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Reversal of drug resistance of trastuzumabresistant human breast cancer cells by recombinant adenovirus carrying PTEN gene
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Abstract:
Abstract Objective: To study the role of adenoviralmediated PTEN gene transfection in reversing drug resistance of trastuzumabresistant human breast cancer. Methods: Recombinant adenoviruses carrying PTEN gene (AdPTEN) were constructed and transfected into trastuzumabresistant human breast cancer cell line BT474. Proliferation and apoptosis of BT474 cells treated with AdPTEN and trastuzumab were measured by MTT assay and FCM. Time course of BT474 cells apoptosis induced by AdPTEN was analyzed by detection of DNA fragmentation. Western blotting was employed to measure phosphorylatedAkt expression levels in AdPTEN treated BT474 cells. The nude mice model of BT474 cells was employed to observe the effects of AdPTEN and trastuzumab on trastuzumabresistant human breast cancer in vivo. The expression of PTEN gene, cancer cells apoptosis and ultrastructural changes were evaluated after the mice were sacrificed. Results: We successfully constructed recombinant AdPTEN; and the titer of the recombinant adenovirus was about 4.2×1011TCID50/ml. PTEN gene expression was identified by PCR, RTPCR and Western blotting assay. Combinatorial AdPTEN and trastuzumab significantly suppressed the proliferation of BT474 cells and induced the apoptosis. The percentage of apopotosis cells treated with AdPTEN was （20.7±5.82）% , companied by cells cycle arrest in G1 phrase (P＜0.01）. DNA of AdPTEN treated BT474 showed a typical DNA ladder at 24-36 h after infection. An obvious downregulation of phosphorylatedAkt expression level in cancer cells was identified by Western blotting. Tumor growth was inhibited in nude mice receiving injection of the recombinant adenoviruses when compared to control mice treated with AdLacZ (P＜0.01）. PTEN protein expression was confirmed by immunohistochemistry. Tumor cell apopotosis was detected by TUNEL and electron microscopy scanning. Conclusion: Our result suggests that PTEN protein can downregulate phosphorylatedAkt and Akt kinase activity, and block the activation of PI3K/Akt pathway, and subsequently reverse trastuzumab resistance and induce cell apoptosis.
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Project Supported:
Surpported by the Shanghai Science and Technology Commisson Foundation (No.06D219509)
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