Experimental Study Of Breast Cancer Treatment With Recombinant EGFR Antisense Adenovirus And Of Effects Of Recombinant EGFR Antisense Adenovirus On Glioma

EGFR is a transmembrane glycoprotein with a tyrosine kinase activity in its intracellular domain. Its overexpression has been detected in many malignant tumors. Therefore EGFR expression plays an important role in regulating the proliferation and differentiation of malignant epithelial cells. In an experimental model, we found that EGFR expression augmented with tumorigenicity increase, and the transformed phenotype could be reversed after transfection with an EGFR antisense RNA expressing vector. To potentiate the therapeutic effectiveness of antisense strategy, we exploited a recombinant antisense adenovirus for EGFR, AdE5. In this experiment we found that AdE5 can inhibit proliferation of human breast cancer cells (MDA-MB-231 cell line) in vivo, suggesting that AdE5 can be used to treat breast cancer as a novel method.We also studied the role of AdE5 on human glioma cells (U251 cell line).Part one: Experimental study of breast cancer treatment with recombinant EGFR antisense adenovirusHuman EGFR cDNA fragment was subcloned in the opposite orientation to the cytomegaloviral promoter and inserted into a E1/E3-deleted type 5 adeno viral vector to obtain AdE5 construct which expresses EGFR antisense RNA. MDA-MB-231 human breast cancer cells were cultured in vitro and then injected s.c. into right flanks of nude mice. Two intra-tumoral injections of 100μl PBS buffer containing 109 pfu of AdE5 or AdCO1 or PBS alone were practised. The growing tumors were measured, and relative gowth index were calculated, we found that the tumor growth was inhibited by AdE5 treatment (relative gowth index < 1). HE staining was performed for histological study, which revealed that AdE5caused an increase of central necrosis in tumor tissues, accompanied with an inhibition of Ki-67 expression determined by immunohistochemical staining compared with control groups. So the transduction of EGFR antisense RNA by adenovlral vector is effective for inhibition of in vivo growth of human breast cancer cells.Part two: Experimental research of effects of recombinant EGFR antisense adenovirus on glioma cellsU251 human glioma cell line was cultured in vitro, and infected with type 5 adenoviral vector containing green fluorescent protein (Ad-GFP). Almost 95% U251 cells expressed GFP after Ad-GFP infection at 300 MOI, demonstrating that type 5 adenovirus can transfect exogenous genes efficiently into U251 cells. We found that EGFR antisense RNA expressed, in a dose- and time- dependent manner, in U251 cells detected by RT-PCR. The protein expression of EGFR was also inhibited after AdE5 infection detected with Western-Blot study. Further more, when U251 cells were stimulated by 2ng/ml EGF, EGFR expression was inhibited more effeciantly byAdE5.