Establecimiento de cultivos primarios de cáncer cervicouterino avanzado, como modelo para terapia antisentido selectiva
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Fecha
2013
Autores
Profesor/a Guía
Facultad/escuela
Idioma
es
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Universidad Andrés Bello
Nombre de Curso
Licencia CC
Licencia CC
Resumen
El cáncer cervicouterino (CaCu) es el segundo cáncer más común en mujeres. Se
estima que alrededor de 530.000 mujeres son diagnosticadas cada año y alrededor
del 50% mueren de esta enfermedad. Por lo tanto, es necesario desarrollar nuevas y
eficientes terapias con bajos efectos secundarios. En nuestro laboratorio se ha
descrito una familia de RNAs mitocondriales no codificantes. Esta familia consta de
dos transcritos: sentido (SncmtRNA) y antisentido (ASncmtRNA). Estos RNA tienen
una expresión diferencial en función del estado proliferativo de la célula. Tanto en
células como biopsias normales proliferantes se detecta la presencia de las moléculas
sentido y antisentido, células en reposo poseen una baja presencia de ambas
moléculas y en células tumorales se detecta la presencia solo de la molécula Sentido
por hibridación in situ. El tratamiento con oligonucleótidos en líneas celulares
tumorales SiHa derivadas de cáncer cervicouterino dirigidos a la molécula antisentido
gatilla la muerte masiva de estas por apoptosis. Por otro lado, este mismo tratamiento
no induce eventos de muerte ni transformación en células normales queratinocitos,
sugiriendo selectividad y bioseguridad . Aunque las líneas celulares de cáncer se
utilizan ampliamente en la investigación del cáncer, es necesario llevar a cabo
estudios en modelos celulares que se parezcan más a la fisiología de las células
tumorales in vivo. Para este propósito, en este trabajo se estableció tres modelos de
cultivos primarios a partir de biopsias de CaCu avanzado que han sido seleccionados
por su capacidad para formar esferas en suspensión. Estos se caracterizan por el uso
de anticuerpos (E-caderina, N-caderina, p161NK4a, citoqueratina 17, survivina,
vimentina y a-SMA), PCR contra HPV, población SP y su capacidad para crecer
independiente de anclaje. El efecto del tratamiento ASO sobre las células de cultivos
primarios se evaluó mediante MTT y ensayos de TUNEL, lo que resultó en un efecto
citotóxico eficiente a 300 nM de ASO para 48 y 72 horas, que también disminuye
significativamente la capacidad de formar colonias en agar blando. Esto sugiere que
el uso de ASOs dirigidos a los ASncmtRNAs puede ser una herramienta terapéutica
eficaz y selectiva contra el cáncer.
Cervical cancer is the second most common cancer in women. lt is estimated that about 530,000 women are diagnosed each year and about 50% die of this disease. Therefore, it is necessary to develop effective therapies to decrease side effects. In our laboratory we have described a family of noncoding mitochondrial RNAs. This family consists of two transcripts: sense (SncmtRNA) and antisense (ASncmtRNA). These RNAs have a differential expression depending on the proliferative state of the cell. Normal proliferating cells in culture, as well as biopsies, show the presence of sense and antisense molecules, resting cells have a low presence of both molecules and in tumor cells only the sense is detected by in situ hybridization. Oligonucleotide treatment targeting the antisense molecules in the tumor cell line SiHa derived from cervical cancer, triggers massive death by apoptosis. On the other hand , the same treatment did not induce death or transformation events in human keratinocytes (HEKn), suggesting selectivity and biosafety. Although cancer cell lines are extensively utilized in cancer research , it is necessary to carry out studies in cellular models that resemble more closely the physiology of tumor cells in vivo. For this purpose, in this work we established three primary culture models from advanced cervical cancer biopsies, which were selected for their ability to form spheres in suspension. These were characterized by the use of antibodies (E-cadherin, N-cadherin, p161NK4a, cytokeratin 17, Survivin, vimentin and a-SMA), PCR for HPV, side population and their ability to grow independent of anchorage. The effect of ASO treatment on cell cytotoxicity of primary cultures was assessed by MTT and TUNEL assays, resulting in an efficient cytotoxic effect at 300 nM ASO for 48 and 72 hours, also significantly decreasing the ability to form colonies in soft agar. This suggest that the use of ASOs targeted to the ASncmtRNAs can be an effective and selective therapeutic tool for cancer.
Cervical cancer is the second most common cancer in women. lt is estimated that about 530,000 women are diagnosed each year and about 50% die of this disease. Therefore, it is necessary to develop effective therapies to decrease side effects. In our laboratory we have described a family of noncoding mitochondrial RNAs. This family consists of two transcripts: sense (SncmtRNA) and antisense (ASncmtRNA). These RNAs have a differential expression depending on the proliferative state of the cell. Normal proliferating cells in culture, as well as biopsies, show the presence of sense and antisense molecules, resting cells have a low presence of both molecules and in tumor cells only the sense is detected by in situ hybridization. Oligonucleotide treatment targeting the antisense molecules in the tumor cell line SiHa derived from cervical cancer, triggers massive death by apoptosis. On the other hand , the same treatment did not induce death or transformation events in human keratinocytes (HEKn), suggesting selectivity and biosafety. Although cancer cell lines are extensively utilized in cancer research , it is necessary to carry out studies in cellular models that resemble more closely the physiology of tumor cells in vivo. For this purpose, in this work we established three primary culture models from advanced cervical cancer biopsies, which were selected for their ability to form spheres in suspension. These were characterized by the use of antibodies (E-cadherin, N-cadherin, p161NK4a, cytokeratin 17, Survivin, vimentin and a-SMA), PCR for HPV, side population and their ability to grow independent of anchorage. The effect of ASO treatment on cell cytotoxicity of primary cultures was assessed by MTT and TUNEL assays, resulting in an efficient cytotoxic effect at 300 nM ASO for 48 and 72 hours, also significantly decreasing the ability to form colonies in soft agar. This suggest that the use of ASOs targeted to the ASncmtRNAs can be an effective and selective therapeutic tool for cancer.
Notas
Tesis (Magister en Biotecnología)
Palabras clave
Cáncer Cérvico-Uterino, Células Cultivadas, Células Cancerosas