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Sensitization to the Cytotoxicity of Cisplatin by Transfection with Nucleotide Excision Repair Gene Xeroderma Pigmentosun Group A Antisense RNA in Human Lung Adenocarcinoma Cells

Institute of Environmental Medicine
¹ and Department of Immunology,
² Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Peoples Republic of China

ABSTRACT

Purpose: The resistance of tumor cells to chemotherapeutic agents, such as cisplatin, remains an important problem to be solved in cancer chemotherapy. One of the mechanisms associated with cisplatin resistance is the enhanced nucleotide excision repair (NER) capacity. Because xeroderma pigmentosum group A (XPA) plays a central role at an early stage in the NER pathway, we are interested in whether down-regulation of XPA gene expression by antisense RNA transfection could reduce DNA repair and thus sensitize tumor cells to cisplatin.

Experimental Design: Human lung adenocarcinoma cells were stably transfected XPA antisense RNA expression vector, and six colonies were selected for determining the XPA mRNA level by Northern blot. The cell viability was measured by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) metabolic assay, and the host cell reactivation was employed to assess the NER capacity of cisplatin-damaged luciferase reporter plasmid. Flow cytometry analysis was used to determine cisplatin-induced apoptosis.

Results: We showed that transfection with antisense XPA RNA could decrease the XPA mRNA level and sensitize tumor cells to cisplatin. This enhanced sensitivity can be attributed to the reduced NER capacity in transfected cells as measured by the host cell reactivation assay. Moreover, the XPA mRNA level is correlated significantly with both cisplatin IC₅₀ value and cellular NER capacity. Furthermore, a more pronounced apoptotic response was observed in transfected cells treated by cisplatin.

Conclusions: Our results suggest that the targeted inhibition of XPA by antisense strategy may provide a valuable tool in clinical cancer chemotherapy.

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