Category Archives: Cancer

Synergism between Rhopalurus junceus venom and cytostatics in HeLa

J Pharm Pharmacogn Res 7(1): 67-76, 2019.

Original Article | Artículo Original

Synergistic effect of Rhopalurus junceus scorpion venom combined with conventional cytostatics in cervical cancer cell line HeLa

[Efecto sinérgico de la combinación del veneno de escorpión Rhopalurus junceus con citostáticos convencionales en la línea celular tumoral HeLa]

Arianna Yglesias-Rivera1, Hermis Rodríguez-Sánchez2, Alexis Díaz-García1*

1Research Department, Laboratories of Biopharmaceuticals and Chemistries Productions (LABIOFAM), Havana, Cuba.
2Microbiology Department, Tropical Medicine Institute “Pedro Kouri”, Havana, Cuba.

*E-mail: alediaz@ipk.sld.cu

Abstract

Context: Venom from endemic scorpion of Cuba, Rhopalurus junceus, decreases the viability of epithelial cancer cells and has negligible cytotoxic effect on normal cells. Conventional chemotherapy induces unspecific cytotoxic effect against cancer and normal cells. Pharmacological interaction of this scorpion venom with conventional cytostatics is unknown.

Aims: To evaluate the cytotoxic effect of combined treatment of Rhopalurus junceus scorpion venom and conventional cytostatics in the cervical cancer and kidney normal cell line HeLa and Vero, respectively.

Methods: Both cell lines were treated alone with different concentrations of 5-fluoruracil (0.5, 5, 50, 500 and 5000 µM), doxorubicin (0.005, 0.05, 0.5, 5 and 50 µM) and cisplatin (3.13, 6.25, 12.5, 25 and 50 µM) or combined with ½IC50 in HeLa (0.5 mg/mL) of scorpion venom. Cell viability was determined by MTT assay. The combination index and dose reduction index at different concentration levels were generated by CompuSyn software.

Results: Rhopalurus junceus scorpion venom exerted synergic effect in HeLa cancer cell line when combined with 5-fluorouracil, cisplatin and doxorubicin at 0.5 – 500 µM, 3.13 – 25 µM and 0.005 – 5 µM, respectively. Meanwhile, higher concentration levels of cytostatics combined with scorpion venom induced antagonist effects. Besides, 5-fluorouracil, cisplatin and doxorubicin as single treatment and combined with scorpion venom did not showed significant differences respect to cell viability in Vero cells.

Conclusions: Rhopalurus junceus scorpion venom is able to potentiate selectively the cytotoxicity, at low concentrations of chemotherapy drugs, against the cervical cancer cell line HeLa.

Keywords: chemotherapy drugs; cytotoxicity; HeLa; Rhopalurus junceus scorpion venom; synergism.

Resumen

Contexto: El veneno del escorpión endémico de Cuba Rhopalurus junceus disminuye la viabilidad de células tumorales epiteliales y no afecta las células normales. La quimioterapia convencional induce efecto citotóxico no específico sobre las células tumorales y normales. Las interacciones farmacológicas del veneno de escorpión con citostáticos convencionales no han sido estudiadas.

Objetivos: Evaluar el efecto citotóxico de los citostáticos, solos y combinados con el veneno de escorpión Rhopalurus junceus, en las células tumorales y normales, HeLa y Vero, respectivamente.

Métodos: Las células se trataron con los citostáticos solos a diferentes concentraciones de 5-fluorouracilo (0,5; 5; 50; 500 y 5000 µM), doxorrubicina (0,005; 0,05; 0,5; 5 y 50 µM) y cisplatino (3,13; 6,25; 12,5; 25 y 50 µM) y combinados con la ½ CI50 (0,5 mg/mL) en HeLa del veneno de escorpión. La viabilidad celular se determinó mediante el ensayo de MTT. El índice de combinación e índice de reducción de la dosis, se determinaron mediante el software CompuSyn.

Resultados: El tratamiento combinado del veneno de escorpión con 5-fluorouracilo, cisplatino y doxorrubicina a 0,5 – 500 µM, 3,13 – 25 µM y 0,005 – 5 µM, respectivamente, ejerció un efecto sinérgico sobre la citotoxicidad de HeLa. Mientras que la mayor concentración de los citostáticos, combinado con el veneno indujo efectos antagónicos. En células Vero no se obtuvieron diferencias significativas, entre el efecto citotóxico del tratamiento individual de los citostáticos 5-fluorouracilo, cisplatino y doxorrubicina o combinados con el veneno, respectivamente.

Conclusiones: El veneno de escorpión Rhopalurus junceus potencia selectivamente la citotoxicidad a bajas concentraciones de los citostáticos, en la línea tumoral cérvico-uterina HeLa.

Palabras Clave: citotoxicidad; HeLa; quimioterapia; sinergismo; veneno de escorpión Rhopalurus junceus.

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Citation Format: Yglesias-Rivera A, Rodríguez-Sánchez H, Díaz-García A (2019) Synergistic effect of Rhopalurus junceus scorpion venom combined with conventional cytostatics in cervical cancer cell line HeLa. J Pharm Pharmacogn Res 7(1): 67–76.

© 2019 Journal of Pharmacy & Pharmacognosy Research (JPPRes)

Cytotoxic activity of Vismia baccifera and V. macrophylla

J Pharm Pharmacogn Res 5(5): 320-326, 2017.

Original Article | Artículo Original

Cytotoxic activity of different polarity fractions obtained from methanolic extracts of Vismia baccifera and Vismia macrophylla (Hypericaceae) collected in Venezuela

[Actividad citotóxica de fracciones de diferentes polaridades obtenidas a partir de extractos metanólicos de Vismia baccifera y Vismia macrophylla (Hypericaceae) colectadas en Venezuela]

Janne del C. Rojas1*, Alexis A. Buitrago1,2, Francisco A. Arvelo3,4, Felipe J. Sojo3,4, Alírica I. Suarez5

1Organic Biomolecules Research Group, Research Inst., Faculty of Pharmacy and Bioanalysis, University of Los Andes, Humberto Tejera Ave., Mérida 5101, Venezuela.
2Analysis and Control Department. Faculty of Pharmacy and Bioanalysis, University of Los Andes, Humberto Tejera Avenue, Mérida 5101, Venezuela.
3Bioscience Center, Fundation of Advanced Studies Institute, Sartenejas Valley, Miranda State, 1080A. Venezuela.
4Tissue Cultivation and Tumor Biology Laboratory, Experimental Biology Institute, Central University of Venezuela, Los Ilustres Avenue, Caracas 1051, Venezuela.
5Natural Products, Faculty of Pharmacy, Central University of Venezuela, Los Ilustres Avenue, Caracas 1051, Venezuela.

*E-mail: janner@ula.ve; janne.rojas24@gmail.com

Abstract

Context: Cancer is a complex disease involving numerous changes in cell physiology and abnormal cell growth, which lead to malignant tumors. Many investigations are still carrying on in different areas including, natural products, to find a possible break point to this pathology.

Aims: To evaluate the cytotoxic activity on different polar extracts from Vismia baccifera and Vismia macrophylla collected in two locations of the Venezuelan Andes.

Methods: Cytotoxic activity assay was carried out following the colorimetric (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) MTT assay. Human tumor cell Lines from breast carcinoma without gene over-expression (MCF-7), breast carcinoma with overexpressed gene (SKBr3), prostate carcinoma (PC3) and cervix epithelial carcinoma (HeLa) were tested with different polarity solvent extracts (hexane, dichloromethane, ethyl acetate, butanol, water) from the two species under investigation. Human dermis fibroblasts were used as control cells. Mean inhibitory concentration (IC50) was calculated.

Results: Extracts from V. macrophylla showed significant inhibition of cervix epithelial carcinoma with values ranging from 6.09 µg/mL to 17.51 µg/mL; breast carcinoma with an overexpressed gene with values from 12.14 µg/mL to 16.90 µg/mL and prostate carcinoma from 10.91 µg/mL to 17.70 µg/mL. V. baccifera extracts showed the strongest activity against prostate carcinoma with an IC50 value of 2.92 µg/mL.

Conclusions: The present study showed evidence for the anticancer activity of Vismia baccifera and Vismia macrophylla extracts since caused growth inhibition in different cell lines at low concentrations, thus, it is considered not only an important contribution to the natural products research but bring supportive data for further investigations on cancer research.

Keywords: cytotoxic activity; HeLa; MCF-7; PC3; SKBr3; Vismia baccifera; Vismia macrophylla.

Resumen

Contexto: El cáncer es una enfermedad que envuelve cambios en la fisiología y crecimiento anormal de las células, conduciendo a la aparición de tumores malignos. Investigaciones están realizándose en diferentes áreas, incluyendo productos naturales, para conseguir el tratamiento que elimine en forma definitiva esta patología.

Objetivos: Evaluar actividad citotóxica de extractos de diferentes polaridades obtenidos de Vismia baccifera y Vismia macrophylla colectadas en dos regiones de los andes venezolanos.

Métodos: La actividad citotóxica se realizó siguiendo el método colorimétrico del MTT. Líneas celulares tumorales de carcinoma de mama sin sobreexpresión del oncogen (MCF-7), carcinoma de mama con sobreexpresión del oncogen (SKBr3), carcinoma de próstata (PC3) y carcinoma de cuello uterino (HeLa) fueron probadas con extractos de diferentes polaridades (hexano, diclorometano, acetato de etilo, butanol, agua) obtenidos de las dos especies en estudio. Fibroblastos de la dermis humana fueron usados como células de control. Se calculó la concentración mínima inhibitoria (IC50).

Resultados: Los extractos de V. macrophylla mostraron inhibición del carcinoma de cuello uterino con valores de IC50 entre 6,09 a 17,51 µg/mL; carcinoma de mama con sobreexpresión del oncogen (12,14 a 16,90 µg/mL) y carcinoma de próstata (10,91 a 17,70 µg/mL). Los extractos de V. baccifera mostraron la mayor actividad frente al carcinoma de próstata con una IC50 de 2,92 µg/mL.

Conclusiones: El presente estudio mostró actividad anticancerígena de los extractos de V. baccifera y V. macrophylla, al causar inhibición del crecimiento en las líneas celulares a bajas concentraciones. Se considera una contribución a la investigación de productos naturales ya que se aportan datos para futuras investigaciones.

Palabras Clave: actividad citotóxica; HeLa; MCF-7; PC3; SKBr3; Vismia baccifera; Vismia macrophylla.

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Citation Format: Rojas JC, Buitrago AA, Arvelo FA, Sojo FJ, Suarez AI (2017) Cytotoxic activity of different polarity fractions obtained from methanolic extracts of Vismia baccifera and Vismia macrophylla (Hypericaceae) collected in Venezuela. J Pharm Pharmacogn Res 5(5): 320–326.

© 2017 Journal of Pharmacy & Pharmacognosy Research (JPPRes)

Antibiotics, cytokine production and cross-talk with cancer cells

J Pharm Pharmacogn Res 4(4): 134-143, 2016.

Original Article | Artículo Original

The effect of antibiotics on cytokine production by mononuclear cells and the cross-talk with colon cancer cells

[Efecto de antibióticos sobre la producción de citocinas por células mononucleares y la interacción con células de cáncer de colon]

Meir Djaldetti1*, Nimrod Nachmias2, Hanna Bessler1

1Laboratory for Immunology and Hematology Research, 2Department of Orthopedics, Rabin Medical Center, Hasharon Hospital, 7, Keren Kayemet St Petah Tiqva, and the Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Israel.

*E-mail: meird@clalit.org.il

Abstract

Context: Antibiotics belong to the powerful weapons applied against microbial infections. It is notable that in addition to their antimicrobial effect they express immunomodulatory and anti-cancer activities.

Aims: To explore the effect of four antibiotics on the immune cross-talk between peripheral blood mononuclear cells (PBMC) and colon carcinoma cells from two human lines.

Methods: Cefotaxime, meropenem, ampicillin and vancomycin were separately added to PBMC co-incubated with cells from two human colon carcinoma cell lines, i.e. HT-29 and RKO. After 24 hours, the level of the following cytokines produced by PBMC was evaluated: IL-6, IL-1ra, IL-1β, TNFα, IFNγ and IL-10.

Results: All four antibiotics did not affect the generation of IL-6 and IL-1ra in both co-cultures. On the other hand all of them restrained the production of IL-1β by PBMC incubated with HT-29 cells. In the same incubation mixture cefotaxime, vancomycin and meropenem decreased IFNγ and IL-10 production, while ampicillin and vancomycin inhibited TNFα. As for PBMC incubated with RKO carcinoma cells, cefotaxime inhibited the production of IL-1β, IFNγ and mildly of IL-10, whereas vancomycin repressed that of IL-1β, TNFα and IFNγ. Notably, vancomycin increased the production of IL-1β and decreased that of TNFα and IFNγ. The results indicate that the four antibiotics examined exert a modulatory effect on the immune cross-talk between PBMC and human colon cancer cells from two lines expressed by a different impact on pro-and anti-inflammatory cytokines generation.

Conclusions: These findings support the conception that antibiotics may express not only an anti-microbial effect, but also possess an anti-cancer activity that may be considered for integration to the therapeutic arsenal against cancer.

Keywords: Ampicillin; cefotaxime; colon carcinoma cells; cytokines; meropenem; mononuclears; vancomycin.

Resumen

Contexto: Los antibióticos son armas poderosas aplicadas contra las infecciones microbianas. Además de su efecto antimicrobiano expresan actividades inmunomoduladoras y contra el cáncer.

Objetivos: Explorar el efecto de cuatro antibióticos sobre la interacción inmune entre las células mononucleares de sangre periférica (PBMC) y células de carcinoma de colon de dos líneas humanas.

Métodos: Cefotaxima, meropenem, ampicilina y vancomicina se añadieron por separado a PBMC co-incubadas con células de dos líneas de carcinoma de colon humano (HT-29 y RKO). Después de 24 horas, se evaluaron las concentraciones de las siguientes citocinas producidas por PBMC: IL-6, IL-1ra, IL-1β, TNFα, IFNγ e IL-10.

Resultados: Los cuatro antibióticos no afectaron la generación de IL-6 e IL-1ra en ambos co-cultivos. Por otra parte, todos contuvieron la producción de IL-1β por PBMC incubadas con células HT-29. En la misma mezcla de incubación cefotaxima, vancomicina y meropenem disminuyeron la producción de IFNγ e IL-10, mientras que la ampicilina y vancomicina inhibieron TNFα. Cuando PBMC se incubaron con células RKO, cefotaxima inhibió la producción de IL-1β, IFNγ y ligeramente de IL-10, mientras que la vancomicina reprimió las de IL-1β, TNFα e IFNγ. En particular, la vancomicina aumentó la producción de IL-1β y disminuyó las de TNFα e IFNγ. Los resultados indican que los cuatro antibióticos examinados ejercieron un efecto modulador sobre la interacción inmune entre PBMC y las células de cáncer de colon humano a partir de dos líneas, expresadas por un impacto diferente sobre la generación de citocinas pro- y anti-inflamatorias.

Conclusiones: Estos hallazgos apoyan la idea de que los antibióticos pueden presentar no sólo un efecto anti-microbiano, también poseen una actividad anti-cancerígena que pudiera ser considerada para la integración de éstos al arsenal terapéutico contra el cáncer.

Palabras Clave: Ampicilina; cefotaxima; células de carcinoma de colon; citocinas; meropenem; mononucleares; vancomicina.

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Citation Format: Meir Djaldetti, Nimrod Nachmias, Hanna Bessler (2016) The effect of antibiotics on cytokine production by mononuclear cells and the cross-talk with colon cancer cells. J Pharm Pharmacogn Res 4(4): 134-143.
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Krishnananthasivam S, Sathkumara HD, Corea E, Natesan M, De Silva AD (2017) Gene expression profile of human cytokines in response to Burkholderia pseudomallei infection. mSphere 2: e00121-17. DOI: 10.1128/mSphere.00121-17

© 2016 Journal of Pharmacy & Pharmacognosy Research (JPPRes)

Cytotoxicity of 1-O-undecylglycerol in melanoma

J Pharm Pharmacogn Res 4(2): 84-94, 2016.

Original Article | Artículo Original

Selective cytotoxic effect of 1-O-undecylglycerol in human melanoma cells

[Efecto citotóxico selectivo del 1-O-undecilglicerol en células de melanoma humano]

Marian Hernández-Colina1*, Alberto Martín Cermeño1, Alexis Díaz García2

1Instituto de Farmacia y Alimentos, Universidad de la Habana, Calle 222, No. 2317, entre 23 y 31, La Coronela, La Lisa, CP 13600, La Habana, Cuba.
2Laboratorios de Producciones Biofarmacéuticas y Químicas (LABIOFAM). Ave. Boyeros Km 16½, Rpto. Mulgoba, Boyeros, La Habana, Cuba.
Abstract

Context: 1-O-alkylglycerols are ether-linked glycerols derived from shark liver oil and found in small amounts in human milk. Previous studies showed antineoplastic activity for this family of compounds, structurally related to alkylphospholipids, but the activity of linear chain synthetic alkylglycerols in cancer cell lines is less documented. Melanoma is a high incidence cancer, highly resistant to potential treatments. Finding new anti-cancer compounds to improve melanoma prognosis is a relevant research issue.

Aims: To study the cytotoxic effect of 1-O-undecylglycerol in primary cultured normal fibroblasts and A375 human melanoma cell line.

Methods: Cells were treated with different concentrations of 1-O-undecylglycerol and viability assessed by MTT assay. Morphological changes were visualized by DAPI and acridine orange-ethidium bromide staining. Mitochondrial membrane potential was evaluated, and gene expression of P53 and BcL-2 was semi-quantified.

Results: 1-O-undecylglycerol decreased viability of A375 cells and exerted very low cytotoxicity on primary cultured normal fibroblasts. Necrosis appeared in A375 cells but not in fibroblasts, and no apoptotic changes were visualized in DAPI staining experiments. After 24 h fibroblasts and melanoma cells developed mitochondrial potential changes similar to valinomycin. The gene expression of P53 and BcL-2 decreased in treated cells.

Conclusions: 1-O-undecylglycerol exhibited selective cytotoxic activity in A375 melanoma cells when compared with primary cultured fibroblast. Its toxicity is mediated by necrosis that may be related with mitochondrial events and decrease in P53 and BcL-2 expression. The results suggest that UDG could be a useful strategy to combine with other chemotherapeutic agents in melanoma treatment.

Keywords: Alkylglycerols, cytotoxicity, melanoma, primary cultured fibroblasts.

Resumen

Contexto: Los 1-O-alquilgliceroles son éteres presentes en el aceite de hígado de tiburón y en la leche materna. Han mostrado actividad antineoplásica, pero está poco documentada para alquilgliceroles sintéticos de cadena lineal. La búsqueda de nuevos antitumorales para mejorar el pronóstico del melanoma resulta relevante, debido a su alta incidencia y resistencia.

Objetivos: Estudiar el efecto citotóxico del 1-O-undecilglicerol en cultivo primario de fibroblastos normales y la línea celular A375 de melanoma humano.

Métodos: En células tratadas con diferentes concentraciones de 1-O-undecilglicerol se monitoreó la viabilidad mediante ensayo con MTT. Los cambios morfológicos se visualizaron por tinción con DAPI y naranja acridina- bromuro de etidio. Se evaluó el potencial de membrana mitocondrial, y se semi-cuantificó la expresión de los genes P53 y BcL-2.

Resultados: El 1-O-undecilglicerol redujo la viabilidad de las células A375, y ejerció poca citotoxicidad en el cultivo primario de fibroblastos normales. Ocurrió necrosis en las células A375 pero no en los fibroblastos, y no se visualizaron cambios apoptóticos en la tinción con DAPI. Luego de 24 h ambas células desarrollaron cambios en el potencial de membrana mitocondrial similar a la valinomicina, y disminuyó la expresión de los genes p53 y BcL-2.

Conclusiones: El 1-O-undecilglicerol mostró actividad citotóxica selectiva en células A375 al compararlo con cultivo primario de fibroblastos humanos. Su toxicidad es mediada por necrosis, probablemente por eventos mitocondriales y disminución en la expresión de P53 y BcL-2. Ello sugiere que el 1-O-undecilglicerol pudiera ser útil en combinación con otros quimioterapéuticos en el tratamiento del melanoma.

Palabras Clave: Alquilgliceroles, citotoxicidad, cultivo primario de fibroblastos melanoma, necrosis.

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Citation Format: Marian Hernández-Colina, Alberto Martín Cermeño, Alexis Díaz García (2016) Selective cytotoxic effect of 1-O-undecylglycerol in human melanoma cells. J Pharm Pharmacogn Res 4(2): 84-94.

© 2016 Journal of Pharmacy & Pharmacognosy Research (JPPRes)

CO 016: CANCER CLINICAL TRIALS: STATE OF THE ART AND THE CUBAN EXPERIENCE

J Pharm Pharmacogn Res 2(Suppl. 1): S8, 2014

Special supplement with the abstract book of LATINFARMA 2013

Oral Communication

CO 016: CANCER CLINICAL TRIALS: STATE OF THE ART AND THE CUBAN EXPERIENCE

Crombet-Ramos T.

Center of Molecular Immunology, La Habana, Cuba.
Abstract

The old paradigm for oncology drug development was based on the experience of classical cytotoxic agents. According it, antitumor activity is connected to toxicity and therefore, treatments must be scaled up to maximal tolerated dose; pharmacokinetics is relevant to define the optimal schedule; an active drug should produce rapid tumor shrinkage; the response rate is a predictor of survival and tumor progression indicates treatment failure. However, the unique characteristics of biologics challenge these dogmas and demand novel developmental guidelines. For immunotherapy, the optimal biologic dose can be far below the maximal tolerated dose, mechanisms of action can be indirect and therefore not related to pharmacokinetics, effect in survival can be seen without tumor shrinkage and therapeutic effect could be delayed in time and continue beyond progression. The new emerging paradigm recommends finding the optimal biologic dose in a “proof of principle trial” according to a pre-defined biological endpoint or biomarker; followed by an efficacy assessment in a randomized trial with long term treatment and survival as the main endpoint. CIM has now 10 projects in clinical development, including biosimilars and proprietary drugs. The original product pipeline concentrates around 3 main targets: the Epidermal Growth Factor Receptor (EGFR) system, the gangliosides and the regulatory loops of the immune system. Four of the innovative products have already transited through clinical trials and received registration in several countries: the anti-EGFR humanized monoclonal antibody nimotuzumab, the anti-CD6 antibody (itolizumab), the EGF conjugated therapeutic vaccine CimaVax-EGF and the anti-idiotypic vaccine racotumomab. The clinical experience with these products illustrates the application of the new development paradigm intended to transform cancer into a chronic disease.

CO 013: BIOMODULINA T: IMMUNOMODULATOR NATURAL PRODUCT OF THE LIFE´S QUALITY

J Pharm Pharmacogn Res 2(Suppl. 1): S7, 2014

Special supplement with the abstract book of LATINFARMA 2013

Oral Communication

CO 013: BIOMODULINA T: IMMUNOMODULATOR NATURAL PRODUCT OF THE LIFE´S QUALITY

Aznar García E1, Pereira Y1, Súarez Fundora S1, Batista Speranza G2, Leyva B3.

1Clinical Study and Sanitaries Register. Centro Nacional de Biopreparados. BioCen. Carretera de Beltrán Km 1 ½ Bejucal, La Habana, Cuba.
2Hosp. Oncológico GB Grassi Costia, Roma, Italia.
3Centro Iberoamericano para la Tercera Edad. MINSAP, La Habana, Cuba.
Abstract

Roundtable is will include a groups of lecture and free topics of one thematic: The role of the Biomoduline T, pharmaceuticals compositions based a natural polypeptide compound made up of specific thymus fractions and include the formulation for the parenteral administration. Others clinical application will be analyzed in oncology; experience in Oncology Hospital Roma, Quality specification and stability was defined. The non clinical and clinical assay was demonstrated effectiveness and safety in geriatrics patient, inflammatory and autoimmune diseases. Study in geriatrics patients with respiratory disease was evaluated clinical and immunological cellular test before and after the beginning of the treatment. This medication is recommended for the immunological dysfunction. The study about the effect of Biomodulin T in children present with recurrent infection and a decrease of thymic area showed that after the treatment decreases of infections and increases the thymic area. The others clinical applications are evaluated: Cancer patient with chemotherapy treatment, rheumatoid arthritis, multiple sclerosis, AIDS, and immunodeficiency atypical.

CO 010: NEOPLASTIC TRANSFORMATION OF HUMAN MESENCHYMAL STEM CELLS CONCERTS IMMUNE-EVASION AND PRO-INFLAMMATORY PROGRAMS IN THE ABSENCE OF IMMUNE-EDITION MEDIATED PREASSURE

J Pharm Pharmacogn Res 2(Suppl. 1): S6, 2014

Special supplement with the abstract book of LATINFARMA 2013

Oral Communication

CO 010: NEOPLASTIC TRANSFORMATION OF HUMAN MESENCHYMAL STEM CELLS CONCERTS IMMUNE-EVASION AND PRO-INFLAMMATORY PROGRAMS IN THE ABSENCE OF IMMUNE-EDITION MEDIATED PREASSURE

Miranda-Rodríguez A1, Sánchez-Castellanos N1, Funes-Quesada JM2, Pérez-Rodríguez R1, de León-Delgado J1.

1Centro de Inmunología Molecular, La Habana, Cuba. E-mail: alexm@cim.sld.cu
2UCL-Cancer Institute, London, United Kingdom.
Abstract

Introduction: The presence of altered molecular pathways associated with oncogenes and tumor suppressor genes as cancer hallmark not only support the self-sufficiency on tumor cells proliferation but also determine the interaction of neoplastic cells with the host, allowing cells to escape from homeostatic control mechanisms.

The aim of this research is to assess the direct impact of cancer progression driven by genetic alterations over immune-surveillance evasion and tumor related inflammation, in a context independent on immune-edition mediated pressure. We took advantage of a model based on in vitro step-wise neoplastic transformation induced on human mesenchymal stem cells (hMSC). The genetic lesions introduced to transform hMSC included hTERT, c-Myc and H-Ras activity while p53 and pRb expression were reduced.

Methods: MSC immunogenicity was evaluated by HLA-ABC gene and protein expression. In vitro co-culture experiments were performed to determine hMSC inhibitory capacity on T cells proliferation. Immunesuppressive mediators, pro-inflammatory molecules and IFNγ signaling were assessed on hMSCby qPCR, western blot, flow cytometry, ELISA, gene micro-array and immune-fluorescence assays. Neoplastic hMSC with reduced expression of IL-beta were obtained by lentiviral infection encoding shRNA targeting IL-beta mRNA. Anchorage independent growth in soft agar was assayed as surrogate of cells tumorigenicity. In vivo tumorigenicity was evaluated by xenogenic transplantation in athymic nude mice.

Results: In vitro step-wise neoplastic transformation reduces hMSC immunogenicity and progressively increases the inhibitory capacity of hMSC on T cell proliferation, whereas a modification in the immunesuppressive mechanism is detected. Transformation leads to the generation of a pro-inflammatory phenotype characterized by increased expression of IL-beta, a mediator that concert tumorigenic and immune-suppressive potential of fully transformed cells.

Conclusions: Overall results suggest that neoplastic transformation can stimulate oncogenic inflammation and immune-surveillance evasion as an intrinsic feature of cancer cells, even in the absence of the immune-edition pressure exerted at tumor microenvironment.

CO 009: A NOVEL PEPTIDE SELECTED FROM AN ALASCANNING REGULATES THE ONCOGENIC ACTIVITY OF NFKB IN CANCER CELLS

J Pharm Pharmacogn Res 2(Suppl. 1): S5, 2014

Special supplement with the abstract book of LATINFARMA 2013

Oral Communication

CO 009: A NOVEL PEPTIDE SELECTED FROM AN ALASCANNING REGULATES THE ONCOGENIC ACTIVITY OF NFKB IN CANCER CELLS

Oliva B, Fernandez Jr, Gil J, Garay H, Reyes O, Guillen G, Vallespi GM.

Center for Genetic Engineering and Biotechnology (CIGB), P.O.Box. 6162, Cubanacan, Playa, La Habana, Cuba.
Abstract

Introduction: The nuclear factor NFkB plays a critical role in diverse cellular processes associated with proliferation, cell death, as well as inflammation. Starting from a LPS-binding peptide from Limulus antiLPS factor, with powerful immunostimulatory activity, we have done alanine substitutions of particular residues and obtained a novel synthetic peptide (CIGB-552) that has lost its LPS binding capacity and is a powerful in vitro and in vivo cytotoxic agent. The potential mechanism responsible for the antitumor activity of CIGB-552 is based on a specific degradation of RelA (p65) and inhibition of NFkB regulated genes.

Material and methods: Cell lines: HT-29 (human colon cancer) and D122 (Lewis lung carcinoma). Female Balb/c mice were purchased from CENPALAB, Cuba. The peptide was synthesized manually using Fmoc/tBu solid phase.

Results: Here we demonstrate that treatment of tumor cells with the peptide CIGB-552 promotes the degradation of RelA (p65) and induces cell cycle arrest and apoptosis. In addition, the peptide inhibits gene products activated by NFkB, such as the anti-apoptotic protein Bcl-2. Here, we demonstrate that the CIGB-552 stabilizes a key protein in tumor cells, it which inhibits the transcription activity of NFkB. These findings provide evidence to support the regulation of transcription factor NFkB by the peptide CIGB- 552 in cancer cells. Furthermore, we demonstrate that CIGB-552 decrease tumor growth and increase survival in the human colon cancer xenograft HT-29 and shows anti-metastatic activity in a spontaneous metastatic model of the Lewis lung carcinoma.

Conclusion: Our finding points out to a novel mechanism by which the anti-apoptotic activity of NFkB can be regulated in cancer cells. Better understanding of the regulation of NFkB will provide a platform for development of specific therapeutic agents targeted towards the cancer-associated inflammation.

CO 008: IMMUNOTRANS-ARTERIAL CHEMOEMBOLIZATION (ITACE) IN HEPATOCELLULAR CARCINOMA PATIENTS TREATED WITH ANTI-EGFR MAB NIMOTUZUMAB PLUS ADRIAMYCIN. PHASE I CLINICAL TRIAL, FINAL RESULTS

J Pharm Pharmacogn Res 2(Suppl. 1): S5, 2014

Special supplement with the abstract book of LATINFARMA 2013

Oral Communication

CO 008: IMMUNOTRANS-ARTERIAL CHEMOEMBOLIZATION (ITACE) IN HEPATOCELLULAR CARCINOMA PATIENTS TREATED WITH ANTI-EGFR MAB NIMOTUZUMAB PLUS ADRIAMYCIN. PHASE I CLINICAL TRIAL, FINAL RESULTS

Ramos-Suzarte M1, Hernández JC2, Roque A2, Ugarte JC2, Jordán J2, Samada M2, Lorenzo P1, Fernández A1, Catalá M2, Fermín E2, Frías A1, Izquierdo M1, Cedeño M1, Rengifo E1, Frómeta M1, Crombet T.1

1Center of Molecular Immunology, Havana, Cuba. E-mail: mayra@cim.sld.cu
2Center for Medical and Surgical Research, Havana, Cuba.
Abstract

Introduction: Hepatocellular carcinoma (HCC) is a common complication of chronic liver disease. EGFR is well validated as a primary contributor of different tumor of epithelial origin included liver cancer in initiation and progression. Nimotuzumab is a humanized monoclonal antibody (mAbs) that recognizes the EGFR extracellular domain. While it has similar preclinical and clinical activity when compared to other anti-EGFR mAbs, it does not induce skin toxicity or hypomagnesaemia.

Methods: Phase I prospective clinical trial (RPCEC00000022) to evaluate safety and optimal biological doses of immunotrans-arterial chemoembolization (ITACE) with nimotuzumab in patients with unresectable HCC. In this trial were enrolled 3 patients in 4 different level doses of Mab: 50 mg, 100 mg, 200 mg and 400 mg maintaining the same concentration of cytostatic drug per level, just one administration per patients. The principal endpoint was evaluate safety and Optimal or Maxima Biological Doses in the treatment of HCC by Trans arterial chemoembolization (ITACE) by injecting nimotuzumab an doxorubicin mixed with a radiopaque contrast (e.g. Lipiodol) and an embolic agent Gel foam into hepatic artery.

Results: The maximum tolerable dose was 200 mg of nimotuzumab, was impossible complete the administration in 400 mg because the administration rout was intra-arterial and the limiting factor was the final volume. Was confirmed a gain for these patients, since the life expectancy of them were about 6 months however median survival of 13.01 months was found to be above expected. Nimotuzumab was safe in terms of intra-arterial administration and the combination with doxorubicin. Not anti-idiotipic response was detected in any treated patients. Was the first time that nimotuzumab was administered by intra-arterial rout but is a perfect way to be used in this tumor.

Conclusions: The maxima tolerated dose was 200 mg of nimotuzumab for ITACE. The administration of nimotuzumab was safety, increasing patient survival, which provides a longer period for the possibility for patients to receive a liver transplant. The combinations with chemotherapy not increase the toxicity of therapy.

CO 005: MOLECULAR MECHANISMS INVOLVED IN THE INHIBITION OF TUMOR CELLS PROLIFERATION EXPOSED TO ELEVATED CONCENTRATIONS OF EPIDERMAL GROWTH FACTOR (EGF)

J Pharm Pharmacogn Res 2(Suppl. 1): S4, 2014

Special supplement with the abstract book of LATINFARMA 2013

Oral Communication

CO 005: MOLECULAR MECHANISMS INVOLVED IN THE INHIBITION OF TUMOR CELLS PROLIFERATION EXPOSED TO ELEVATED CONCENTRATIONS OF EPIDERMAL GROWTH FACTOR (EGF)

Camacho H, Guillen IA, Berlanga J, Fernández-de-Cossio ME, Pérez L, Palenzuela D, Díaz T, Guillen GE, Herrera L, Cosme K, Gorovaya L, Mendoza O, Fernández JR, Ancizar JA, Suarez J, Tuero A, Ochagavia ME, Roca J, Gavilondo J, García del Barco D, Martin J and Novoa LI.

Center for Genetic Engineering and Biotechnology, CIGB, Havana, Cuba. E-mail: hamlet.camacho@cigb.edu.cu
Abstract

Introduction: The EGF promotes inhibition of cell proliferation in vitro and in vivo models depending on its concentration, application schema and the type of tumor cells on which it acts. Our research hypothesis was based on the fact that the EGF varies the expression of genes involved in a negative regulation of tumor cell lines proliferation carrying high levels of its receptor (EGFR). Our objectives were, to obtain information about the effect of EGF on tumor cell proliferation in vitro and in vivo models and, know the gene expression patterns of a group of genes involved in cancer signaling pathways and EGFR.

Material and methods: We used six cancer cell lines (K562, MCF-7, U1906, LS-174T, H125 and A431) with different concentrations of EGFR. An animal model was obtained in Nude mice xenografts with the cell line A431 over-expressing the levels of EGFR The real time PCR technique was used to explore in models in vitro and in vivo, the differential expression of 44 genes involved in EGF signaling pathway and cancer in cells treated with EGF and controls. Relative quantification of gene expression was performed using the software REST 2009 v2.0.13 (QIAGEN GmbH All rights reserved).

Results: The results showed that EGF at nanomolar concentrations inhibits the tumor cells proliferation bearing high levels of EGFR and, promotes the survival of treated animals, establishing a direct relationship between the inhibition of cell proliferation, high concentrations of EGF and, high amount of EGFR in the cells. The differential gene expression profile showed a variation in a group of genes which exert a powerful control over the cell cycle progression, gene transcription and apoptosis.

Conclusions: The inhibition of tumor cell proliferation by the action of EGF is due to activation of molecular mechanisms controlling cell cycle progression.