ONCOLOGY REPORTS 32: 1513-1520, 2014 Downregulation of phosphoglycerate kinase 1 by shRNA sensitizes U251 xenografts to radiotherapy YI-JUN CHENG1, HAO DING1, HUA-QING DU2, Hua YAN1, JIN-BING ZHAO1, WEN-BIN ZHANG1, YUAN-JIE ZOU1, HONG-YI LIU1 and HONG XIAO2 1Department of Neurosurgery and 2Neuro-Psychiatric Institute, Nanjing Brain Hospital Affiliated to Nanjing Medical University, Nanjing, Jiangsu, P.R. China Received April 6, 2014; Accepted July 9, 2014 DOI: 10.3892/or.2014.3353 Abstract. Phosphoglycerate kinase 1 (PGK1) has been demon- radiotherapy is one of the standard treatments for glioma. Yet, strated to be involved in radioresistance. The present study was the prognosis remains dismal due to the ability of gliomas to designed to investigate the effect of PGK1 on the radioresis- infiltrate diffusely into the normal brain parenchyma, a direct tance in vivo. U251 glioma cells were transfected with the short consequence of the transformation into genetic higher-grade hairpin RNA (shRNA)-PGK1 and pcDNA3.1-PGK1 using gliomas and recurrence (4-6). As known, radioresistance is Lipofectamine 2000. The radiosensitivity of U251 xenografts a common phenomenon in gliomas and, to date there are no was observed by tumor growth curve following radiotherapy. valid biomarkers for evaluating radiosensitivity. Quantitative PCR, western blot analysis and immunohisto- In fact, radiotherapy which relies on the generation of chemistry were performed to evaluate PGK1 expression in the oxygen super-radicals, often fails to kill tumor cells, due to xenografts from the different tumor models. The expression inadequate oxygen stress within the tumor cell mass (7,8). of PGK1 was maximally inhibited in response to shRNA4 at However, in tumor microenvironments where oxygen is scarce 24 h after the transfection in vitro. Tumor growth of the U251 and glucose consumption is high, as within the expanding xenografts was significantly inhibited following treatment tumor mass, a metabolic pathway shift occurs in order to with shRNA-PGK1 and radiotherapy. The expression of PGK1 maintain local homeostasis. This phenomenon is known as the in vivo at the mRNA and protein levels was downregulated by Warburg effect. Transcription of numerous enzymes such as the treatment of shRNA1 when compared to levels following phosphofructokinase-1 (PFK-1), lactate dehydrogenase (LDH) treatment with shNC and PBS after radiotherapy. The results and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is showed that suppression of PGK1 enhanced the radiosensi- activated in the glycolytic pathway. Among these enzymes, tivity of U251 xenografts and suggest that PGK1 may serve as PGK1, an ATP-generating glycolytic enzyme, plays a vital role. a useful target in the treatment of radioresistant glioma. It catalyzes the transfer of a high-energy phosphoryl group from the acyl phosphate of 1,3-diphosphoglycerate to ADP to Introduction produce ATP. PGK1 has also been proven to have an increased expression level in many malignant tumors (9-16). The possible The annual incidence of gliomas which account for more than role of PGK1 in radioresistance has been previously studied. 50% of all brain tumors in adults is 6/100,000 (1). Based on Data from our laboratory showed that expression of PGK1 is the histopathologic subgroups characterized by different levels significantly upregulated in radioresistant astrocytomas and of aggressiveness and malignancy, astrocytomas account for radioresistant glioma U251 cells (17,18). As an initial step toward 60-70% of gliomas. Extensive surgical resection, which corre- identification of a clinically applicable method for regulating lates with patient survival independently (2,3), followed by PGK1 for combination with radiotherapy, we investigated the effects of shRNA-PGK1 on the in vitro radiosensitivity of human glioma U251 cells (18). Downregulation of PGK1 by shRNA combined with radiotherapy significantly decreased Correspondence to: Professor Hong-Yi Liu, Department of cell viability and reduced the ability of migration and inva- Neurosurgery, Nanjing Brain Hospital Affiliated to Nanjing Medical sion in U251 cells. This indicates that PGK1 may be closely University, 264 Guangzhou Road, Nanjing, Jiangsu, P.R. China associated with glioma radioresponse and may be a potential E-mail: [email protected] target with which to sensitize conventional radiotherapy. Professor Hong Xiao, Department of Neuro-Psychiatric Institute, However, the exact effect of PGK1 on the radiosensitivity Nanjing Brain Hospital Affiliated to Nanjing Medical University, in vivo using animal models has not yet been clarified. To 264 Guangzhou Road, Nanjing, Jiangsu, P.R. China further evaluate the potential radioresistance of PGK1, we E-mail: [email protected] extended these in vitro results to an in vivo xenograft model. Mice bearing U251 xenografts were exposed to shRNA-PGK1 Key words: glioma, U251 xenograft, PGK1, radiosensitivity and the level of PGK1 was determined. Radiation was then delivered in a single exposure, and shRNA-PGK1 treatment 1514 CHENG et al: Downregulation of PGK1 by shRNA sensitizes U251 xenografts to radiotherapy resulted in a significant enhancement in tumor response. These (3x107) which were diluted in 100 µl phosphate-buffered saline findings suggest a possible approach for the rational design of (PBS) containing 50% Matrigel (BD Biosciences, Franklin a clinical protocol combining downregulation of PGK1 and Lakes, NJ, USA) by using 0.1 ml/inoculation in the lower back radiotherapy. region of the mice. Materials and methods Statement of ethics. Animal studies were carried out in accor- dance with the US National Institutes of Health Guidelines Cell lines and culture. Human U251 cells were obtained and followed the rules of the National Animal Protection of from KeyGen Biotech (Nanjing, China), and radioresistant China. The present study was approved by the Medical Ethics U251 cells (RR-U251 cells) were established according to a Committee of Nanjing Brain Hospital Affiliated to Nanjing previously described method (18). The cells were cultured in Medical University (ethic no. 2011KY001). All efforts were Dulbecco's modified Eagle's medium (DMEM) supplemented made to minimize suffering and to reduce the number of mice with 10% fetal calf serum (both from Gibco-Invitrogen, used. Carlsbad, CA, USA), 100 U/ml of penicillin and 100 µg/ml of streptomycin in a 5% CO2 humidified incubator at 37˚C. Delivery of shRNA in vivo and radiotherapy. When the s.c. tumors of the nude mice were ~60-80 mm3 in size, shRNA- Plasmid molecules and stable transfection in vitro. The PGK1 (500 pmol/tumor, 25 µl injection volume) was slowly shRNA-PGK1 plasmid and the high expression plasmid injected into the tumors at different sites. Then, the tumors pcDNA3.1-PGK1 were synthesized commercially by were immediately electrically pulsed (electric field strength, GenePharma Co. Ltd. (Shanghai, China). shRNA1 (5'-GCAAG 180 V/cm; pulse duration, 20 msec; pulse number, 6; frequency GATGTTCTGTTCTTGA-3'), shRNA2 (5'-GCTCAACAAC of pulses, 1 Hz) using Square Wave pulse (Ningbo Scientz ATGGAGATTGG-3'), shRNA3 (5'-GGATGTCTATGTCAA Biotechnology, Ningbo, China) though 2 parallel stainless TGATGC-3') and shRNA4 (5'-GAAGATTACCTTGCCTGT steel electrodes on each side of the long diameter of the TGA-3') were designed to target the coding region of the tumor in situ. The negative control group was injected with human PGK1 sequence (gene ID: 5230). A negative control shRNA-NC and the control group with PBS. Fifty-four nude shRNA-NC (5'-GTTCTCCGAACGTGTCACGT-3') was also mice were divided randomly and equally into 9 groups, obtained from Shanghai GenePharma. U251 cells were tryp- and the groups were as follows: i) U251+shRNA-PGK1; sinized and harvested from a monolayer to a cell suspension in ii) U251+shNC; iii) U251+PBS; iv) RR-U251+shRNA-PGK1; DMEM containing 10% fetal bovine serum without antibiotics v) RR-U251+shNC; vi) RR-U251+PBS; vii) overexpressed- and allowed to seed overnight. When the cell confluency was PGK1 U251+shRNA-PGK1; viii) overexpressed-PGK1 70-80%, complexes of shRNAs or pcDNA3.1-PGK1 duplexes U251+shNC; and viiii) overexpressed-PGK1 U251+PBS. and Lipofectamine 2000 (Invitrogen) were prepared as Groups were administered the indicated treatment every 24 h follows: 4 µg shRNAs or pcDNA3.1-PGK1 and 8 µl of for 14 days. After the first treatment, groups were treated with Lipofectamine 2000 were diluted in 240 µl Opti-MEM I a dose of 5 Gy of radiation by a 60Co source at 0.5 Gy/min for medium (Gibco) separately, and after 5 min standing at room 10 min every 3 days until the cumulative dose of radiotherapy temperature, gentle mixing was conducted. After a 20-min reached 20 Gy. The tumor size was measured every other day incubation of this mixture, the complexes were added to each with calipers, and the tumor volume (V) was measured with well (final concentration for DNA plasmids was 2 µg/ml). The all measurements in millimeters using the following formula: medium for transfection was replaced by complete medium at V = 0.523 x length x width2. 6 h after transfection and observed under a fluorescence microscope (Axiovert 40 CFL; Carl Zeiss, Oberkochen, Real-time polymerase chain reaction (RT-PCR). Total Germany) to observe the transfection efficiency at 24 h after RNA was extracted from an equal number of cells in vitro transfection. The expression of PGK1 was analyzed, respec- and the tumor samples with the SV Total RNA Isolation tively, at 24 and 48 h after transfection. A control and a negative System (Promega, Madison, WI, USA) according to the control were also placed in the 6-well plates. In addition, manufacturer's protocol and quantified by UV absorbance complete medium with G418 (400 µg/ml) (Keygen, Nanjing, spectroscopy. RNA was reverse transcribed into cDNA with China) was used to select the stable clones which were trans- a reverse transcription system (Promega). mRNA expression fected with pcDNA3.1-PGK1. was determined by RT-PCR using GoTaq® qPCR Master Mix (Promega) under standard thermocycler conditions (Eppendorf Establishment of the nude mouse tumor xenograft models.