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Original Article Adenovirus-Delivered PDCD5 Counteracts Adriamycin Resistance of Osteosarcoma Cells Through Enhancing Apoptosis and Inhibiting Pgp

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Original Article Adenovirus-Delivered PDCD5 Counteracts Adriamycin Resistance of Osteosarcoma Cells Through Enhancing Apoptosis and Inhibiting Pgp Int J Clin Exp Med 2014;7(12):5429-5436 www.ijcem.com /ISSN:1940-5901/IJCEM0002682 Original Article Adenovirus-delivered PDCD5 counteracts adriamycin resistance of osteosarcoma cells through enhancing apoptosis and inhibiting Pgp Hui Zhao1, Changliang Peng2, Guorui Ruan3, Junlin Zhou1, Yihan Li1, Yong Hai1 1Department of Orthopedics, Beijing Chaoyang Hospital, Capital Medical University, Beijing, China; 2Department of Orthopedics, Second Affiliated Hospital, Shandong University, Jinan, China; 3Peking University People’s Hospital and Institute of Hematology, Beijing, China Received September 21, 2014; Accepted November 25, 2014; Epub December 15, 2014; Published December 30, 2014 Abstract: In the present study, we investigated the roles of PDCD5 (programmed cell death 5) in multidrug re- sistance (MDR) of osteosarcoma cells and the possible lurking mechanisms. An adenovirus expression vector of PDCD5 was constructed and transfected into human adriamycin-resistant osteosarcoma cell line Saos-2/ADM. We found that up-regulation of PDCD5 could significantly enhance the sensitivity of Saos-2/ADM cells towards vincris- tine, methotrexate, cisplatin and arsenic trioxide (As2O3), and could decrease the capacity of cells to efflux adria- mycin. PDCD5 could significantly down regulate the expression of P-glycoprotein (Pgp), but not affect the expression of multidrug resistance associated protein (MRP) or the glutathione S-transferase (GST). PDCD5 was also able to significantly increase the apoptotic activity of modified osteosarcoma cells. Further study of the biological functions of PDCD5 might be helpful in the understanding of the mechanisms of multidrug resistance (MDR) in osteosarcoma and exploring PDCD5 based adjuvant genetic therapy. Keywords: Osteosarcoma, MDR, PDCD5, apoptosis Introduction drug-induced apoptosis, up-regulation of lipids and other biochemical changes [5-9]. However, Osteosarcoma is the most common primary the precise mechanisms of MDR have not been malignant bone tumor, mainly occurring in chil- fully elucidated to this day. dren and adolescents. Despite current treat- ment strategies, including a combination of PDCD5 (programmed cell death 5), earlier limb salvage surgery and neo-adjuvant chemo- named TF-1 apoptosis-related gene 19 (TFAR- therapy, long-term disease-free survival rates 19), is a novel apoptosis-related gene cloned as are between 60% and 76% in patients with an increased expression gene during the apop- localized disease. The patients whose tumors totic process of TF-1 cells induced by cytokine respond poorly to chemotherapy are at a higher withdrawal using a cDNA-RDA method. The risk of relapse and adverse outcome [1, 2]. The human PDCD5 gene encodes a protein ex- most challenging problem that orthopedics pressed in tumor cells, which is translocated oncologists must deal with is multidrug resis- rapidly from the cytoplasm into the nuclei of tance (MDR) induced by the classic chemother- cells during apoptosis. The appearance of apeutic agents, such as vincristine, methotrex- PDCD5 in the nuclei of apoptotic cells precedes ate, adriamycin and cisplatin [1-4]. Previous the externalization of phosphatidylserine and reports revealed a diverse range of possible fragmentation of chromosomal DNA. And the mechanisms of MDR, such as extrusion of drug nuclear translocation of PDCD5 is a universal by cell membrane pumps, increase of drug early event of the apoptotic process, and may detoxification, DNA damage repair, redistribu- be a novel early marker for apoptosis [10]. tion of intracellular drug accumulation, modifi- Several pieces of evidence have suggested that cation of drug target molecules, suppression of the expression of PDCD5 protein is down-regu- Adenovirus-delivered PDCD5 enhances apoptosis and inhibiting Pgp lated in some human tumors, such as breast manufacturer’s instructions. Reverse transcrip- cancer [11], hepatocellular carcinoma [12] and tion was performed and cDNAs were amplified gastric cancer [13]. with the following primer pairs. PDCD5, for- ward: 5’-GTGATGCGGCCCAACAG-3’; reverse: In the previous study, we have firstly found that 5’-ATCCAGAACTTGGGCTAAGATACTG-3’; and be- PDCD5 would facilitate the sensitivity of K562 ta-actin, forward: 5’-AGCGGGAAATCGTGCGT- cells to idarubicin in vitro and in vivo, suggest- G-3’; reverse: 5’-CAGGGTACATGGTGGTGCC-3’. ing that the PDCD5 might transfer high sensitiv- All of the PCR products were analyzed on 2.0% ity to other anticancer drugs through apoptosis agarose gels [16]. [14]. Here the potential roles of PDCD5 in Adriamycin resistant osteosarcoma cell line Western blotting and the possible underlying mechanisms were further investigated. The results showed that Cells were plated at 5 × 105 per well in six well PDCD5 might mediate Adriamycin resistance of plates. The following day, cells were treated osteosarcoma through regulation of Pgp and with different anticancer drugs. After treat- apoptosis. ment, for whole-cell extracts, cells were washed with PBS and lysed in the culture dishes using Methods and materials lysis buffer (150 mM NaCl, 50 mM Tris-HCL, pH Cell lines and culture 7.4, 2 mM EDTA, 1% NP-40) containing prote- ase inhibitors. Then the total cellular proteins The human osteosarcoma cell line Saos-2, was were quantified by Bradford method. Equal obtained from Memorial Sloan-Kettering Can- amounts of total protein (50-100 μg per lane) cer Center. Human Adriamycin-resistant osteo- were electrophoresed in 12% SDS-PAGE and sarcoma cell line Saos-2/ADM was selected in blotted on a nitrocellulose membrane (0.45 vitro by growing them in progressively increas- μm, Millipore, USA) in 25 mM Tris-base, 190 ing drug concentrations in the medium as mM glycine, and 20% methanol using a semi- described previously [15]. All the cells were rou- dry blotter. Membranes were blocked with 8% tinely maintained in high-glucose Dulbecco’s fat-free milk powder at room temperature for 2 Modified Eagle’s Minimum Essential Medium hours and incubated overnight with primary (DMEM) (Invitrogen Co., Carlsbad, CA) supple- antibody at 4°C. After three washes for 15 min- mented with 10% heat-inactivated fetal calf utes in PBS-Tween 20, the membrane was incu- serum (Gibco) in 37°C humidified incubator bated with the horseradish peroxidase-conju- with a mixture of 95% air and 5% CO2, fed every gated goat anti-mouse IgG antibody for 1 hour 3 days with complete medium, and subcultrued at room temperature. The membrane was when confluence was reached. washed again in PBS-Tween 20; enhanced che- miluminescence (Amersham, NJ) was added Adenovirus infection and monitored for the development of color. The construction and purification of Ad-null and The following antibodies were used: anti-Pgp, Ad-PDCD5 (E1, E3 deleted, CMV promoter) anti-MRP, anti-GST, anti-Bcl-2, anti-cleaved were performed by the AGTC Gene Technology caspase-3, anti-PDCD5, and anti-beta-actin. All Company Ltd. (Beijing, China). For infections, these antibodies were from Santa Cruz Bio- cells were plated in DMEM supplemented with technology (Santa Cruz, CA). The density of the 10% heat-inactivated fetal calf serum and cul- bands was assessed using the BioRad Mul- tured until they achieved 70-80% confluence. tianalyst software (BioRad, Hercules, CA). Culture medium was then replaced with low- In vitro drug sensitivity assay serum media (containing 0.5% FBS), and then infected 24 h later with 400 multiplicities of Different anticancer drugs were freshly pre- infection (MOI). After 36 h, cells were washed pared before each experiment. Drug sensitivity with PBS, and fresh standard medium was was assessed using 3-(4,5-dimethylthiazol- added for subsequently study. 2-yl)-2,5-diphenyltetrazolium bromide (MTT) RT-PCR assay [17]. Briefly, cells were diluted with the standard culture medium to the seeding densi- Total RNA was extracted from cells using TRIzol ty (1 × 104/well), suspended in 96-well plates reagent (Invitrogen, Carlsbad, CA), following the (200 μl/well), and incubated at 37°C for 24 5430 Int J Clin Exp Med 2014;7(12):5429-5436 Adenovirus-delivered PDCD5 enhances apoptosis and inhibiting Pgp hours. Then, cells were incubated for 72 hours tion in a water bath at 37°C for 30 minutes, the in the absence or presence of various concen- DNA samples were analyzed on 2.0% agarose trations of the anticancer agents in 200 μl gel containing 0.1 μg/ml ethidium bromide. medium. Each assay was performed according to the manufacturer’s instructions. All samples Annexin V staining and standards were run in triplicate. After cells being cultured for 72 hours, 50 μl of 2 mg/ml After incubation with 2 μg/ml of Adriamycin, MTT (Sigma) was added into each well, and Adriamycin resistant cells were washed twice cells were cultured for another 4 hours. Then, with cold PBS and resuspended in 100 μl bind- 6 supernatants were discarded and 150 μl DMSO ing buffer at a concentration of 1 × 10 /ml. (Sigma) was added into each well to dissolve Then, 5 μl Annexin V-FITC (BD Biosciences, crystals. Absorbance was determined by spec- USA) and 10 μl of 20 μg/ml propidium iodide trophotometry (Biohit, BP800, Finland) using a (50 μg/mL, Sigma) were added to these cells. wavelength of 490 nm. Cell survival rates were After incubation at room temperature for 15 calculated according to the formula: survival minutes, 400 μl Annexin-binding buffer was rate = (mean A490 of treated wells/mean A490 added to each sample, and the samples were of untreated wells) × 100%. Finally, the IC50s kept on ice for counting the stained cells by flow of different
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