Tesis presentada a la Universidad de Chile para optar al Grado de Doctor en Farmacología / La mitocondria ha sido recientemente reconocida como potencial blanco molecular en el tratamiento del cáncer, dado que exhibe un potencial de transmembrana (Ψm) significativamente mayor en células tumorales que en células normales y por tanto sería más susceptible de ser atacada con cationes lipofílicos deslocalizados con trifenilfosfonio (TPP+). Resultados previos de nuestro laboratorio mostraron que derivados TPP+ del ácido gálico son citotóxicos y eficaces desacoplantes de la generación de Ψm en células de cáncer de mama de ratón (TA3/Ha). Con el objetivo de incrementar la eficiencia y la eficacia citotóxica, se sintetizaron nuevos derivados TPP+ de los ácidos gentísico (GA) y protocatecuico (PCA), y se evaluó su potencial citotoxicidad en varias líneas celulares humanas de cáncer de mama, que difieren tanto en su expresión de receptores hormonales y de factores de crecimiento, como en su metabolismo basal. Las líneas celulares utilizadas representan además 4 subtipos distintos de CM: Luminal A (MCF-7; ER+/HER2/neu-), Luminal B (MDA-MB-361 y BT-474; ER+/HER2/neu+), HER2/neu sobreexpresado (AU565; ER-/HER2/neu+) y triple negativo (MDA-MB-231; ER-/HER2/neu-). Los resultados muestran que GA-TPP+ y PCA-TPP+ poseen una alta potencia citotóxica en todas las líneas celulares tumorales ensayadas, y que dicho efecto es significativamente menor en la línea celular MCF 10F de epitelio mamario humano normal. Se observó, sin embargo, que algunas líneas celulares mostraron leves diferencias en su sensibilidad (posiblemente dadas por su capacidad de activar vías metabólicas alternativas o por su mayor expresión de transportadores dependientes de ATP). Se demostró además que ambos compuestos son capaces de incrementar el consumo de oxígeno celular, desacoplando la fosforilación oxidativa e induciendo una importante pérdida de Ψm, junto con un incremento significativo en el estrés oxidativo celular y una caída en la razón GSH/GSSG, tanto en células altamente oxidativas como en aquellas preferentemente glicolíticas. Ambos derivados indujeron, además, una caída leve de ATP, la cual fue exacerbada tras la co-incubación con inhibidores de AMPK o adenilato quinasa. Esto último sugiere que ambas enzimas podrían activarse como respuesta a la acción de los compuestos a nivel mitocondrial. Adicionalmente, se observó que ambos compuestos incrementan la biogénesis mitocondrial, lo que podría representar un mecanismo compensatorio alternativo frente al efecto de los ácidos unidos a TPP+. Todos los efectos antes mencionados desencadenarían la activación de la muerte celular apoptótica. Finalmente, se demostró que PCA-TPP+ y GA-TPP+ son capaces de inhibir de manera importante la migración de células metastásicas de cáncer de mama humano. En base a nuestros resultados, postulamos que GA-TPP+ y PCA-TPP+ alteran de manera importante el funcionamiento mitocondrial, y que tal efecto podría subyacer su citotoxicidad selectiva frente a células de cáncer de mama. Lo anterior nos permite afirmar que ambos compuestos presentan un interesante potencial como posibles agentes farmacológicos anti-tumorales, e invita a continuar la investigación en torno a su efecto citotóxico selectivo en modelos animales de cáncer mamario / Mitochondria has been recently acknowledged as a potential molecular target in the treatment of cancer, especially considering that such organelle exhibits a significantly higher transmembrane potential (Ψm) in cancer cells than in normal cells, and is thus more susceptible to be targeted with lipophilic delocalized cations such as triphenylphosphonium (TPP+) derivatives. Previous work conducted by our laboratory has shown that TPP+-derivatives of gallic acid were cytotoxic and effective uncouplers of the Ψm generation in TA3/Ha mouse breast cancer cells. Therefore, and aiming to further expand our knowledge on the possible mechanisms underlying the cytotoxic effect of TPP+ derivatives, gentisic acid (GA) and protocatechuic acid (PCA), both linked to the TPP+ moiety, were evaluated as potential cytotoxic agents in several human breast cancer cell lines, that differ primarily in their expression of estrogen and epidermal growth factor receptors and in their metabolic profile. In addition, the cell lines represent the four clinical subtypes of breast cancer: Luminal A (MCF-7; ER+/HER2/neu-), Luminal B (MDA-MB-361 y BT-474; ER+/HER2/neu+), HER2/neu overexpressed (AU565; ER-/HER2/neu+) and triple negative (MDA-MB-231; ER-/HER2/neu-). Results obtained show that GA-TPP+ and PCA-TPP+ are highly cytotoxic to all tumoral cell lines tested, and that such effect is significantly lower in the normal human breast epithelial cell line MCF 10F. It was observed, nonetheless, that some cell lines showed minor differences in their sensitivity towards the cytotoxic effect (possibly due to their ability to activate alternative metabolic pathways or to their higher expression of ATP-dependent transporters). Additionally, GA-TPP+ and PCA-TPP+ were shown to increase oxygen consumption, uncoupling oxidative phosphorylation and inducing a significant loss of Ψm, together with an increase in cellular oxidative stress and a drop in the GSH/GSSG ratio, both in highly oxidative and highly glycolytic human breast cancer cells. Moreover, both derivatives were able to induce a minor drop in ATP, which was exacerbated upon the co-incubation with inhibitors of either AMPK or adenylate kinase. The latter suggests that the activation of both enzymes could represent a compensatory mechanism in response to the mitochondrial effect of TPP+ derivatives. In the same line, it was shown that GA-TPP+ and PCA-TPP+ are able to enhance mitochondrial biogenesis, an event that could represent another compensatory mechanism against the damage induced by both compounds. All the effects mentioned above would eventually trigger the activation of apoptotic cell death, evidenced as an increase in annexin V-staining. Finally, we demonstrated that both acids linked to TPP+ strongly inhibit the migration of highly metastatic breast cancer cells. Based on our results, we postulate that the GA-TPP+ and PCA-TPP+ derivatives are able to strongly alter mitochondrial functioning, and that such effect would underlie their selective cytotoxicity against cells that belong to different clinical subtypes of breast cancer. Such contention allows us to postulate GA-TPP+ and PCA-TPP+ as molecules with pharmacological potential in breast cancer treatment, and warrants future research regarding their Cytotoxicity and selectivity in “in vivo” breast cancer models
| Identifer | oai:union.ndltd.org:UCHILE/oai:repositorio.uchile.cl:2250/142374 |
| Date | January 2016 |
| Creators | Sandoval Acuña, Cristián |
| Contributors | Ferreira Parker, Jorge, Speisky Cosoy, Hernán |
| Publisher | Universidad de Chile |
| Source Sets | Universidad de Chile |
| Language | Spanish |
| Detected Language | English |
| Type | Tesis |
| Rights | Attribution-NonCommercial-NoDerivs 3.0 Chile, http://creativecommons.org/licenses/by-nc-nd/3.0/cl/ |
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