Leukotoxin (LtxA/Leukothera) induces ATP expulsion via pannexin-1 channels and subsequent cell death in malignant lymphocytes.

Leukotoxin (LtxA) (Trade name, Leukothera) is a protein that is secreted from the oral bacterium Aggregatibacter actinomycetemcomitans, which targets and kills activated white blood cells (WBCs) by binding to lymphocyte function associated antigen-1 (LFA-1). Interaction between LtxA and Jurkat T-cells results in cell death and is characterized by increased intracellular Ca2+, activation of caspases, clustering of LtxA and LFA-1 within lipid rafts, and involvement of the Fas death receptor. Here, we show that LtxA can kill malignant lymphocytes via apoptotic and necrotic forms of cell death. We show that LtxA causes activation of caspases and PARP, cleavage of pannexin-1 (Panx1) channels, and expulsion of ATP, ultimately leading to cell death via apoptosis and necrosis. CRISPR-Cas9 mediated knockout (K/O) of Panx1 in Jurkat cells prevented ATP expulsion and resulted in resistance to LtxA for both apoptotic and necrotic forms of death. Resistance to necrosis could only be overcome when supplementing LtxA with endogenous ATP (bzATP). The combination of LtxA and bzATP promoted only necrosis, as no Panx1 K/O cells stained positive for phosphatidylserine (PS) exposure following the combined treatment. Inhibition of LtxA/bzATP-induced necrosis was possible when pretreating Jurkat cells with oATP, a P2X7R antagonist. Similarly, blockage of P2X7Rs with oATP prevented the intracellular mobilization of Ca2+, an important early step in LtxA induced cell death. We show that LtxA is able to kill malignant lymphocytes through an apoptotic death pathway which is potentially linked to a Panx1/P2X7R mediated necrotic form of death. Thus, inhibition of ATP release appears to significantly delay the onset of LtxA induced apoptosis while completely disabling the necrotic death pathway in T-lymphocytes, demonstrating the crucial role of ATP release in LtxA-mediated cell death.

Cytotoxic activity of standardized extracts, a fraction, and individual secondary metabolites from fenugreek seeds against SKOV-3, HeLa and MOLT-4 cell lines.

CONTEXT: Trigonella foenum-graecum L. (Fabaceae) has many therapeutic properties and anticancer potential. OBJECTIVE: The cytotoxic activities of standardized extracts and a fraction from fenugreek seeds and their compounds (sapogenins, flavone C-glycosides, alkaloid trigonelline) against human cancer SKOV-3, HeLa and MOLT-4 cells were evaluated. MATERIALS AND METHODS: Fenugreek seeds were extracted with 70% methanol (A) or water (B). Furthermore, the seeds were purified with petroleum ether and chloroform and next extracted with methanol to obtain fraction (C). The quantitative analysis of saponins and flavonoids in the extracts was done with HPLC methods. The extracts (5-120 µg/mL) and compounds (1-50 µg/mL) were tested on the cells by MTT assay and RTCA system. The effect of a fraction on ROS production, mitochondrial membrane potential and caspase-3/7 activity in HeLa and SKOV-3 cells was also evaluated by flow cytometry. RESULTS: The strongest cytotoxic activity on cancer cells showed the fraction C (IC50 was 3.91 ± 0.03 for HeLa, 3.97 ± 0.07 for SKOV-3, and 7.75 ± 0.37 for MOLT-4) with the highest content of steroidal saponins (163.18 ± 11.03 µg/mg) and flavone C-glycosides (820.18 ± 0.05 µg/mg). The fraction significantly increased ROS production (up to four times higher than in keratinocytes as control) and caspases activity in the cells. The examined flavonoids did not exhibit the cytotoxic activity in contrast to yamogenin, tigogenin, and diosgenin. CONCLUSIONS: The obtained results complement the data on the cytotoxic activity of Foenugraeci Semen and synergistic effect of flavonoids and saponins complex contained in the plant.

Eficacia y seguridad de obinutuzumab más clorambucil en pacientes adultos mayores de 65 años con leucemia linfocítica crónica sin tratamiento sistémico previo que no son tributarios a fludarabina / Efficacy and safety of obinutuzumab plus chlorambucil in adult patients older than 65 years with chronic lymphocytic leukemia without prior systemic treatment who are not candidates for fludarabine

INTRODUCCIÓN: El presente dictamen expone la evaluación de la eficacia y seguridad de obinutuzumab más clorambucil (O + C) en comparación con rituximab más clorambucil (R + C) en pacientes adultos mayores de 65 años con leucemia linfocítica crónica (LLC) sin tratamiento sistémico previo que no son tributarios a fludarabina. La leucemia linfocítica crónica (LLC) es una neoplasia caracterizada por una acumulación progresiva de linfocitos B monoclonales en sangre periférica. Constituye la leucemia más común en adultos en los países occidentales (edad media para el diagnóstico de 70 años), y representa el 25 % al 30 % de todas las leucemias. Aproximadamente, la mediana de sobrevida global (SG) es de 10 años y la tasa de SG del 80 %. El inicio del tratamiento del paciente con LLC depende de la progresión y del estado general de salud del individuo, iniciándose terapia bajo criterios específicos. El Petitorio Farmacológico de EsSalud dispone de fludarabina, clorambucil y rituximab para el tratamiento sistémico de primera línea de los pacientes mayores de 65 años con LLC que cuentan con indicación de inicio de tratamiento sistémico. No obstante, fludarabina cuenta con contraindicaciones de uso en su inserto, entre los que se listan: la hipersensibilidad al principio activo o excipientes, enfermedad renal crónica avanzada, anemia hemolítica descompensada, y la lactancia. En ese sentido, en los pacientes que no son tributarios al tratamiento con fludarabina, en EsSalud se opta por el uso de rituximab (R) asociado a clorambucil (C) (R + C). No obstante, los especialistas han sugerido la evaluación de uso de obinutuzumab (O) asociado a clorambucil (O + C), bajo la hipótesis de que éste tendría un mejor perfil de eficacia y seguridad que (R + C), como tratamiento sistémico de primera línea en la población de pacientes mayores de 65 años con LLC que no son tributarios de fludarabina. METODOLOGÍA: Se realizó una búsqueda sistemática de literatura con el objetivo de identificar evidencia científica sobre la eficacia y seguridad de (O + C), en comparación con (R + C), en pacientes adultos mayores de 65 años con LLC sin tratamiento sistémico previo que no son tributarios a fludarabina. La búsqueda se inició revisando la información sobre el uso del medicamento de acuerdo con entidades reguladoras como FDA, EMA, y DIGEMID en el Perú. Se realizó tanto una búsqueda sistemática en las principales bases de datos, tales como MEDLINE vía PubMed y en Cochrane Library. Asimismo, se realizó una búsqueda manual en las páginas web de grupos dedicados a la investigación y educación en salud que elaboran GPC y ETS: National Institute for Health and CareExcellence (NICE), Canadian Agency for Drugs and Technologies in Health (CADTH), Scottish Medicines Consortium (SMC), Haute Authorité de Santé (HAS), Institute for Quality and Efficiency in HealthCare (IQWiG), Institute for Clinical and EconomicReview (ICER), la Base Regional de Informes de Evaluación de Tecnologías en Salud de las Américas (BRISA), la Organización Mundial de la Salud (OMS), el Ministerio de Salud del Perú (MINSA) y el Instituto de Evaluación de Tecnologías en Salud e Investigación (IETSI). Además, se realizó una búsqueda de las GPC de las principales sociedades o instituciones especializadas en cáncer, tales como el Instituto Nacional de Enfermedades Neoplásicas (INEN) del Perú, National Comprehensive Cancer Network (NCCN), la American Society of Clinical Oncology (ASCO), European Society for Medical Oncology (ESMO), American Society of Hematology (ASH), y The European Hematology Association (EHA). Adicionalmente, se buscaron ensayos clínicos en desarrollo o que no hayan sido publicados aún en la página web www.clinicaltrials.gov que contengan estudios acerca de la tecnología evaluada y así disminuir el sesgo de publicación. Finalmente, se consideró extraer información con una estrategia de "bola de nieve" mediante la revisión de las listas de referencias de las revisiones sistemáticas, estudios primarios y revisiones narrativas seleccionadas que sean relevantes para responder a la pregunta PICO del presente dictamen preliminar. RESULTADOS: En la presente sinopsis se describe la evidencia disponible según el tipo de publicación, siguiendo lo indicado en los criterios de elegibilidad. CONCLUSIONES: En el presente documento, se evaluó la evidencia científica disponible hasta la actualidad sobre la eficacia y seguridad de (O + C), en comparación con (R + C), en pacientes adultos mayores de 65 años con LLC sin tratamiento sistémico previo que no son tributarios a fludarabina. La evidencia científica identificada para la presente evaluación corresponde a dos GPC (NCCN y ESMO), dos ETS (NICE y pCODR) y un ECA de fase 3 denominado CLL11. Los panelistas de las GPC de NCCN y ESMO recomiendan, en general, el uso de (O + C) y (R + C) como alternativas terapéuticas para los pacientes adultos mayores de 65 años con LLC sin tratamiento sistémico previo, sin las mutaciones del(17p) o TP53, con una alta calidad de evidencia. El panel de la GPC de NCCN establece como tratamiento de preferencia el uso de (O + C). Ambos paneles basaron sus recomendaciones en los beneficios que a su criterio se habría evidenciado con (O + C) y (R + C) en el desenlace de SLP en el ECA CLL11. Los comités que elaboraron las ETS de NICE y pCODR recomendaron el empleo de (O + C) para pacientes con LLC sin tratamiento sistémico previo que no pueden recibir terapias basadas en fludarabina. Ambas ETS basaron sus recomendaciones en los beneficios que a su criterio se observó en el desenlace de SLP en el ECA CLL11. Además, NICE señaló que los análisis económicos mostraron que (O + C) era un tratamiento costo-efectivo en comparación con (R + C) para el sistema de salud inglés. Por otro lado, el comité de pCODR argumentó que el esquema (O + C), comparado con (R + C), no ofrecía beneficio adicional en la SG y calidad de vida; pero sí observó diferencias favorables al compararse con (C) en monoterapia. Estos escenarios deben ser interpretados tomando en cuenta las diferencias entre los sistemas de salud inglés y canadiense con el sistema de salud peruano al cual pertenece EsSalud, por tratarse de diferentes contextos económicos y sanitarios. Las recomendaciones de las GPC y ETS incluidas en este dictamen se apoyan en los resultados del ECA CLL11; el cual es un ensayo clínico fase 3, multicéntrico, de etiqueta abierta cuyo objetivo fue comparar la eficacia y seguridad de (O + C) con los esquemas de (R + C) y (C) en monoterapia en pacientes adultos con LLC sin tratamiento sistémico previo. La población de interés de este ECA es más amplia que la planteada en la pregunta PICO del presente dictamen preliminar; por lo tanto, sus resultados deben tomarse como evidencia indirecta. Se desconoce qué proporción de pacientes del ECA CLL11 corresponde a la población de interés del presente dictamen. Los resultados disponibles a la fecha muestran que, luego de una mediana de seguimiento de 59.4 meses, la SG26 y la calidad de vida de los esquemas (R + C) y (O + C) eran similares. Sin embargo, el análisis de los EA mostró que (O + C) tiene un perfil de seguridad desfavorable en comparación con (R + C), dado que presentó una mayor proporción de EA totales, EA de grado 3 a 5, y EA serios. El diseño de etiqueta abierta del ECA CLL11 introduce riesgo de sesgo de realización y de detección en los desenlaces de eficacia y seguridad. Además, la alta proporción de uso de terapias subsecuentes en los grupos (O + C) y (R + C) -las cuales quedaron a criterio del investigador- aumenta la incertidumbre sobre la existencia de verdaderas diferencias entre los grupos de tratamiento en evaluación. Por último, cabe incidir en el hecho del alto riesgo de error del tipo I en los análisis del ECA CLL11, al evidenciarse múltiples comparaciones estadísticas para el desenlace de interés del presente dictamen (SG), para el cual no se llevaron a cabo procedimientos de ajuste por multiplicidad. De este modo, la evidencia disponible a la fecha, sugiere que el balance riesgo-beneficio sería desfavorable con (O + C), en comparación con (R + C), en la población de interés del presente dictamen preliminar. Esto porque los resultados del ECA CLL11 no han mostrado que (O + C) ofrezca un beneficio adicional, en comparación con (R + C), respecto a los desenlaces de eficacia de SG y calidad de vida, en la población total del estudio. Mientras que, los análisis de los EA mostraron que (O + C) tendría un peor perfil de seguridad que (R + C), en la población total del estudio. En se sentido, no es posible sustentar técnicamente una recomendación favorable para (O + C) en la población de interés del presente dictamen preliminar. Además, se debe tener en cuenta que las GPC también recomiendan el uso de esquemas basados en (R + C) como alternativas terapéuticas para una población similar a la población de interés del presente dictamen, estando estos medicamentos actualmente disponibles en la institución. Por lo expuesto, el IETSI no aprueba el uso de (O + C) en pacientes adultos mayores de 65 años con LCC sin tratamiento sistémico previo que no son tributarios a fludarabina.

Eficacia y seguridad del uso de ibrutinib en el tratamiento en primera línea o recaída de pacientes adultos con leucemia linfocítica crónica/linfoma de células pequeñas con deleción 17p o mutación TP53 / Efficacy and safety of the use of ibrutinib in the first-line or relapse treatment of adult patients with chronic lymphocytic leukemia/small cell lymphoma with 17p deletion or TP53 mutation

INTRODUCCIÓN: El presente dictamen expone la evaluación de tecnología de la eficacia y seguridad de ibrutinib en el tratamiento en primera línea o recaída de pacientes adultos con leucemia linfocítica crónica o linfoma linfocitico de células pequeñas. La leucemia linfocítica crónica (LLC) y el linfoma de células pequeñas (LCP) son neoplasias hematológicas de linfocitos B consideradas incurables y que al ser manifestaciones distintas de una misma enfermedad, presentan un abordaje terapéutico similar. La prevalencia de LLC/LCP en el Perú no se ha estimado, sin embargo, un estudio en un hospital de Lima muestra que la frecuencia de casos es baja. La sobrevida global (SG) a 5 años en pacientes con LLC/LCP se estima alrededor de 80 %, aunque es menor en pacientes de alto riesgo, como los que presentan la deleción 17p o mutación TP53. Actualmente el Petitorio Farmacológico de EsSalud cuenta con el esquema de quimioinmunoterapia que incluye fludarabina, ciclofosfamida, y rituximab (FRC) para el tratamiento de primera línea de pacientes con LLC/LCP en general. Sin embargo, los especialistas locales manifiestan que la subpoblación específica de pacientes con deleción 17p o mutación TP53 podrían beneficiarse más de recibir tratamiento con ibrutinib, en términos de SG y calidad de vida. METODOLOGIA: Se llevó a cabo una búsqueda de la literatura con respecto a la eficacia y seguridad de ibrutinib en el tratamiento de pacientes adultos con LLC/LCP y deleción 17p o mutación TP53 en las bases de datos de PubMed, Cochrane Library, el metabuscador TRIP database y el sitio web www.clinicaltrials.gov. Adicionalmente, se realizó una búsqueda de evaluaciones de tecnologías y guías de práctica clínica en las páginas web de grupos dedicados a la investigación y educación en salud en general como The National Institute for Health and Care Excellence (NICE), Canadian Agency for Drugs and Technologies in Health (CADTH), Scottish Medicines Consortium (SMC), Instituto de Evaluación de Efectividad Clínica y Sanitaria (IECS), Instituto de Evaluación de Tecnología en Salud (IETS); en grupos especializados en oncología como European Society for Medical Oncology (ESMO), National Comprehensive Cancer Network (NCCN) y American Society of Clinical Oncology (ASCO) y, en hematología como The American Society of Hematology (ASH). Para la búsqueda de información que pueda ser empleada para responder a la pregunta PICO, se utilizaron estrategias de búsqueda que incluyeron términos relacionados a la población de interés, la intervención, el fármaco comparador y los tipos de estudio priorizados. Se emplearon términos MeSH2 y términos generales de lenguaje libre para la búsqueda en PubMed (Ver estrategia de búsqueda en la Tabla 1 del Material suplementario). En la selección de los estudios se priorizaron las guías de práctica clínica (GPC), evaluaciones de tecnología sanitaria (ETS), revisiones sistemáticas (RS), meta-análisis (MA) y ensayos clínicos aleatorizados (ECA) de fase III de acuerdo con la pregunta PICO. De no contar con evidencia de dichas categorías, se podría recurrir a un ensayo clínico de fase II o de menor nivel metodológico que puedan ayudar a responder a la pregunta PICO. RESULTADOS: De acuerdo con la pregunta PICO, se llevó a cabo una búsqueda de evidencia científica relacionada al uso de ibrutinib en el tratamiento de pacientes con LLC/LCP y deleción 17p o mutación TP53. En la presente sinopsis se describe la evidencia disponible según el tipo de publicación, siguiendo lo indicado en los criterios de elegibilidad (GPC, ETS, RS, MA y ECA fase III). Siendo que no se identificó ninguna RS, MA ni ECA de fase III que permita responder a la pregunta PICO, se recurrió a reportar los resultados de la evidencia disponible a la fecha correspondiente a un ensayo clínico de fase II. CONCLUSIONES: A la fecha no se ha publicado ningún ensayo clínico de la comparación requerida por la pregunta PICO. Por lo que se ha incluido únicamente un ensayo de fase II, de un solo brazo y etiqueta abierta a manera de evidencia indirecta, por ser la única evidencia disponible al momento de la evaluación. Se han considerado también dos GPC (NCCN y ESMO), y tres ETS (NICE, CADTH y SMC). Fue relevante únicamente la GPC de NCCN, la cual recomienda el uso de ibrutinib como una alternativa de tratamiento en la población específica de pacientes con deleción 17p o mutación TP53, tanto en primera línea como en refractariedad o recaída. Sin embargo, la evidencia que la respalda es de estudios de bajo nivel. Las ETS identificadas no permitieron responder a la pregunta PICO del presente dictamen preliminar, sin embargo, muestran que el uso de ibrutinib en la población de pacientes con LLC está condicionado a un descuento o ajuste en el precio del medicamento. Adicionalmente, dentro del documento de ETS realizado por CADTH, se menciona que no es posible definir de manera sólida el beneficio neto de ibrutinib ante la ausencia de comparaciones directas o indirectas frente a las alternativas consideradas como el estándar de tratamiento en Canadá. Se tomó como evidencia central e indirecta en torno al uso de ibrutinib un ensayo de fase II, de un solo brazo que evalúa a la población de interés: RESONATE-17. Dicho ensayo reporta únicamente una estimación de SG a 24 meses de 75 % (IC 95 %: 67-81) en el brazo que recibió ibrutinib, es decir, evidencia descriptiva que no permite concluir con respecto al beneficio neto de ibrutinib con respecto a quimioinmunoterapia. Con respecto a los eventos adversos. Se observó un perfil de seguridad desfavorable, donde 18 pacientes de 144 murieron a causa de eventos adversos, y 22 descontinuaron el tratamiento por eventos adversos. Esta información es de gran relevancia considerando que los pacientes incluidos en el estudio fueron pacientes sanos, de acuerdo con los criterios de exclusión. Asimismo, con la información obtenida de clinicaltrials.gov, se observa que más de la mitad de los pacientes incluidos en el RESONATE-17 presentaron eventos adversos serios (52.78 %, 76/144 pacientes), los cuales ponen al paciente en riesgo de hospitalización o muerte. En conclusión, a la fecha no se cuenta con evidencia de estudios comparativos entre ibrutinib y quimioinmunoterapia para la población específica de pacientes con deleción 17p o mutación TP53 que permita tener certeza del beneficio neto de ibrutinib en estos pacientes. La incertidumbre con respecto al beneficio de ibrutinib se ve acentuada por su perfil de seguridad, donde se observa una frecuencia importante de muertes durante el tratamiento y eventos adversos serios en más de la mitad de los pacientes. Así, con la evidencia disponible a la fecha no se cuenta con argumentos técnicos suficientes para sustentar una recomendación positiva sobre el uso de ibrutinib en el tratamiento de pacientes con LLC/LCP con deleción 17p o mutación TP53. Por lo expuesto, el Instituto de Evaluación de Tecnologías en Salud e Investigación (IETSI) no aprueba el uso de ibrutinib para el tratamiento de pacientes con LLC/LCP con deleción 17p o mutación TP53.

Los ritmos circadianos en cáncer y la cronoterapia / The circadian rhythms in cancer and chronotherapy / Os ritmos circadianos em câncer e a cronoterapia

Los ritmos circadianos, períodos fisiológicos de 24 horas aproximadamente, coordinan la actividad temporal de la mayoría, si no, de todos los seres vivos del planeta. Uno de los procesos más importantes del cuerpo, la proliferación celular, también está regulado por el reloj biológico cuya alteración puede tener repercusiones directas en el desarrollo del cáncer. El concepto de cronoterapia ha surgido a partir de la evidencia que tanto la proliferación de las células, como los mecanismos responsables de la farmacocinética y la farmacodinamia de los antineoplásicos ocurren a horas específicas del día. En esta revisión se presentan las generalidades del ciclo circadiano y su relación con el ciclo celular y el cáncer. Además, se expone evidencia del uso de la cronoterapia en pacientes con leucemia linfocítica aguda y en estudios clínicos de cáncer de colon, endometrio y ovario con asignación aleatoria. Se concluye que la hora de administración de la quimioterapia debe tener en cuenta los ritmos circadianos de los pacientes. Se enfatiza en la necesidad de hacer estudios clínicos enfocados en la quimioterapia cronomodulada, con el fin de aumentar la tolerancia y efectividad de los medicamentos con los protocolos existentes.

Circadian rhythms, physiological periods of about 24 hours, coordinate the temporal processes of most, or maybe all, living beings on the planet. Cell proliferation, one of the most important events in the body, is also regulated by the biological clock, whose alteration may have a direct impact on cancer development. The concept of chronotherapy comes from evidence showing that both cell proliferation and the mechanisms responsible for pharmacokinetics and pharmacodynamics of antineoplastic drugs occur at specific times of the day. This review presents an overview of the circadian cycle and its relation to cell cycle and cancer. Also, it presents evidence for the use of chronotherapy in patients with acute lymphocytic leukemia and in randomized clinical trials for colon, endometrial and ovarian cancer. As a conclusion, the time of chemotherapy administration should take into account the circadian rhythms of patients. We emphasize on the need to conduct clinical trials focused on chronomodulated chemotherapy, in order to increase the tolerance and effectiveness of drugs under the existing protocols.

Os ritmos circadianos, períodos fisiológicos de 24 horas aproximadamente, coordenam a atividade temporal da maioria, se não, de todos os seres vivos do planeta. Um dos processos mais importantes do corpo, a proliferação celular, também está regulada pelo relógio biológico cuja alteração pode ter repercussões diretas no desenvolvimento do câncer O conceito de cronoterapia há surgido a partir da evidência que tanto a proliferação das células, como os mecanismos responsáveis da farmacocinética e a farmacodinâmica dos antineoplásicos ocorrem a horas específicas do dia. Nesta revisão se apresentam as generalidades do ciclo circadiano e sua relação com o ciclo celular e o câncer. Ademais, se expõe evidência do uso da cronoterapia em pacientes com leucemia linfocítica aguda e em estudos clínicos de câncer de cólon, endométrio e ovário com designação aleatória. Se conclui que na hora de administração da quimioterapia deve ter em conta os ritmos circadianos dos pacientes. Se enfatiza na necessidade de fazer estudos clínicos enfocados na quimioterapia cronomodulada, com o fim de aumentar a tolerância e efetividade dos medicamentos com os protocolos existentes.

Efficacy and safety of vitamin D supplementation in patients with chronic lymphocytic leukemia.

BACKGROUND: Vitamin D (VD) deficiency in chronic lymphocytic leukemia (CLL) is associated with inferior prognosis, shorter time to treatment and worse overall survival. VD deficiency is the first potentially modifiable prognostic factor in CLL. Currently, however, there is a lack of studies concerning VD supplementation in CLL patients. AIM: To evaluate the efficacy and safety of VD supplementation in patients with CLL. METHODS: A 6-month interventional study was conducted in CLL patients with lower serum 25-OH-D3 concentrations (< 30 ng/ml) than currently recommended. Patients with VD insufficiency (20-30 ng/ml) received 2000 IU of cholecalciferol/day, patients with moderate deficiency (10-19.9 ng/ml) received 4000 IU/day, and patients with severe VD deficiency (<10 ng/ml) received 6000 IU/day. RESULTS: In the analyzed group of 13 CLL subjects, only 1 patient had a VD level within the optimal range (30-80 ng/ml), 7 had an insufficient concentration, 4 had moderate deficiency, and 1 had severe deficiency. Secondary hyperparathyroidism was diagnosed in 4 subjects. Cholecalciferol supplementation (mean dose of 3384 ± 1211 IU) was followed by a significant increase in 25-OH-D3 concentration (from 17.3 ± 5.8 to 41.4 ± 17.5 ng/ml; p<0.05) and decrease in PTH (p<0.05). Five patients did not achieve the recommended 25-OH-D3 concentration. Calcium level remained unchanged and no patients developed hypercalcemia. CONCLUSIONS: VD replenishment is safe and can be effectively achieved by means of the employed cholecalciferol dosage in the majority of patients. However, some subjects may require higher doses to obtain the optimal level and immune function.

Use of Chamomilla recutita in the Prevention and Treatment of Oral Mucositis in Patients Undergoing Hematopoietic Stem Cell Transplantation: A Randomized, Controlled, Phase II Clinical Trial.

BACKGROUND: Oral mucositis is a common inflammatory complication among patients undergoing hematopoietic stem cell transplantation (HSCT). Among its therapeutic properties, Chamomilla recutita has anti-inflammatory effects. OBJECTIVE: The aim of this study was to identify the dosage of the liquid extract of C recutita in mouthwash that is needed to reduce the incidence and intensity of oral mucositis in adult patients undergoing allogenic HSCT. METHODS: In a randomized phase II clinical trial, 40 patients were randomized to receive routine care plus mouthwash containing a liquid extract of C recutita at 0.5%, 1%, or 2% (experimental groups) or standard care alone (control group). Daily evaluation was performed using the measurement scale for oral toxicity defined by the World Health Organization. Statistical analysis was performed, in which the incidence, intensity, and duration of oral mucositis were compared between each experimental group and the control group. RESULTS: The experimental group at the 1% dosage demonstrated reduced incidence, intensity, and duration of oral mucositis compared with the control group. The formulation was well tolerated by patients and was safe, as no moderate or severe adverse effects were identified. CONCLUSIONS: In this study, the use of mouthwash containing 1% C recutita extract can be associated with reduced incidence, intensity, and duration of mucositis in adults patients undergoing allogenic HSCT. IMPLICATIONS FOR PRACTICE: The results of this investigation will help nurses and other professionals in selecting the C recutita dosage used to manage oral mucositis in patients undergoing HSCT.

Evaluation of selected honey and one of its phenolic constituent eugenol against L1210 lymphoid leukemia.

People affected with leukemia are on the rise and several strategies were employed to thwart this deadly disease. Recent decade of research focuses on phenolic constituents as a tool for combating various inflammatory, cancer, and cardiac diseases. Our research showed honey and its phenolic constituents as crusaders against cancer. In this work, we explored the antileukemic activity of selected honey and one of its phenolic constituent eugenol against L1210 leukemia animal model. Results of this experiment showed that the selected honey samples as well as eugenol after intraperitoneal injection could not increase the median survival time (MST) of animals. Further, there was only slight marginal increase in the %T/C values of honey and eugenol treated groups. The number of phenolics present in the honey may not be a prime factor to promote antileukemic effect since there was no difference in the MST of two different honeys tested. This study limits the use of selected honey and eugenol against leukemia animal model.

Antitumor screening of Pterodon pubescens terpenic fraction indicates high sensitivity for lymphocytic leukemia cells.

Cancer is the second leading cause of human mortality worldwide. Therefore, the search for new drugs or alternative therapy strategies has been required. Anticancer agents have been developed from plants since the 1950s and natural products still represent an important source of new and promising bioactive molecules. This work describes the cytotoxic effects of SF5 on tumor cells of high prevalence in the world and investigated some mechanisms of its antitumor action. The antitumor screening was performed with human lung carcinoma (A549), human breast (MCF-7) and prostate (PC-3) adenocarcinoma and chronic myeloid and acute lymphocytic leukemia cell lines. The acute lymphocytic leukemia Jurkat cells presented high sensitivity to the cytotoxic effects of SF5 (inhibition of 85-90%), compared with either the chronic myeloid leukemia K562 or solid tumor cell lines (lung, breast and prostate). SF5 arrested the cell cycle in G1 phase, which may be related with the observed downregulation of mRNA expression of c-Myc transcription factor at 24 h and 36 h. SF5 treatment induced cytochrome c release from mitochondria to cytosol, leading the Jurkat cells into apoptosis, which was evidenced by the internucleosomal fragmented DNA and increased number of annexin V-FITC positive cells. The SF5 showed high cytotoxicity for lymphocytic leukemia cells and low or none for solid tumor cells, without toxicity for peripheral mononuclear cells of healthy humans. SF5 altered gene expression, arrested the cell cycle and induced apoptosis via the mitochondrial pathway, similar to traditional antineoplastic chemotherapeutic drugs.

Telomerase contributes to fludarabine resistance in primary human leukemic lymphocytes.

We report that Imetelstat, a telomerase inhibitor that binds to the RNA component of telomerase (hTR), can sensitize primary CLL lymphocytes to fludarabine in vitro. This effect was observed in lymphocytes from clinically resistant cases and with cytogenetic abnormalities associated with bad prognosis. Imetelstat mediated-sensitization to fludarabine was not associated with telomerase activity, but with the basal expression of Ku80. Since both Imetelstat and Ku80 bind hTR, we assessed 1) if Ku80 and Imetelstat alter each other's binding to hTR in vitro and 2) the effect of an oligonucleotide complementary to the Ku binding site in hTR (Ku oligo) on the survival of primary CLL lymphocytes exposed to fludarabine. We show that Imetelstat interferes with the binding of Ku70/80 (Ku) to hTR and that the Ku oligo can sensitize CLL lymphocytes to FLU. Our results suggest that Ku binding to hTR may contribute to fludarabine resistance in CLL lmphocytes. This is the first report highlighting the potentially broad effectiveness of Imetelstat in CLL, and the potential biological and clinical implications of a functional interaction between Ku and hTR in primary human cancer cells.

Trans fatty acid intake is associated with increased risk and n3 fatty acid intake with reduced risk of non-hodgkin lymphoma.

We evaluated the association of dietary fat and protein intake with risk of non-Hodgkin lymphoma (NHL) in a clinic-based study in 603 cases (including 218 chronic lymphocytic leukemia/small lymphocytic lymphoma, 146 follicular lymphoma, and 105 diffuse large B-cell lymphoma) and 1007 frequency-matched controls. Usual diet was assessed with a 128-item food-frequency questionnaire. Unconditional logistic regression was used to estimate ORs and 95% CIs, and polytomous logistic regression was used to assess subtype-specific risks. trans Fatty acid (TFA) intake was positively associated with NHL risk [OR = 1.60 for highest vs. lowest quartile (95% CI = 1.18, 2.15); P-trend = 0.0014], n3 (ω3) fatty acid intake was inversely associated with risk [OR = 0.48 (95% CI = 0.35, 0.65); P-trend < 0.0001], and there was no association with total, animal, plant-based, or saturated fat intake. When examining intake of specific foods, processed meat [OR = 1.37 (95% CI = 1.02, 1.83); P-trend = 0.03], milk containing any fat [OR = 1.47 (95% CI = 1.16, 1.88); P-trend = 0.0025], and high-fat ice cream [OR = 4.03 (95% CI = 2.80, 5.80); P-trend < 0.0001], intakes were positively associated with risk, whereas intakes of fresh fish and total seafood [OR = 0.61 (95% CI = 0.46, 0.80); P-trend = 0.0025] were inversely associated with risk. Overall, there was little evidence for NHL subtype-specific heterogeneity. In conclusion, diets high in TFAs, processed meats, and higher fat dairy products were positively associated with NHL risk, whereas diets high in n3 fatty acids and total seafood were inversely associated with risk.

Immune activation and antitumor response of ar-turmerone on P388D1 lymphoblast cell implanted tumors.

Aromatic turmerone (ar-turmerone) has been reported to have a cytotoxic effect on L-1210 and HL-60 cells. In the present study, we investigated the anticancer responses and immune activities in implanted tumor cells. Our study found that ar-turmerone inhibited the increase in the number of white blood cells, which normally increase by the injection of lymphoblast cells, or P388D1, and ar-turmerone increased lymphocyte percentage compared to the control. Tumor inhibition rate in the ar-turmerone-treated group was 11.79%, and the apoptosis indexes of the control, ar-turmerone and Glivec groups were 4.22±1.02, 5.45±1.46 and 10.01±2.01, respectively, in which only the Glivec-treated group showed a significance. The positive rates of Bcl-2 and Bax proteins which were treated by ar-turmerone did not show marked differences compared to the control group, but the Bax protein in the Glivec-treated group increased compared to the control group. The density of caspase-1, -3, -6, -9, Bcl-2, Bax, p21 and p53 mRNA in the control, ar-turmerone and Glivec groups did not change considerably, but the Bax mRNA of the Glivec-treated group increased compared to the control group. The ar-turmerone-treated group increased T-lymphocyte and B-lymphocyte proliferation activities compared to the control group, which was more significant in T-lymphocyte than in B-lymphocyte proliferation activity. The interleukin-2 (IL2) production activity of the ar-turmerone group increased compared to the control group. These findings suggest that ar-turmerone does not have a chemotherapeutic effect on tumor incidence, but it has a repressive effect on P388D1 lymphocytic leukemia. Furthermore, this protective effect of ar-turmerone from P388D1 lymphocytic leukemia resulted from the increased activity of tumor immunogenicity through increased T-lymphokine production and increased percentage of lymphocytes.

Apoptosis of K562 leukemia cells by Abnobaviscum F®, a European mistletoe extract.

Evidence suggests that mistletoe extract has the potential to be used as an anticancer agent. Abnobaviscum F® is a European mistletoe extract from the host tree Fraxinus. We investigated the effect of Abnobaviscum F on the growth and survival of different leukemia cell lines. Abnobaviscum F treatment strongly reduced survival and induced apoptosis of K562 (human myeloid leukemia), RPMI-8226 (human plasmacytoma) and L1210 (murine lymphocytic leukemia) cells in culture. Using K562 cells to further investigate the mechanism of action of Abnobaviscum F, we showed that Abnobaviscum F-induced cell death was associated with the activation of caspase-9, JNK-1/2 and p38 MAPK, as well as with the downregulation of Mcl-1, and inhibition of ERK-1/2 and PKB phosphorylation. Moreover, Abnobaviscum F treatment led to both a reduction of cellular glutathione (GSH) and the induction of ER stress (GRP78 and CHOP induction and eIF-2α phosphorylation). By contrast, Abnobaviscum F did not impact the expression of the DR4 and DR5 death receptors. The Abnobaviscum F-induced apoptosis of K562 cells was blocked by pretreatment with either GSH, z-VAD-fmk or SP600125. Our results, therefore, show that Abnobaviscum F induces apoptosis of K562 cells through the activation of the intrinsic caspase pathway, the phosphorylation of JNK-1, the reduction of cellular GSH, and the induction of ER stress.

Anti-tumor and pro-apoptotic activity of ethanolic extract and its various fractions from Polytrichum commune L.ex Hedw in L1210 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Polytrichum commune L.ex Hedw is a traditional Chinese herb for treatment of fever, hemostatic, uterine prolapse and especially for lymphocytic leukemia, but the antitumor effect and its potential mechanism remains unclear. AIMS OF THE STUDY: The present study was to investigate the possible anti-proliferative activity of ethanolic extract and the organic fractions from P. commune on murine leukemia L1210 cells. MATERIALS AND METHODS: The content of ethanolic extract and its fractions was performed on HPLC analysis with gradient elution. L1210 cells were treated with different concentrations of ethanolic extract and its fractions at different time intervals. Cell viability was evaluated using MTT assay. Apoptotic cell death was monitored by nuclear condensation and confirmed by exposure of phosphatidylserine to outer leaflet of plasma membrane. Loss of mitochondrial membrane potential was analyzed by flow cytometry using rhodamine 123 staining. RESULTS: The obtained results showed that the cell viability of L1210 cells was reduced by ethanolic extract of P. commune in a concentration-dependent manner, and the IC(50) value was about 77.22 µg/ml at 24h post treatment. The ethylacetate fraction displayed higher anti-tumor effect than that of chloroform and butanol fractions with 32.29 µg/ml (IC(50) value, 48 h). Microscopy studies revealed that ethanolic extract and ethylacetate fraction treated cells showed morphological characteristics of apoptosis such as chromatin condensation and DNA aggregation. Further, Annexin V-PE/ 7-AAD double staining showing the out leaflet of phosphatidylserine and the decline of mitochondrial membrane potential by flow cytometry confirmed that the extracts do, in fact, induce apoptosis in L1210 cells. CONCLUSION: This is the first report on anti-tumor and pro-apoptotic effect of P. commune in cultured leukemia cells, which provides scientific basis for its usefulness as traditional medicine. Further studies are needed to confirm the precise mechanism not only the crude extract but as well the monomeric chemical substances of Polytrichum commune L.ex Hedw.

Effects of bioactive compounds from carrots (Daucus carota L.), polyacetylenes, beta-carotene and lutein on human lymphoid leukaemia cells.

New therapies for leukaemia are urgently needed. Carrots have been suggested as a potential treatment for leukaemia in traditional medicine and have previously been studied in other contexts as potential sources of anticancer agents. Indicating that carrots may contain bioactive compounds, which may show potential in leukaemia therapies. This study investigated the effects of five fractions from carrot juice extract (CJE) on human lymphoid leukaemia cell lines, together with five purified bioactive compounds found in Daucus carota L, including: three polyacetylenes (falcarinol, falcarindiol and falcarindiol-3-acetate) and two carotenoids (beta-carotene and lutein). Their effects on induction of apoptosis using Annexin V/PI and Caspase 3 activity assays analysed via flow cytometry and inhibition of cellular proliferation using Cell Titer Glo assay and cell cycle analysis were investigated. Treatment of all three lymphoid leukaemia cell lines with the fraction from carrot extracts which contained polyacetylenes and carotenoids was significantly more cytotoxic than the 4 other fractions. Treatments with purified polyacetylenes also induced apoptosis in a dose and time responsive manner. Moreover, falcarinol and falcarindiol-3-acetate isolated from Daucus carota L were more cytotoxic than falcarindiol. In contrast, the carotenoids showed no significant effect on either apoptosis or cell proliferation in any of the cells investigated. This suggests that polyacetylenes rather than beta-carotene or lutein are the bioactive components found in Daucus carota L and could be useful in the development of new leukemic therapies. Here, for the first time, the cytotoxic effects of polyacetylenes have been shown to be exerted via induction of apoptosis and arrest of cell cycle.

Antileukemic activity of genistein, a major isoflavone present in soy products.

Soy has been used in traditional medicine for the treatment of various diseases, including cancer. The isoflavones present in soy have been shown in animal models to have cancer-preventing activity. However, the therapeutic effects of isoflavones against cancer are still unclear. We have evaluated the in vitro and in vivo antileukemic activity of genistein (1), a major isoflavone present in soy. We observed that it produced a dose- and time-dependent antineoplastic activity against myeloid and lymphoid leukemic cell lines. In addition, genistein treatment of the leukemic cells reactivated tumor suppressor genes that were silenced by aberrant DNA methylation. A genistein-enriched diet produced a moderate, but significant, antileukemic effect in mice. The limited extent of this in vivo response may have been due to the rapid metabolic inactivation of genistein in mice. Due to the longer half-life of genistein in humans, a soy-enriched diet has the potential to produce plasma levels of this isoflavone in the range of the concentrations used in vitro that produced an antileukemic activity.

Timing of apoptosis onset depends on cell cycle progression in peripheral blood lymphocytes and lymphocytic leukemia cells.

Apoptosis results in cell death within 10 min after initiation by Bcl-2 family proteins and mitochondria; however, cells enter the apoptotic pathway at different elapsed times after being triggered. Intrinsic factors related to chemical or physical cell damage can initiate apoptosis at a specific cell cycle phase; it is not clear whether cells insulted via an extrinsic pathway also die at a specific cell cycle phase, or how apoptosis is related to cell cycle progression in cells. To illustrate the kinetic changes of apoptosis during cell cycle progression, we examined both intrinsically and extrinsically induced apoptosis in MOLT-4 and Jurkat lymphocytic leukemia cells and in cultured peripheral blood lymphocytes (PBLs) using a recently modified annexin V and propidium iodide method, which detects cell cycle-specific apoptosis. Apoptosis predominantly occurred at a specific cell cycle phase. Leukemia cells were sensitive to induction by both intrinsic (X-rays, UV light, camptothecin, arsenic trioxide, and the traditional Chinese medicine Jinke, which is an extract of Auricularia auricula) and extrinsic factors (via Fas and TNF receptor pathways). The phase at which leukemia cells entered apoptosis depended on the nature of the insult (X-ray or UV, G1-phase; camptothecin, S-phase; arsenic, G1/S phases; Jinke, G1/S phases; and TNF or Fas ligand, G1/S phases), whereas PBLs did not exhibit such insult-dependent differences. PHA-stimulated PBLs entered apoptosis, and additional cells were recruited following additional insults. Unstimulated PBLs remained unresponsive to apoptosis, and proliferating cells became insensitive to the insults after the cell cycle checkpoint was abolished by caffeine. Confluent or starving PBLs were also unresponsive to apoptotic triggers. Thus, apoptotic cell death is a cell cycle event with most, if not all, apoptosis being initiated during a particular cell cycle phase, and changes in the cell cycle result in changes in the apoptotic pattern and schedule. The coordination of apoptosis and proliferation in cells offers a mechanism for the integration of both cell cycle and apoptotic signals.