad en el paciente y hace necesario reducir las dosis de estos fármacos a niveles que son poco efectivos. La poca eficacia de la terapia anticancerosa en el tratamiento de pacientes con metástasis se refleja en los bajos porcentajes de supervivencia en estos pacientes. Por ejemplo, las estadísticas indican que el cáncer que más se diagnostica y más muertes causa a nivel mundial es el cáncer de pulmón, que en países desarrollados aproximadamente el 50% de los cánceres de pulmón se diagnostican cuando el paciente ya tiene metástasis distantes, y que únicamente el 4% de estos pacientes logra sobrevivir más de 5 años2. Estos datos indican que es necesario buscar nuevas estrategias selectivas para el tratamiento de cánceres con metástasis, y especialmente para el cáncer de pulmón. En la búsqueda de estrategias anticancerosas selectivas es importante encontrar diferencias entre las células cancerosas y las células normales que puedan explotarse desde el punto de vista terapéutico. Datos recientes indican que las células tumorales tienen defectos en la reparación de determinados tipos de daño genético. La utilización de compuestos que induzcan daños específicos en el ADN causaría la muerte de las células tumorales que carezcan de los mecanismos necesarios para repararlos. Las células normales, que no tienen esos defectos en la reparación del daño genético, repararían el daño y sobrevivirían al tratamiento con esos compuestos3-5. También se ha demostrado recientemente que el metabolismo de las células cancerosas está alterado. A diferencia de las células normales, las células cancerosas activan la glicólisis de forma mantenida incluso cuando los niveles de oxígeno son adecuados (efecto Warburg), y generan niveles elevados de especies reactivas de oxígeno de forma constitutiva. Estas diferencias metabólicas hacen que las células cancerosas puedan ser más susceptibles que las células normales al tratamiento con inhibidores de la glicólisis y/o con compuestos prooxidantes6-12. Tras revisar brevemente información general sobre el cáncer y aspectos básicos sobre el cáncer de pulmón, este trabajo analiza la literatura que sugiere que es posible matar células tumorales de forma selectiva explotando sus defectos en la reparación del daño en el ADN y sus alteraciones metabólicas. Fruto de este análisis se seleccionaron más de 100 compuestos (o estrategias), y su posible actividad anticancerosa selectiva se evaluó mediante el ensayo MTT en células cancerosas de pulmón (A549) y células no cancerosas de pulmón (MRC-5). Posteriormente se evaluó la posible implicación de la inducción de daño en el ADN, de la inhibición de la glicolisis y de la capacidad de generar especies reactivas de oxígeno (capacidad prooxidante) en la actividad anticancerosa de los compuestos más selectivos. El daño en el ADN se evaluó mediante el ensayo del cometa y mediante las técnicas de inmunofluorescencias TARDIS y focos de ¿H2AX. El tipo de daño genético y la implicación de dicho daño en la citotoxicidad de los compuestos activos se evaluaron mediante la utilización de líneas celulares deficientes en las principales rutas de reparación del daño en el ADN (VC8, EM9, EM9-V, UV4, UV5, UV61, KO40 y V3-3) y sus correspondientes líneas celulares no deficientes (VC8-B2, EM9-XH y AA8). La inhibición de la glicólisis se evaluó midiendo los niveles de glucosa (producto inicial de la glicólisis) y de lactato (producto final de la glicólisis) en el medio extracelular tras el tratamiento con el compuesto ensayado. La capacidad prooxidante se analizó evaluando si la actividad citotóxica del compuesto se reducía en presencia de compuestos antioxidantes (catalasa, N-acetilcisteína y MnTMPyP). También se emplearon los ensayos XTT, SRB, clonogénico y Anexina V-FITC para profundizar en los mecanismos citotóxicos de algunos compuestos activos. Finalmente, para confirmar la selectividad anticancerosa de los compuestos más activos, se utilizaron células cancerosas de mama (MCF7), piel (UACC-62) y colon (HCT116) frente a células normales de mama (MCF10) y piel (VH10). Varios compuestos demostraron una selectividad anticancerosa destacable. El epoxialquil galactopiranósido (2S,3S)-2,3-epoxidecil 4,6-O-(S)-bencilideno-ß-D-galactopiranósido (compuesto 35) mostró actividad anticancerosa selectiva en células de cáncer de pulmón, mama y melanoma. Este compuesto indujo daño genético, en cuya reparación estuvo involucrada la vía de reparación por escisión de nucleótidos (NER). La aziridina derivada de galactosa 2-metil-2,3-[N-(4-metilbencenosulfonil)imino]propil 2,3-di-O-bencil-4,6-O-(S)-bencilideno-ß-D-galactopiranósido (compuesto 22) también indujo selectividad anticancerosa en células de pulmón, mama y melanoma, que puede estar mediada por su capacidad de inducir daño genético que requiere NER para su reparación. Es de destacar que las dosis de esta nueva aziridina requeridas para matar células de cáncer de mama fueron aproximadamente 50 veces inferiores a las necesarias para matar células normales de mama. El compuesto dicarbonílico fenilglioxal presentó actividad anticancerosa selectiva sobre células cancerosas de pulmón. Esta actividad fue debida, al menos en parte, a la inducción de daño en el ADN y a la generación de peróxido de hidrógeno. Células deficientes en NER, recombinación homóloga (HR) y unión de extremos no homólogos (NHEJ) fueron más sensibles al efecto citotóxico de este compuesto. Estos datos indican que fenilglioxal produce aductos en el ADN y roturas de doble cadena, y sugieren que tumores con defectos en estas vías de reparación pueden ser hipersensibles al efecto citotóxico de este compuesto13. Las lactonas ¿,ß-insaturadas 2-furanona y 2-pirona mostraron actividad citotóxica selectiva sobre células de cáncer de pulmón, e indujeron daño en el ADN que requiere HR para su reparación. El nuevo antagonista del receptor de neurokinina-1 INKB300b demostró una selectividad anticancerosa marcada sobre células de cáncer de pulmón, mama y melanoma. Resultados preeliminares sugieren que esta selectividad anticancerosa puede deberse, en parte, a su capacidad de inducir daño genético reparado por HR. Los compuestos fenólicos pirogalol (presente en el compuesto mayoritario del té verde epigalocatequina-3-galato), ácido clorogénico (presente en el café) y dodecil hidroxitirosil éter (derivado sintético del compuesto presente en el aceite de oliva hidroxitirosol) también mostraron selectividad frente a células cancerosas de pulmón14-16. La citotoxicidad de estos compuestos estuvo mediada, al menos en parte, por la producción de especies reactivas de oxígeno y por la generación de daño en el ADN. Las vías de reparación HR, NHEJ y Anemia de Fanconi (FA) estuvieron implicadas en la reparación del daño inducido por pirogalol, y la vía de reparación por escisión de bases (BER) en la de dodecil hidroxitirosil éter. La combinación de este derivado de hidroxitirosol con 5-fluorouracilo indujo un efecto sinérgico en células de cáncer de pulmón y un efecto antagónico en células normales de pulmón. Este compuesto también demostró actividad anticancerosa selectiva frente a células de cáncer de mama. Debido a la posible implicación del transportador Na+-K+-ATPasa en la elevada capacidad glicolítica de las células cancerosas17, y sabiendo que los heterósidos cardiotónicos son conocidos inhibidores de este transportador, se evaluó la actividad anticancerosa selectiva de varios heterósidos cardiotónicos (digitoxina, digoxina y ouabaína) y de un extracto hidroalcohólico de las hojas de una planta rica en heterósidos cardiotónicos (Nerium oleander). Los 3 heterósidos cardiotónicos y el extracto vegetal mostraron una elevada selectividad frente a células cancerosas de pulmón e indujeron una marcada inhibición de la glicólisis en estas células. También potenciaron la citotoxicidad de cisplatino en células de cáncer de pulmón al administrarse tras este fármaco anticanceroso. El extracto de Nerium oleander indujo daño en el ADN que requiere HR para su reparación. Cabe destacar que digitoxina inhibió el crecimiento de células cancerosas de pulmón a concentraciones más bajas que las presentes en el plasma de pacientes que utilizan este fármaco para el tratamiento de su enfermedad cardiaca18,19. En conclusión, los resultados obtenidos en este trabajo indican que es posible matar células cancerosas de forma selectiva explotando sus defectos en la reparación del daño en el ADN y sus alteraciones metabólicas. La marcada selectividad anticancerosa mostrada por algunos de los compuestos ensayados requiere confirmación en modelos animales de cáncer. La actividad anticancerosa selectiva de digitoxina en células de cáncer de pulmón a concentraciones terapéuticas apoya la posible realización de ensayos clínicos en pacientes con este tipo de cáncer.Premio Extraordinario de Doctorado U

The Cardiac Glycosides Digitoxin, Digoxin and Ouabain Induce a Potent Inhibition of Glycolysis in Lung Cancer Cells

Cardiac glycosides are promising anticancer drugs. We have recently shown that the cardiac glycosides digitoxin, digoxin and ouabain induce selective killing of lung cancer cells, and that the cytotoxicity of digitoxin against these cells occurs at concentrations below those observed in the plasma of cardiac patients treated with this drug (Oncogene, 2013. doi: 10.1038/onc.2013.229). Here we report that digitoxin, digoxin and ouabain induce a potent inhibition of glycolysis (glucose consumption and lactate production) in A549 cells at nanomolar concentrations. This inhibition was comparable to that observed with millimolar concentrations of the glycolysis inhibitor dichloroacetate, which is currently undergoing clinical trials for the treatment of cancer. Because platinum compounds are commonly used in the treatment of lung cancer, we tested the cytotoxicity of several combinations of cisplatin with each cardiac glycoside; these combinations induced synergistic, antagonistic or additive effects mainly depending on the order at which the drugs were added to the cells

Effect of DNA repair deficiencies on the cytotoxicity of resveratrol

Numerous preclinical studies have shown that the naturally-occurring polyphenol resveratrol may produce health-beneficial effects in a variety of disorders, including cancer, diabetes, Alzheimer, and cardiovascular diseases. Resveratrol has entered clinical trials for the prevention and treatment of several of these disorders. This polyphenol is also available in the market as a dietary supplement. Experimental data have shown, however, that resveratrol induces DNA damage in a variety of cells. Here we review such evidence and evaluate the cytotoxicity of resveratrol (MTT assay) in cells deficient in several major DNA repair pathways (i.e., homologous recombination, non-homologous end joining, base excision repair, nucleotide excision repair, mismatch repair, and Fanconi anemia repair). Cells deficient in base excision repair (EM9), nucleotide excision repair (UV4 and UV5) and Fanconi Anemia (KO40) were slightly hypersensitive to resveratrol-induced cytotoxicity with respect to their parental cells (AA8). Our results suggest that these pathways may participate in the repair of the DNA damage induced by resveratrol and that deficiencies in these pathways may confer hypersensitivity to the genotoxic activity of this dietary constituen

Vitamin C and the Red Wine Polyphenol Resveratrol - but not Curcumin and the Glycolysis Inhibitors 2- Deoxyglucose, Dichloroacetate and 3-Bromopyruvate - Induce Selective Cytotoxicity against Lung Cancer Cells.

Cancer statistics show that the most commonly diagnosed cancer in the world is lung cancer, that over 50% of patients diagnosed with this cancer have distant metastasis, and that only 4% of these patients manage to survive more than 5 years. The limited selective cytotoxicity of the drugs used for the treatment of these patients probably accounts for these high mortality rates. In this work, we have assessed the selective anticancer activity of several drugs currently undergoing clinical trials by using human A549 lung cancer cells and human MRC5 non-malignant lung fibroblasts. Vitamin C and the red wine polyphenol resveratrol induced selective cytotoxicity towards the cancer cell line. Vitamin C (1 mM) induced higher selective cytotoxicity than the anticancer agents cisplatin, oxaliplatin, etoposide and 5-fluorouracil. A lyophilized red wine extract, but not a hydroalcoholic extract from red grapes, also showed certain selectivity against lung cancer cells. Neither the curry polyphenol curcumin nor the glycolysis inhibitors 2-deoxyglucose, dichloroacetate and 3-bromopyruvate displayed selective cytotoxicity. We also report that A549 lung cancer cells have higher glycolytic rates (higher glucose consumption and higher lactate production) than human MRC5 non-malignant lung fibroblasts, and that the combination of each glycolytic inhibitor with the pro-oxidant agents pyrogallol and hydrogen peroxide does not result in a significant increase in their cytotoxicity or selectivity against the cancer cell line. Our results support the possible evaluation of vitamin C and resveratrol in clinical trial for the treatment of metastatic lung cancers, and suggest that curcumin and the glycolysis inhibitors 2-deoxyglucose, dichloroacetate and 3-bromopyruvate have a limited potential (at least as single agents) for the treatment of patients with this type of cance

A hydroalcoholic extract from the leaves of Nerium oleander inhibits glycolysis and induces selective killing of lung cancer cells

Recent evidence suggests that cardiac glycosides might be used for the treatment of cancer. The ornamental shrub Nerium oleander has been used in traditional medicine for treating several disorders including cancer, and extracts from the leaves of this plant have already entered phase I clinical trials. In this communication, we have prepared a hydroalcoholic extract from the leaves of Nerium oleander (containing 4.75 ± 0.32 % of cardenolides) and have assessed its cytotoxic activity in A549 lung cancer cells vs. MRC5 nonmalignant lung fibroblasts. The results showed that the cytotoxicity of the Nerium oleander extract against the cancer cell line was significantly higher than that against the nonmalignant cell line, with a potency and selectivity similar to those of the anticancer drug cisplatin. Pretreatment of A549 cells with the antioxidants N-acetylcysteine and catalase slightly prevented the cytotoxicity of the extract, therefore suggesting that the formation of reactive oxygen species participates in its cytotoxic activity but does not play a major role. Nerium oleander extract-induced cytotoxicity and DNA damage (gamma-H2AX focus formation) were slightly higher in cells lacking BRCA2 (deficient in homologous recombination repair) than in parental cells; this indicates that the induction of DNA damage may also play a role in the cytotoxicity of the extract. Nerium oleander extract induced a marked inhibition of glycolysis (glucose consumption and lactate production) in A549 cells, comparable to that of the glycolysis inhibitor dichloroacetate (currently in clinical development for cancer therapy). Because platinum compounds are widely used in the treatment of lung cancer, we tested the cytotoxicity of several combinations of cisplatin with the extract and found a moderate synergism when Nerium oleander extract was administered after cisplatin but a moderate antagonism when it was added before cisplatin. Our results suggest that extracts from Nerium oleander might induce anticancer effects in patients with lung cancer and support their possible advancement into phase II clinical trials for the treatment of this type of cancer

Consumption of the Dietary Flavonoids Quercetin, Luteolin and Kaempferol and Overall Risk of Cancer - A Review and Meta-Analysis of the Epidemiological Data

Numerous epidemiological and preclinical studies suggest that flavonoids may play an important role in the decreased risk of cancer associated with a diet rich in plant-derived foods. In this article, we have reviewed the epidemiological studies assessing the relationship between the consumption of three of the most common flavonoids, i.e. quercetin, luteolin and kaempferol, and the risk of developing cancer. We have also performed a meta-analysis on the consumption of these three flavonoids (alone and combined) and overall risk of cancer. The analysis of data from 18 case-control studies (8585 cases with cancer and 9975 control subjects) revealed that a high consumption of these three flavonoids (combined) was associated with a statistically significant reduction of overall cancer risk (OR: 0.73; 95% CI: 0.63, 0.84; p<0.01). A reduction of overall cancer risk was also observed for quercetin (OR: 0.73; 95% CI: 0.62, 0.86; p<0.01), kaempferol (OR: 0.86; 95% CI: 0.73, 1.11; p>0.05) and luteolin (OR: 0.90; 95% CI: 0.69, 1.18; p>0.05), which was statistically significant for quercetin. A high intake of these three flavonoids (combined) was also associated with a statistically significant reduction of lung cancer risk (OR: 0.67; 95% CI: 0.49, 0.91; p<0.05) and colon cancer risk (OR: 0.75; 95% CI: 0.57, 0.98; p<0.05). The analysis of data from 14 cohort studies (385033 individuals and 10809 cancer cases) showed a statistically significant reduction of overall cancer risk for the three flavonoids combined (RR: 0.89; 95% CI: 0.80, 1.00; p<0.05), for quercetin (RR: 0.82; 95% CI: 0.71, 0.96; p<0.05) and for kaempferol (RR: 0.88; 95% CI: 0.78, 0.99; p<0.05), and a non-statistically significant reduction for luteolin (RR: 0.95; 95% CI: 0.67, 1.34; p>0.05). These results suggest that consumption of foods rich in the flavonoids quercetin, kaempferol and luteolin may reduce the risk of developing cance

Cells Deficient in the Fanconi Anemia Protein FANCD2 are Hypersensitive to the Cytotoxicity and DNA Damage Induced by Coffee and Caffeic Acid

Epidemiological studies have found a positive association between coffee consumption and a lower risk of cardiovascular disorders, some cancers, diabetes, Parkinson and Alzheimer disease. Coffee consumption, however, has also been linked to an increased risk of developing some types of cancer, including bladder cancer in adults and leukemia in children of mothers who drink coffee during pregnancy. Since cancer is driven by the accumulation of DNA alterations, the ability of the coffee constituent caffeic acid to induce DNA damage in cells may play a role in the carcinogenic potential of this beverage. This carcinogenic potential may be exacerbated in cells with DNA repair defects. People with the genetic disease Fanconi Anemia have DNA repair deficiencies and are predisposed to several cancers, particularly acute myeloid leukemia. Defects in the DNA repair protein Fanconi Anemia D2 (FANCD2) also play an important role in the development of a variety of cancers (e.g., bladder cancer) in people without this genetic disease. This communication shows that cells deficient in FANCD2 are hypersensitive to the cytotoxicity (clonogenic assay) and DNA damage (γ-H2AX and 53BP1 focus assay) induced by caffeic acid and by a commercial lyophilized coffee extract. These data suggest that people with Fanconi Anemia, or healthy people who develop sporadic mutations in FANCD2, may be hypersensitive to the carcinogenic activity of coffee

Evaluating the cancer therapeutic potential of cardiac glycosides

Cardiac glycosides, also known as cardiotonic steroids, are a group of natural products that share a steroid-like structure with an unsaturated lactone ring and the ability to induce cardiotonic effects mediated by a selective inhibition of the Na(+)/K(+)-ATPase. Cardiac glycosides have been used for many years in the treatment of cardiac congestion and some types of cardiac arrhythmias. Recent data suggest that cardiac glycosides may also be useful in the treatment of cancer. These compounds typically inhibit cancer cell proliferation at nanomolar concentrations, and recent high-throughput screenings of drug libraries have therefore identified cardiac glycosides as potent inhibitors of cancer cell growth. Cardiac glycosides can also block tumor growth in rodent models, which further supports the idea that they have potential for cancer therapy. Evidence also suggests, however, that cardiac glycosides may not inhibit cancer cell proliferation selectively and the potent inhibition of tumor growth induced by cardiac glycosides in mice xenografted with human cancer cells is probably an experimental artifact caused by their ability to selectively kill human cells versus rodent cells. This paper reviews such evidence and discusses experimental approaches that could be used to reveal the cancer therapeutic potential of cardiac glycosides in preclinical studie

Artificial Diets with Selective Restriction of Amino Acids and Very Low Levels of Lipids Induce Anticancer Activity in Mice with Metastatic Triple-Negative Breast Cancer

Current treatments for patients with metastatic triple negative breast cancer (TNBC) are generally ineffective. This manuscript shows for the first time that the survival of mice with metastatic TNBC can be markedly increased through dietary manipulation. Our study revealed that the survival of some mice with metastatic TNBC was increased by replacing their normal diet with artificial diets in which the levels of amino acids (AAs) are manipulated, and the levels of lipids are markedly reduced. The anticancer activity of this non-pharmacological strategy was higher than that of drugs currently used in the treatment of patients with metastatic TNBC. This anticancer strategy also increased the survival of mice with other types of metastatic cancers. Manipulation of AA and lipid levels with artificial diets may be a useful strategy to treat patients with metastatic TNBC and other types of disseminated cancer.Patients with metastatic triple negative breast cancer (TNBC) need new therapies to improve the low survival rates achieved with standard treatments. In this work, we show for the first time that the survival of mice with metastatic TNBC can be markedly increased by replacing their normal diet with artificial diets in which the levels of amino acids (AAs) and lipids are strongly manipulated. After observing selective anticancer activity in vitro, we prepared five artificial diets and evaluated their anticancer activity in a challenging model of metastatic TNBC. The model was established by injecting 4T1 murine TNBC cells into the tail vein of immunocompetent BALB/cAnNRj mice. First-line drugs doxorubicin and capecitabine were also tested in this model. AA manipulation led to modest improvements in mice survival when the levels of lipids were normal. Reducing lipid levels to 1% markedly improved the activity of several diets with different AA content. Some mice fed the artificial diets as monotherapy lived much longer than mice treated with doxorubicin and capecitabine. An artificial diet without 10 non-essential AAs, with reduced levels of essential AAs, and with 1% lipids improved the survival not only of mice with TNBC but also of mice with other types of metastatic cancers.Junta de Andalucía 2017/CTS-657, 2019/CTS-657, 2021/CTS-657Universidad de Sevilla VIPPIT-2019-I.5, VIPPIT-2020-I.5, VIPPIT-2021-I.

Artificial Diets with Altered Levels of Sulfur Amino Acids Induce Anticancer Activity in Mice with Metastatic Colon Cancer, Ovarian Cancer and Renal Cell Carcinoma

Sulfur-containing amino acids methionine (Met), cysteine (Cys) and taurine (Tau) are common dietary constituents with important cellular roles. Met restriction is already known to exert in vivo anticancer activity. However, since Met is a precursor of Cys and Cys produces Tau, the role of Cys and Tau in the anticancer activity of Met-restricted diets is poorly understood. In this work, we screened the in vivo anticancer activity of several Met-deficient artificial diets supplemented with Cys, Tau or both. Diet B1 (6% casein, 2.5% leucine, 0.2% Cys and 1% lipids) and diet B2B (6% casein, 5% glutamine, 2.5% leucine, 0.2% Tau and 1% lipids) showed the highest activity and were selected for further studies. Both diets induced marked anticancer activity in two animal models of metastatic colon cancer, which were established by injecting CT26.WT murine colon cancer cells in the tail vein or peritoneum of immunocompetent BALB/cAnNRj mice. Diets B1 and B2B also increased survival of mice with disseminated ovarian cancer (intraperitoneal ID8 Tp53−/− cells in C57BL/6JRj mice) and renal cell carcinoma (intraperitoneal Renca cells in BALB/cAnNRj mice). The high activity of diet B1 in mice with metastatic colon cancer may be useful in colon cancer therapy.Junta de Andalucía 2017/CTS-657, 2019/CTS-657, 2021/CTS657Universidad de Sevilla VIPPIT2019-I.5, VIPPIT-2020-I.5, VIPPIT-2021-I.5, VIPPIT-2020-II.