Influence of the MACC1 Gene on Sensitivity to Chemotherapy in Human U251 Glioblastoma Cells

Glioma is the most general primary tumors in nervous system, and show the highest mortality and mortality among endocranial tumors, because of the marked characteristics of malignant proliferation and invasion (Kuhnt et al., 2011). Gliomas are rarely curable. Treatment for a gioblastoma is customized to the individual patient and may include surgery, radiation therapy, chemotherapy, or observation. So far, surgery is the most important initial approach. Chemotherapy and radiotherapy after initial surgical resection is regarded as an effective treatment plan to prevent recurrence and metastasis (Mrugala et al., 2013). However, chemotherapy does not work for everyone with a glioma. It helps about half the people treated. For this reason, the prognosis for patients especially with high-grade glioma is still gloom, despite being given prompt and comprehensive treatment. The median survival time for adults with an anaplastic astrocytoma is about two to three years, and for those with more aggressive glioblastomas, median survival drops off to about 12~14.6 months with a two-year median survival rate of 30% (Nieder et al., 2008).


Introduction
Glioma is the most general primary tumors in nervous system, and show the highest mortality and mortality among endocranial tumors, because of the marked characteristics of malignant proliferation and invasion (Kuhnt et al., 2011). Gliomas are rarely curable. Treatment for a gioblastoma is customized to the individual patient and may include surgery, radiation therapy, chemotherapy, or observation. So far, surgery is the most important initial approach. Chemotherapy and radiotherapy after initial surgical resection is regarded as an effective treatment plan to prevent recurrence and metastasis (Mrugala et al., 2013). However, chemotherapy does not work for everyone with a glioma. It helps about half the people treated. For this reason, the prognosis for patients especially with high-grade glioma is still gloom, despite being given prompt and comprehensive treatment. The median survival time for adults with an anaplastic astrocytoma is about two to three years, and for those with more aggressive glioblastomas, median survival drops off to about 12~14.6 months with a two-year median survival rate of 30% (Nieder et al., 2008).

Influence of the MACC1 Gene on Sensitivity to Chemotherapy in Human U251 Glioblastoma Cells
Chao Shang 1 , Yang Hong 2 , Yan Guo 3 , Yun-Hui Liu 2 , Yi-Xue Xue 1 * In this new era, investigating the molecular mechanisms in carcinogenesis might shed light on this deadly disease (Vlachostergios et al., 2013;Burgio et al., 2014;Ugur et al., 2014). In our previous researches, the gene chips were used to detecte the up-regulation of metastasisassociated in colon cancer-1 (MACC1) gene in human glioma. MACC1 gene was firstly identified by Stein et al in 2009(Stein et al., 2009. MACC1 could activate the HGF/MET signal pathway and mediate the metastasis and recurrence of colorectal cancer . The current reports suggested that MACC1 participated in regulation of cell proliferation, apoptosis, migration and invasion Zhang et al., 2014;Zhen et al., 2014). Proximally, MACC1 has been identified to act as a key biomarker for the prognosis of kinds of cancer, including colorectal cancer, gastric carcinoma and nonsmall cell lung cancer (Ma et al., 2013;Yamamoto et al., 2014). Therefore, the object of this study was to first determine the correlation between the expression abnormality of MACC1 and the carcinogenesis and development of human glioma, and then search for its possible influence on chemotherapy sensitivity of human glioblastoma U251 cells.

Materials
The study was approved by the Ethics Committees, and we obtained patient's permission before surgery. Total 49 glioma and corresponding paracancerous tissues provided by the Department of Neurosurgery, Shengjing Hospital of China Medical University from May 2011 to April 2014. The tumors with at least 1cm margin from the corresponding peri-cancerous tissues were obtained from all patients through surgical resection and further histologically proven to be gliomas.
All patients had not experienced radiation or chemotherapy before surgery. The patients include 32 men and 17 women (mean age: 54.1±3.8 years, age range: 47-69 years); and included 23 cases of astrogliomas (Grade I-II), 14 of anaplastic gliomas (Grade III), 12 of glioblastomas (GBM, Grade IV). Human glioblastoma cell line U251 was obtained from Biological Sciences Cell Resource Center (China). Real-time PCR reagents were from Takara Bio (Japan), TransMessenger from Qiagen (Germen), siRNA-MACC1 and siRNA-control from Invitrogen (Carlsbad, CA, USA), DDP from Sigma (USA). The MTT Cell Proliferation Assay Kit was from Beyotime Company (Shanghai, China), Annexin V Cell Apoptosis Assay Kit from Biosea Company (Beijing, China). The PCR primers of MACC1 gene were synthesized by Takara Bio (Japan).

Cell transfection
24h before transfection, appropriate concentration (about 80%) resuspended U251 cells were seeded on 6-well plates. Then 1 mg of siRNA-MACC1 or siRNA-control was mixed with Enhancer R, followed by mixing with 4 μl TransMessenger, and then 900μl Serum-free medium was further added for incubating the non-transfected U251 cells. The transfected cells were incubated for 4h, and normal media was added. After 48h, the cells were harvested to detect further.

Western blot analysis
Cells were harvested and extracted protein. SDS-PAGE electrophoresis and antibody hybridization were practiced as describe previously. The ECL analysis system (Santa Cruz, USA) was used for detection in accordance with the manufacture's protocol. Western blot quantification was performed using Image Processing and Analysis software. GAPDH was selected to be reference protein.

Flow cytometry detection
Cells (5×10 6 ) were harvested, and the apoptosis detection kit (Biosea, China) was used to examine the apoptosis rate in accordance with manufacturer's instruction. Then the cells were read by flow cytometry (BD, USA) (Ex 488 nm, Em 635 nm), and the obtained numerical values were analyzed with CELLQuest 3.0 software (BD, USA). Annexin V positive cells were regarded as apoptosis cells. The cells were counted by a dual-color flow cytometric method.

Cell proliferation assay
Cells were seeded in 96-well plate with 2×10 4 cells per well. 20 μl of 0.5mg/ml MTT solution (Sigma, USA) was added to each well, and the 96-well plate was incubated at 37˚C. We cleaned up the media after 4 hr, and added 0.2 ml DMSO to each well. The 96-well plate was incubated 30 min, and read on an enzyme-labeled instrument (Bio-Rad, USA) with 570 nm wavelength. The obtained numerical values were used to construct the cell growth curve.

U251 cells treated by DDP
U251 cells were treated with DDP of three different concentrations (1, 3, 5μg/mL) (Jiang et al., 2000;Zhou et al., 2013), and the cell growth inhibitory rate and apoptosis rate was detected after 24h of incubation.

Statistical analysis
All results were obtained from independent experiments in three times. All numerical data were presented as mean±standard deviation (SD) and dealt with SPSS 13.0 software. A Student's t-test was performed to determine the significant differences between two groups. Pearson's correlation was analyzed between the grade of glioma and relative expression levels of MACC1 mRNA. One-way ANOVA and post hoc comparisons (LSD test) were used to determine the significant differences among multiple groups. p<0.05 was considered as significant

MACC1 mRNA up-regulated in human brain glioma specimens
Amplification of the MACC1 gene was shown in human brain gliomas using the primer melting curve analysis. The qRT-PCR analysis showed the ΔCt of glioma and peri-cancerous tissues were 2.947±0.314 and 3.614±0.297 respectively, and the ΔΔCt was -0.667. Compared with corresponding peri-cancerous tissues, the MACC1 expression increase 158.78% in the glioma (p<0.05) (Figure 1). Further, there is an positive relationship between the MACC1 expression and the pathologic grades of gliomas (Table 1).

Influence of MACC1 silencing on apoptosis and growth inhibition of U251 cell
The western blot images showed clear bands of MACC1 protein in all groups (Figure 2). The analytical results confirmed that there was no significant difference between two control groups (p>0.05). However, compared DOI:http://dx.doi.org/10.7314/APJCP.2015.16.1.195 Influence of MACC1 Expression on Chemotherapy Sensitivity in Human U251 Glioblastoma Cells No significant difference was found between two control groups (p>0.05). The apoptosis rate in MACC1 silence group was significantly elevated relative to two control groups (p<0.05).
The growth inhibitory rate increased from 1.5% in negative control group to 17.8% in MACC1 silence group (Fig.3B), presenting significantly higher inhibition of cell proliferation in MACC1 silence group (p<0.05).

Discussion
MACC1 gene was a new carcinogenesis and metastasis related gene which firstly found in colorectal cancer. The recent researches affirmed MACC1 gene act as an oncogene and metastasis-inducing gene, it could activate Met to promote HGF/Met signal pathway which had been identified necessary for carcinogenesis and metastasis. Furthermore, overexpression of MACC1 gene had been reported in many kinds of cancers, including glioma (Hagemann et al., 2013;Yang et al., 2014). However, there was not a conclusive report about chemotherapy sensitivity of MACC1 gene in glioma.
In this study, we performed qRT-PCR and observed the up-regulation of MACC1 mRNA in brain glioma, indicating the underlying correlation between MACC1 and the carcinogenesis of glioma. Furthermore, the MACC1 expression remarkably correlated with the pathologic grade of glioma. Our data demonstrated that overexpression of MACC1, which promoted tumor growth and migration, correlated with poor differentiation and high grade of glioma patients. This suggests that MACC1 plays an important role in glioma carcinogenesis and development. Consistent with these results, our study further demonstrates that MACC1 genes are positively correlated with pathologic grades, and functions as an oncogene in tumors. Moreover, we silenced MACC1 gene expression in U251 cells and investigated the apoptosis rate and growth inhibitory rate. Our results showed that the gene silencing of MACC1 induced remarkable apoptosis and reduced the proliferation ability of gliomas. Studies on the correlation between MACC1 and apoptosis are few in literature, calling for future efforts to investigate the mechanisms of how MACC1 influences cell apoptosis and proliferation.
Up to now, therapeutic approaches are tailored for individual patients, depending on the nature of the tumor, the growth rate, the location and the patient's state. Upon initial diagnosis of glioma, standard treatment consists of maximal surgical resection, combined with optional chemotherapy and radiotherapy. Chemotherapy and radiotherapy are recommended for reducing the risk of recurrence and metastasis. Chemotherapy is the treatment of cancer with one or more cytotoxic anti-neoplastic drugs ("chemotherapeutic agents") as part of a standardized regimen. At present, dosage of chemotherapy can be difficult due to a compromise between toxicity and efficacy. For this reason, chemotherapy sensitivity is of vital importance to give guidance on customized dose for individuals to maximize the effectiveness and minimize side-effects (Yang et al., 2009).
MACC1 is proven to be involved in regulating cell apoptosis and proliferation, and MACC1 silencing could induce remarkable apoptosis and reduced the proliferation ability of U251 cells. Therefore, we hypothesized that MACC1 might influence the effect of chemotherapy on glioma. And, there is not a conclusive report about the influence of MACC1 gene on chemotherapy sensitivity in glioma. For this reason, the MACC1-silenced U251 cells were treated with different concentrations of DDP (1, 3, 5μg/ml) (Jiang et al., 2000;Zhou et al., 2013) and then characterized. The apoptosis rate and growth inhibitory rate increased dramatically as compared with normal U251 cells, indicating the inhibition of cell proliferation and indicating the possibility of better chemotherapy for patients. On these grounds, we presumed that silencing or low-expression of MACC1 gene could enhance the chemotherapy effectiveness in the same concentration of DDP. In addition, if MACC1 gene was down-regulated or silenced, the chemotherapy with lower concentration DDP could obtain similar effectiveness, in order to reduce the side-effects of chemotherapy.
In conclusion, MACC1 functions as an oncogene in glioma carcinogenesis. The silencing of MACC1 could enhance the chemotherapy sensitivity of DDP in U251 cells. In this context, MACC1 would be a new potential  DOI:http://dx.doi.org/10.7314/APJCP.2015.16.1.195 Influence of MACC1 Expression on Chemotherapy Sensitivity in Human U251 Glioblastoma Cells target gene for glioma treatment, or provide guidance to show chemosensitivity for chemotherapy.