Abolishing multidrug resistance in cultured lung cancer cells with RNA interference

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dc.contributor.advisor Vann, Carolyn N.
dc.contributor.author Prajapati, Kamal
dc.date.accessioned 2011-06-09T15:25:45Z
dc.date.available 2011-06-09T15:25:45Z
dc.date.created 2011-06-09 en_US
dc.date.issued 2010-07-24
dc.identifier.uri http://cardinalscholar.bsu.edu/handle/123456789/193322
dc.description.abstract The gene, cox-1, is over-expressed in cultured GLC4 small cell lung cancer cells concurrent with the development of multi-drug resistance (MDR) as a result of the use of the chemotherapeutic agent used to combat the cancer, doxorubicin. Prevention of MDR has been a tremendous challenge in cancer research and this research is concerned with abolishment of MDR as a cancer survival strategy. RNA-mediated interference technology (RNAi) was employed using siRNA to decrease cox-1 expression and temporarily restore the susceptibility of the cells to doxorubicin. GLC4 cells are of three types: S (sensitive cells never exposed to doxorubicin); ADR (MDR cells cultured in doxorubicin), and; REV (revertant cells previously cultured in presence of doxorubicin but no longer). REV and ADR cells were transfected with cox-1 siRNA. After 24 h, 1x106cells were used for RNA isolation and 1 μg of RNA was used for RT-PCR to assess down-regulation of cox-1 RNA. RT-PCR results indicated that cox-1 RNA was down-regulated to basal levels seen before exposure to doxorubicin. Ct values for GLC4/ADR and cox-1 down-regulated GLC4/ADR cells were 23 and 34, respectively. The result indicated abundant levels and moderate levels of cox-1 mRNA in the ADR cells and the transfected ADR cells respectively. The relative expression level of cox-1 mRNA was 33% higher in the non-transfected GLCR/ADR cells as compared to the transfected GLCR/ADR cells as shown by the curve. Two hundred thousand cells were used for hemacytometer cell counts in the presence of trypan blue to assess cell viability. cox-1 down-regulation in ADR cells resulted in a significantly higher percentage of non-viable cells (25.4%) as compared to its non-transfected control (20.5%) using a Student’s t-test (*P <0.05). Similarly, fluorescence microscopy confirmed that apoptosis was significantly increased in the ADR cells treated with doxorubicin and cox-1 siRNA simultaneously (69.4%) as compared to its non-transfected control (56.7%) (*= P <0.01). A Western blot analysis performed by Fernando Cuadrado indicated that siRNA transfection decreased the expression of COX-1 by 66% in GLC4/ ADR cells as compared to the non-transfected control using densitometry. However, no conclusive results were obtained using flow cytometry as the flow cytometer was incapable of analyzing the mixed cell population (adherent and suspension) which is a characteristic of this cell line, GLC4. Thus, we have clearly demonstrated that MDR cancer cells can be altered temporarily to become susceptible to doxorubicin, a potentially important finding for the treatment of cancer patients.
dc.description.sponsorship Department of Biology
dc.subject.lcsh Small cell lung cancer -- Gene therapy.
dc.subject.lcsh Gene silencing.
dc.subject.lcsh Cyclooxygenases -- Inhibitors.
dc.subject.lcsh Multidrug resistance.
dc.subject.lcsh RNA interference.
dc.subject.lcsh Doxorubicin -- Effectiveness.
dc.title Abolishing multidrug resistance in cultured lung cancer cells with RNA interference en_US
dc.description.degree Thesis (M.S.)
dc.identifier.cardcat-url http://liblink.bsu.edu/catkey/1610827

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  • Master's Theses [5510]
    Master's theses submitted to the Graduate School by Ball State University master's degree candidates in partial fulfillment of degree requirements.

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