PTEN gene therapy induces apoptosis in tumor cells and enhances sensitivity to oxaliplatin in colorectal cancer.
PTEN gene therapy induces apoptosis in tumor cells and enhances sensitivity to oxaliplatin in colorectal cancer.
Narjes Nakhaee,1Mahboubeh Kabiri,2,*Sirous Zeinali,3Ladan Teimoori-Toolabi,4
1. Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran 2. Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran 3. Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran 4. Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran
Introduction: Colorectal carcinoma (CRC) is the most common malignancy of the gastrointestinal tract and the third most common cancer worldwide (1). Nowadays, surgical resection and systematic chemotherapy are applied for CRC treatment. Oxaliplatin is the first platinum derivative that is effective in the treatment of CRC (2). However, due to drug resistance, advances in chemotherapy for CRC are limited. A tumor suppressor gene on chromosome 10q23, PTEN, has been reported to be mutated or deleted in a variety of tumors, including colon cancer (3). The initial identification of PTEN as a phosphatase suggested its functional role as a regulator of mitogenic signaling pathways. Specifically, PTEN acts as a phosphatidylinositol phosphatase with a possible role in phosphatidylinositol 3 -kinase (PI3-K)-mediated signal transduction. Activation of the proto-oncogene Akt, a serine-threonine kinase downstream in the PI3-K pathway, promotes cell survival by inhibiting the function of pro-apoptotic proteins. In the nucleus, PTEN-mediated PPIs enhance the activity of numerous protein substrates such as the E3 ubiquitin ligase APC/C and p53 (4). In general, these nuclear interactions have been shown to benefit chromosomal stability and healthy cell-cycle progression (4). Introduction of PTEN into PTEN deficient cells inhibits the activation of Akt (5). Activating mutations, amplifications and rearrangements in the upstream members of the pathway, e.g. RAS and PTEN are the most common events in cancers (6) . In the present study, we show that PTEN gene therapy of human colorectal cancer cells inhibit tumor growth and exhibits increased efficacy when combined with oxaliplatin.
Methods: SW480 Cell lines were cultured in DMEM with 10% heat-inactivated FBS. The cells were cultured in 5% CO2 and 37 °C. PTEN was cloned into pCMV-Tag2 plasmid, purchased from Addgene. When the confluency of SW480 cells reached to 80%, they were transfected by calcium phosphate method. Neomycin was used for the selection of transfected cells. Cells were treated with 2𝜇M oxaliplatin 24h post transfection and harvested 48h post treatment.
Results: Cells transfected with PTEN show 33% more cell death in flowcytometry, compare to cells transfected with control plasmid. Similar comparison was conducted between cells transfected with PTEN and treated with oxaliplatin and cells with control plasmid and oxaliplatin treatment which indicate 60% increase in cell death in the PTEN-transfected cells.
In cells transfected with PTEN, real-time PCR data reveal an increase of the caspase 9 gene, demonstrating the protein's function in the intrinsic route of apoptosis.
Conclusion: First reported by Tanaka et al. in 2000, PTEN was exogenously introduced via an adenoviral vector into PTEN-null human bladder cancer cells where it was found to suppress tumor cell growth through the abrogation of AKT signaling (7). Subsequent studies involving human colorectal and prostate cancer cells yielded similar results (8,9).
This research demonstrates that forced expression of PTEN can provide effective therapy for some colorectal cancers. It enhanced the sensitivity to oxaliplatin in SW480 cells by promoting apoptosis. Combining PTEN gene therapy with oxaliplatin can result in greater efficacy.