Impact of O 6 -methylguanine-DNA methyltransferase expression on the drug resistance of clear cell renal cell carcinoma

Objective: The deoxyribonucleic acid-repair protein O 6 -methylguanine-deoxyribonucleic acid methyltransferase is a major determinant of resistance of cells to various alkylating drugs. Its expression profile is different in different cancer types. Here, we studied the expression and function of O 6 -methylguanine-deoxyribonucleic acid methyltransferase in clear cell renal cell carcinoma. Methods: The expression of O 6 -methylguanine-deoxyribonucleic acid methyltransferase was evaluated in clear cell renal cell carcinoma tissues and cell lines by quantitative real-time polymerase chain reaction and immunohistochemistry. The relationship between O 6 -methylguanine-deoxyribo-nucleic acid methyltransferase expression and clinicopathological characteristics was analyzed. To further investigate the function of O 6 -methylguanine-deoxyribonucleic acid methyltransferase in clear cell renal cell carcinoma resistance to alkylating agents, siRNA targeting O 6 -methylguanine-deoxyribonucleic acid methyltransferase were used to silence the O 6 -methylguanine-deoxyribonucleic acid methyltransferase expression. Results: We found that O 6 -methylguanine-deoxyribonucleic acid methyltransferase is over-expressed in clear cell renal cell carcinoma tissues and cell lines. O 6 -methylguanine-deoxyribonucleic acid methyltransferase expression is related with tumor progression in clear cell renal cell carcinoma patients. Up-regulation of O 6 -methylguanine-deoxyribonucleic acid methyltransferase plays a critical role in primary resistance to alkylating agents. Conclusions: The overexpression of O 6 -methylguanine-deoxyribonucleic acid methyltransferase contributes to resistance of clear cell renal cell carcinoma to standard chemotherapy. Our results have significance for understanding a new pathway of the development of drug resistance of clear cell renal cell carcinoma.

ccRCC is thought to arise from the proximal tubular epithelial cells and it can be either familial or sporadic (4). RCC is notoriously refractory to radiation therapy and standard chemotherapy. Until recently, the outcome of medical treatment with cytotoxic agents for RCC was disappointing (5). To date, many mechanisms have been proposed to induce drug resistance of cancer cells. These include overexpression of P-glycoprotein, decreased expression of DNA topoisomerase II, overexpression of an antiapoptotic gene bcl-2, overexpression of DNA repair proteins and so on (5). In order to improve chemotherapy for patients with ccRCC, it is essential to identify factors in their tumor cells that contribute to resistance to anti-cancer drugs.
A large number of environmental alkylating carcinogens, endogenous alkylating species and various tumor chemotherapeutic agents attack DNA at the O 6 position of guanine, forming O 6 -alkylguanine (6). This lesion is considered to be a major cause of mutations and malignant transformation induced by O 6 -alkylating agents (7).
It also provokes genotoxicity and cell death by inducing apoptosis and, therefore, represents the underlying reason for the antineoplastic effect of O 6 -alkylating agents (8). O 6 -alkylguanine is repaired by the DNA repair protein O 6 -methylguanine-DNA methyltransferase (MGMT). It catalyzes the transfer of mutagenic and cytotoxic adducts from O 6 -guanine in DNA (9). Following the incorporation of the alkyl group by MGMT, the enzyme is irreversibly inactivated and degraded by the proteasome, thus requiring de novo protein synthesis to sustain the enzyme activity. If the methyl group is not removed from guanine, guanine can pair with thymine during DNA replication which leads to transition of guaninecytosine to adenine-thymine (10). Hence, through removal of alkyl groups from guanines, MGMT safeguards the cells against malignant transformation and alkylating agents (11).
The expression pattern of MGMT is different in different types of tumors. The expression level of MGMT is found to be lower than their tissues of origin in some tumors. Su et al. (12) found that MGMT was expressed at a low level in esophageal squamous cell carcinoma than in normal tissue. The loss of MGMT expression has been reported with a higher frequency among more aggressive pituitary tumors (13). Mokhtar et al. (14) found that loss of expression of MGMT protein was significantly more frequent in thymic carcinoma than in thymoma. In testicular tumor samples MGMT was lower than that in the normal tissue from the same patient (15). But in other studies, MGMT was found to be overexpressed in many types of human tumors and correlate with the progression of the cancer. In breast cancers it has been shown that the MGMT activity increases with disease progression and is accompanied by a reduction in the frequency of MGMT-deficient cells (16,17). In ovarian cancer, tumor progression was clearly associated with increase in MGMT activity (18). However, the MGMT expression profile in ccRCC remains unclear. In this study, we sought to investigate the expression profile of MGMT in ccRCC and analyzed the role of MGMT in the drug resistance of ccRCC.

Patient samples
Samples of ccRCC from 60 patients who had undergone surgery for ccRCC were obtained from the surgical department of The Second Hospital of Shandong University between 2011 and 2014. None of these patients received antitumor treatment before the operation, and the diagnosis as ccRCC was histologically confirmed. ccRCC specimens were fixed in 10% buffered formalin and were embedded in paraffin for immunohistochemistry. Of these specimens, 20 pairs of ccRCC and corresponding normal tissues were collected and immediately frozen in liquid nitrogen after resection and stored at −80°C before RNA extraction. The patients had a median age of 58 (range 51-78 years).

Cell culture
The ccRCC cell lines ACHN and 786-0 were purchased from the Institute of Biochemistry and Cell Biology and the human kidney tubular epithelial cells (HK-2) were purchased from ATCC. Cells were cultured in Eagle's Minimum Essential Medium or 1640 medium containing 10% Gibco fetal bovine serum. The human kidney tubular epithelial cells (HK-2) were cultured in KSF medium with epidermal growth factor. All cells were cultured at 37°C in a humidified incubator with 5% CO 2 .

Quantitative real-time PCR
Total RNA was extracted from fresh-frozen tumors tissues using Trizol Reagent according to the manufacturer's protocol (Invitrogen, San Diego, CA, USA). RNA concentration and purity were determined by measuring the UV absorbance at 260/280 nm. One microgram of total RNA was reverse-transcribed into cDNA using M-MLV reverse transcriptase (Takara, Japan). Quantitative real-time polymerase chain reaction (qRT-PCR) for MGMT was performed using the ABI PRISM 7000 detection system. The primers of MGMT were as follows: 5′-TGG AGC TGT CTG GTT GTG AG-3′, 5′-AGG GCT GCT AAT TGC TGG TA-3′. The PCR conditions were as follows: enzyme activation at 95°C for 10 s, followed by 40 cycles of denaturing at 95°C for 10 s, extension at 60°C for 15 s. The reaction was performed with 20 μl SYBR Green reaction system. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the endogenous control and amplified using the following primers: 5′-CCA TGG AGA AGG CTG GGG-3′ (forward) and 5′-CAA AGT TGT CAT GGA TGA CC-3′ (reverse). All assays were performed in triplicate. Each plate included multiple non-template controls. Determination of gene expression was performed using the 2 −ΔΔCt method.

Immunohistochemistry
Immunohistochemistry was performed according to standard procedures. Briefly, tissue sections were deparaffinized and rehydrated through graded alcohols to water. Sections were immersed in 10 mM sodium citrate buffer (pH 6.0) and subjected to heat-induced antigen retrieval. Endogenous peroxidase activity was blocked by incubating sections in 3% hydrogen peroxide for 10 min. Sections were then treated with goat serum in phosphate buffered saline to block non-specific protein binding. After blocking, tissue sections were incubated with the monoclonal antibody against MGMT (Abcam, 1:50) overnight at 4°C. After brief rinsing, sections were treated with biotinylated rabbit antimouse IgG for 30 min at room temperature, rinsed, and then incubated with streptavidin biotin complex for 15 min at room temperature. After brief washing, sections were incubated with diaminobenzidine for 5 min. Sections were then lightly counterstained with hematoxylin, dehydrated in graded alcohols, cleared with xylene and cover slipped. The MGMT staining intensity was scored as follows: '−' for negative staining, '+' for weak staining,'++' for moderate staining and '+++' for strong staining.

RNA interference of MGMT
ACHN and 786-0 cells were seeded in six-well plates and incubated overnight. A control random small interfering RNA (siRNA) or MGMT-targeted siRNA (Invitrogen; 1 nmol per well) was transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. After 48 h transfection, cells were treated with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or temozolomide (TMZ) for an additional 24 h. Then cells were collected and cell lysates were subjected to immunoblotting of MGMT.

Cell viability measurement
Cell viability was determined via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) assay. Cells were seeded in 96-well plates at a density of 1 × 10 4 cells and incubated for 24 h. After knocking down the expression of MGMT by siRNA for 48 h, the cells were treated with the desired concentrations of BCNU or TMZ for additional 24 h, then 10 µl of MTT (5 mg/ml) was added to each well and incubated in a humidified 5% CO 2 atmosphere at 37°C for 4 h. Crystals were dissolved in 100 µl of DMSO. The absorbance of the solution was read spectrophotometrically at 570 nm using a microtitre plate reader (Bio-Rad). Cell viability was calculated according to the following formula: Cell viability (%) = A570 (tested group − blank group)/A570 (vehicle-treated control group − blank group) × 100. At least three replicates were used for each treatment.

Immunoblotting assay
After treatment, cells were lysed with a solution containing Tris-HCl (50 mmol/l, pH 6.8), sodium dodecyl sulphate (SDS) (2% w/v), glycerol (10%), and dithiothreitol (10 mmol/l), supplemented with protease inhibitor mix (Thermo Fisher). Cell lysates were centrifuged at 12 000×g for 30 min. Equal amounts of proteins (50 µg) were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto a nitrocellulose membrane. The membranes were blocked with 5% non-fat dry milk in TBS for 1 h at room temperature and then incubated with primary antibodies against MGMT (Abgent), GAPDH (Santa Cruz Biotechnology) at 4°C overnight. Membranes were washed and treated with appropriate secondary antibodies for 1 h at room temperature. The immunocomplexes were detected using the enhanced chemiluminescence plus kit.

MGMT expression is up-regulated in ccRCC tissues
We examined MGMT protein expression in 60 formalin-fixed specimens by immunohistochemistry. MGMT was detected in the nucleus of ccRCC cells. MGMT protein expression in tumors was in general up-regulated compared with that in normal kidney tissues. The patients were divided into the MGMT low or none-expression group (staining score is '−' or '+') and MGMT high-expression group (staining score is '++' or '+++') based on immunohistochemistry scores. The high-expression rate of the MGMT was 62% (37/60) in ccRCC samples. The representative pictures of immunostaining are shown in Fig. 1A and B.
Next we confirmed the up-regulation of MGMT by qRT-PCR. The results revealed that the level of MGMT mRNA was up-regulated by 2-fold in 17/20 (85%) ccRCC tissues compared with their adjacent normal tissues. The paired Student t test showed that the MGMT mRNA expression was significantly higher in ccRCC tissues than that in the matched normal tissues (P < 0.05) (Fig. 1C). Moreover, we analyzed the correlation between the level of MGMT mRNA and the level of MGMT protein. We found that the level of MGMT mRNA was significantly correlated with the level of MGMT protein (R = 0.75) in 20 cases of primary ccRCC tissues (Fig. 1D).

The MGMT expression is up-regulated in ccRCC cell lines
We also detected MGMT expression level in two ccRCC cell lines and one normal kidney cell lines HK-2. We found that MGMT mRNA expression level was higher in all two ccRCC cell lines than that in HK-2 cells (Fig. 2A). Western blot analysis confirmed high expression of MGMT protein in two ccRCC cell lines (Fig. 2B).

MGMT expression is associated higher grades/stages of ccRCC
Immunohistochemistry analysis was performed to detect MGMT expression level in 60 ccRCC samples. The correlation between MGMT protein expression and ccRCC clinical-pathological feature was listed in Table 1

siRNA targeting MGMT sensitizes ACHN and 786-0 cells to BCNU or TMZ
To study the role of MGMT in the resistance of ccRCC cells to alkylate agents, we analyzed cell survival of ACHN cells treated with alkylate agents after knockdown of MGMT. To confirm the specificity of siRNA-mediated silencing of MGMT, western blotting assay was used to detect the MGMT protein expression. As shown in Fig. 3A, after treatment with siRNA for 48 h, MGMT protein expression in ACHN cells was significantly decreased compared with that in control siRNA-treated cells.
The MTT method was used to assess cell viability. As indicated in Fig. 3B and C, knockdown of MGMT decreased cell survival at all doses of BCNU or TMZ, as compared with the random control. This result indicated that inhibition of MGMT expression sensitized ACHN cells to alkylate agents and MGMT plays a protective role against alkylation-induced cell killing.
The protective role of MGMT against alkylation-induced cell killing was further studied in ccRCC line 786-0. Treatment with siRNA for 48 h could significantly decrease MGMT protein expression in 786-0 cells (Fig. 4A). As shown in Fig. 4B and C, knockdown of MGMT also significantly increased the sensitivity of 786-0 cells to BCNU or TMZ.

Discussion
RCC is the most common form of kidney cancer in the world. There are at least five subtypes of RCC currently recognized and the most common form is clear cell cancer, which accounts for ∼70-80% of cases. The unpredictable outcome of RCC following diagnosis is a major obstacle to the effective management of this disease. Despite major advances in the diagnosis and treatment of RCC, mortality has changed very little over the last three decades, with an overall  5-year survival of ∼40% (19,20). Considerable effort has therefore been devoted to obtain a better understanding of molecular and biological mechanisms involved in the development and progression of RCC. The identification of such molecular markers may help patients, for whom specific adjunct therapies may be appropriated, thereby improving outcomes.  MGMT, also known as O 6 -alkylguanine-DNA alkyltransferase, has been studied extensively because of its role in the response of tumor cells to alkylating chemotherapeutic agents (21). The downregulation of MGMT has been found in esophageal squamous cell carcinoma, pituitary, testicular tumor (12,13,15). In contrast, the overexpression of MGMT has been found in several types of tumors including breast cancer and ovarian cancer (16,18). The expression profile of MGMT in ccRCC remains unclear. In this study, by analyzing paired samples from the same patients, we demonstrated for the first time that MGMT mRNA was up-regulated by 2-fold in 17/20 (85%) ccRCC tissues compared with their adjacent normal tissues and MGMT protein was also up-regulated in ccRCC tissues. In addition, the epigenetic alteration of MGMT has been investigated in the ccRCC as a tumor suppressor gene in the previous studies. Onay et al. (22) reported that methylation rates for MGMT was 33% and Morris et al. (23) reported that promoter methylation occurs in the 9% RCC tissues . Promoter hypermethylation always reduce the expression of the gene. So MGMT expression may be down-regulated in a small subset of ccRCC by the methylation in the MGMT promoter region. In the study by Morris et al. (23) also showed that the frequency of MGMT methylation was higher in Stages 1 and 2 Wilms' tumors (50%) than in Stages 3 and 4 tumors (17%). This result may also support the tumor promotion function of MGMT.
In order to investigate the role of MGMT in the tumorigenesis of ccRCC, the association of MGMT expression with clinicopathological features was further analyzed. We found that MGMT expression was positively correlated with the tumor pathologic grade and clinical stage. These results suggest that MGMT overexpression may play an important role in carcinogenesis and progression of ccRCC. Our results are in agreement with observations from other tumor models. In breast tumors it has been shown that the MGMT activity increases with disease progression and is accompanied by a reduction in the frequency of MGMT-deficient cells (16,17). In ovarian cancer, tumor progression was clearly associated with increase of MGMT activity. Thus the average MGMT activity level increased with FIGO stage, grading of the tumor (18). It has been postulated that the majority of human cancers arise from spontaneous mutagenesis (24). There are many environmental alkylating agents which can induce DNA mutation. The most commonly found environmental alkylating agents are N-nitroso compounds formed during industrial processes, incomplete combustion of tobacco products and food preparation. These N-nitroso compounds have been shown to be mutagenic and carcinogenic (25)(26)(27). Endogenous N-nitroso compounds forms in the human body after ingestion of quantities of nitrate and amines consistent with normal dietary intakes. N-nitroso compounds may be one of the carcinogenic factors that cause ccRCC and the elevation of MGMT may be a response to these carcinogenic agents.
Pre-existing and acquired drug resistance of tumor cells is a major threat in cancer therapy. ccRCC is notoriously refractory to many types of chemotherapeutic drugs including alkylating agents. Alkylating agents are used in chemotherapy of various cancers such as glioblastoma, malignant melanoma, Hodgkin's lymphoma, soft tissue sarcomas and brain metastasis from solid tumors (28). MGMT was known to exert protection against treatment with alkylating agents. The most relevant antineoplastic drugs against which MGMT exerts protection are methylating agents such as temozolomide (TMZ) as well as chloroethylating agents such as BCNU (29). The clinical effectiveness of BCNU and TMZ is attributed, in part, to the potentially cytotoxic DNA lesions O 6 -chloroethylguanine, formed by BCNU, and O 6 -methylguanine (O 6 -meG) formed by the methylating agents. O 6 -methyl/chloroethylguanine incorrectly pairs with thymine, which triggers the mismatch repair system, leading to a double strand break of the genome that results in the arrest of the cell cycle and induction of apoptosis (30,31). To further investigate the function of MGMT in ccRCC resistance to alkylating agents, siRNA targeting MGMT were used to inhibit MGMT expression. Our result showed that silencing of MGMT induced reversal of TMZ and BCNU resistance in ccRCC cell line ACHN and 786-0. Together with these observations, our findings support that high MGMT expression in ccRCC cells facilitates and provides cells with more resistant phenotypes towards alkylating agents.
The regulation of MGMT gene expression is incompletely understood. Recent studies have shown that the expression of MGMT could be regulated by several signal pathways. MGMT expression is highly regulated via promoter methylation (29,32). Overall 97 CpG islands have been identified in the MGMT promoter. But the exact molecular mechanism responsible for the methylation of the MGMT promoter is still unclear. Another factor shown to be involved in MGMT regulation is p53. It can exert a negative effect on MGMT expression and wild-type p53 down-modulates MGMT promoter activity (33). In addition, MGMT expression is transcriptionally controlled by the growth-factor signaling protein kinase C-mediated pathway involving AP-1 (34). An understanding of the mechanism of overexpression of MGMT, particularly in tumor cells, has clinical significance because reversal of such overexpression may lead to sensitization of the tumor to alkylating antineoplastic drugs. Further study will therefore be required to elucidate molecular mechanisms through which MGMT was up-regulated in ccRCC.
In summary, we found for the first time that MGMT is overexpressed in ccRCC tissues and cell lines. MGMT expression is related with tumor progression in ccRCC patients. Up-regulation of MGMT plays a critical role in primary resistance to alkylating agents. Since MGMT was checked in a small amount of samples, more cases will be included to confirm the role of MGMT in the progression of ccRCC in the future.

Funding
This study was supported by Shandong Province Natural Science Foundation (Y2008C14).