Glioma is one of the most common types of primary brain tumor in humans, accounting for 52% of all parenchymal brain tumor cases and 20% of all intracranial tumors. The average life expectancy for patients with malignant glioma is 14 months, and only 5% survive longer than 5 years. Symptoms of a glioma are similar to those produced by other malignant brain tumors. Common symptoms include seizures, nausea, vomiting, headache, hemiparesis, deteriorating memory, and personality changes. These symptoms may change, according to which part of the brain is affected. Symptoms usually appear soon after the disease but occasionally, symptoms may not appear until the tumor achieves a considerably higher scale. Malignant gliomas are highly invasive and the boundary between gliomas and brain parenchyma is difficult to discern. It is very difficult to treat glioma because the tumor cells are resistant to conventional therapies, the brain is susceptible to damage due to conventional therapies and has a very limited capacity to repair itself, and many anti-cancer drugs cannot cross the blood-brain-barrier to act on the tumor. Clinically, the main therapeutic startegy for advanced glioma is alleviative therapy (Guo et al., 2012; Theeler et al., 2012; Yu et al., 2012; Woehrer et al., 2013; Xing et al., 2013).
With the development of molecular biology, a number of new types of gene therapy strategies for treatment of cancer have also been developed. Melanoma differentiation associated gene-7 (mda-7), also known as interleukin (IL)-24, is primarily generated by the megakaryocytes. It has been confirmed that mda-7/IL-24 can induce apoptosis, inhibit growth, and enhance sensitivity to chemotherapy in a variety of tumor cells including glioma (Yacoub et al., 2010; Eulitt et al., 2011; Whitaker et al., 2011). Meanwhile, it has also been demonstrated that the Akt signaling pathway is a key regulator of drug resistance-related genes such as, MPR, bcl-2, survivin, caspase-3 and -8 (Hamed et al., 2010). In this study, we constructed a drug resistant human glioma cell line U87. The effects of mda-7/IL-24 on glioma cells sensitivity to cisplatin and the relationship between mda-7/IL-24 and phosphorylation levels of Akt were investigated in order to find a predictive biomarker and potential therapeutic target for glioma.
MATERIALS AND METHODS
Cell Lines and Cell Culture
The human glioma cell line U87 was obtained from the American Type Culture Collection and the cisplatin-resistant cell line U87/DDP was established by gradually increasing doses of cisplatin in our laboratory. Cells were grown and maintained in DEME (Life Technologies, Inc.) medium supplemented with 10% fetal bovine serum (FBS; Life Technologies) at 37°C with 5% CO2, and 0.25% trypsin-EDTA was used in digestion and passage. All tests were performed using the logarithmic growth phase cells.
Transfection and Establishment of Stable Cell Lines
The primers of mda-7/IL-24 were: sense 1: 5′-GGAACACGAGACTGAGAGATG-3′, sense 2: 5′-TTGGATCCGAGAGATGAATTTTCAACAGAGG-3′, antisense 1: 5′-ACAGGGAACAAACCAGTGCCA-3′ and antisense 2: 5′l-GCCTCGAGTCCTGGTCTAGACATTCAGAGC-3′ (Fang et al., 2012. The EcoRI and SalI restriction endonuclease cleavage sites were introduced to 5′ end, and inverse transcription of cDNA was carried out using random primers. The mda-7/IL-24 gene amplification template was cDNA. Then the gene was digested with EcoRI and SalI, and cloned to the eukaryotic expressing vector pEGFP-C1. Human glioma cells were cultured in RPMI 1640 medium supplemented with 10% FBS at 37°C in humified air with 5% CO2. The day before transfection, cells were selected in the logarithmic growth phase, and appropriate cell density achieved the degree of integration of 90-95% overnight. Transfections were performed using Lipofectamine 2000 according to the manufacturer's instructions. The groups were as follows: (1) empty vector control group: pEGFP-C1 vector; (2) experimental group: pEGFP-C1- mda-7/IL-24 vector. Stably expressing mda-7/IL-24 cell line was obtained by using 400 µg/L of G418.
The Cytotoxicity Assay
Cells (4,000 per well) were seeded in 96-well plates and treated with different concentrations of cisplatin for 24 hr after being adhered overnight. After treatment, medium containing drug was removed. The CellTiter-Glo® Luminescent Cell Viability assay (Promega) assay was performed according to the manufacturer's instructions on a microplate reader and then the cell inhibition rate was calculated as follows: inhibition rate = (1 - OD value of experimental group/OD value of control group) × 100%. Taking logarithm of the concentration of cisplatin as x-axis and the inhibition rate as y-axis to fit the inhibition curves, the corresponding inhibitory 50% concentration (IC50) was determined.
Cell Apoptosis Assay
Cells were treated with 1 μg/ml cisplatin, incubated for 24 hr, and then the supernatant and adherent cells were collected. The cell concentration was adjusted to 106 cells/mL and the cells were stained with an apoptosis detection kit (BD Biosciences) and apoptosis positive control detection kit (Beyotime). The apoptotic rate was determined by flow cytometry with annexin V/PI at the excitation wavelength of 488 nm.
Rhodamine-123 (Rh-123) Content and Expression of Glutathione and P-glycoprotein (P-gp) Assays
Cells (106 cells) were collected in 0.1 mL medium of each sample, and 0.1 mL of 10 μM Rh-123 dye was added. The cells were cultured and collected after 1 hr. The fluorescence intensity of Rh-123 in cells was detected using flow cytometry (excitation light: 488 nm, emitted light: 560 nm). Cells were collected in 0.1 mL medium of each sample, dyed in accordance with the manufacturer's instructions (eBioscience), and fluorescence intensity of cells were detected by flow cytometry (excitation light: 488 nm, emitted light: 560 nm) to reflect the expression of P-gp. Cells were collected in 0.1 mL medium of each sample and operated in accordance with the manufacturer's instructions (Beyotime), and chemiluminescence was detected by ELISA to reflect the expression of Glutathione.
Western Blotting Analysis
Cells were disrupted and the proteins were collected. The protein concentration in cell lysates was measured with BCA kit. Isolated proteins were separated by 12% SDS-PAGE and transferred to PVDF membranes, and then incubated with the appropriate monoclonal antibody at room temperature to detect the target protein (MPR, 1:800, Santa Cruz; bcl-2, 1:800, Santa Cruz; caspase-3, 1:500, Santa Cruz; caspase-8, 1:500, Santa Cruz; survivin, 1:600, Santa Cruz; p-Akt, 1:400, Santa Cruz; total-Akt, 1:800, Santa Cruz; β-actin, 1:2000, Sigma). After washing the primary antibody, membranes were incubated with the secondary antibody conjugated with HRP for 1 hr at room temperature, and then washed and subjected to enhanced chemiluminescence (ECL) detection kit. β-actin was used as a loading control.
Real-Time PCR Assay
Total RNA was extracted with Trizol reagent from each sample in the logarithmic growth phase according to the manufacturer's protocol, and cDNA was synthesized by reverse transcription using Real-Time PCR kit. The following primers were used: MPR sense: 5′-TCTCTCCCGACATGACCGAGG-3′, antisense: 5′-CCAGGAATATGCCCCGACTTC-3′; bcl-2 sense: 5′-TTCTTTGAGTTCGGTGGGGT-3′, antisense: 5′-TGCATATTTGTTTGGGGCAGG-3′ survivin sense: 5′-TTGGCAGGTGCCTGTTGAAT-3′, antisense: 5′-AGCCAGTCCCCCACAGCAT-3′; β-actin sense: 5′-AGCATCCTAGAACTCTGTGC-3′, antisense: 5′-ATTTCGGACCCCTGAACATA-3′. The total RNA was degenerated at 94°C for 3 min, and amplified for 40 cycles under the following conditions: 95°C for 5 sec, 65°C for 35 sec, 72°C for 60 sec, 72°C for 5 min cycle.
The experimental data were expressed as mean ± standard deviation, and analyzed by SPSS 13.0 software. A one-way analysis of variance (ANOVA) was used to compare the data. P < 0.05 indicated a statistically significant difference. Each experiment was carried out for three times, and achieved similar results.
Expression of Mda-7/IL-24 in Stably Established Human Glioma Cell Lines
To explore the effects of mda-7/IL-24, U87/DDP cells were transfected with mda-7/IL-24 overexpressing plasmids to establish the stable mda-7/IL-24 overexpressing cell lines. The untransfected cells were the parent group, the cells transfected with empty vector plasmids were the negative control group, and the cells transfected with mda-7/IL-24 plasmids were the mda-7/IL-24 over-expressing groups (Ad-mda-7/IL-24-1 and Ad-mda-7/IL-24-2). The mda-7/IL-24 expression levels in these cell lines were measured by Western blotting (Fig. 1).
Mda-7/IL-24 Enhances Cisplatin Sensitivity of Human Glioma Cell Lines In Vitro
The cisplatin sensitivity of human glioma cells in vitro was measured by CellTiter-Glo® Luminescent Cell Viability assay. The results revealed that the mda-7/IL-24 overexpressing group had significantly higher cisplatin sensitivity, compared with the negative control and parent groups, indicating that mda-7/IL-24 could significantly enhance the sensitivity of U87/DDP cell lines to cisplatin (Fig. 2). The IC50 values of the U87/DDP parent group, Ad-mda-7/IL-24-1 group and Ad-mda-7/IL-24-2 group were 7.23 μg/mL, 4.11 μg/mL, and 1.72 μg/mL, respectively. The sensitivity of the U87/DDP cell line to cisplatin was enhanced by 1.76-fold while, the sensitivity of mda-7/IL-24 over-expressing groups was enhanced by 4.20-folds.
Mda-7/IL-24 Enhances Apoptotic Rate of Human Glioma Cell Lines In Vitro
The apoptotic rate of human glioma cell lines in vitro was measured by flow cytometry. The results revealed that the mda-7/IL-24 over-expressing groups had a significantly higher apoptotic rate, compared to the negative control and parent groups, indicating that mda-7/IL-24 could significantly enhance the apoptotic rate in U87/DDP cell lines (Fig. 3).
Mda-7/IL-24 Regulates Rh-123 Content and Expression of P-gp and Glutathione in Human Glioma Cell Lines in Vitro
P-gp acts as a efflux pump in tumor cells which could expulse the drug from cells and lead to drug resistance. Rh-123 is a fluorescence substrate which could reflect the drug content in cells. Glutathione is one of the major antioxidant molecules and is thought to play a role in decreasing drug effect. The Rh-123 content and expression of glutathione and P-gp in human glioma cell lines in vitro were measured by flow cytometry. The results revealed that the mda-7/IL-24 over-expressing groups had a significantly higher intracellular Rh-123 content, compared with the negative control and parent groups, indicating that mda-7/IL-24 could significantly enhance the intracellular Rh-123 content of U87/DDP cell line whereas, the glutathione and P-gp expression levels were lower in the mda-7/IL-24 over-expressing groups, as compared with the negative control and parent groups (Fig. 4).
Mda-7/IL-24 Regulates Drug Resistance-Related Genes Expression of Human Glioma Cell Lines In Vitro
The drug resistance-related genes expression of human glioma cell lines in vitro was measured by Western blotting and real-time PCR. The results revealed that the mda-7/IL-24 over-expressing groups had a significantly lower protein expression levels of MPR, Bcl-2, survivin and p-Akt, compared to the negative control and parent groups whereas, the protein expression levels of caspase-3 and -8 in the mda-7/IL-24 over-expressing groups were higher than the negative control and parent groups (Fig. 5). In addition, the mRNA expression levels of MPR, Bcl-2 and survivin reflects the same trend in U87/DDP cell line (Fig. 6).
Gliomas are the most common type of brain tumors. Platinum-based chemotherapy is a first-line treatment choice for malignant glioma. However, the tumor often becomes resistant to treatment. Therefore, further studies are required to elucidate the mechanisms of drug resistance and new treatment strategies are needed to improve clinical benefit and survival in patients with glioma. A series of changes in the molecular regulatory network of tumor cells that increase the anti-apoptotic ability and decrease cell apoptosis are the main mechanisms of tumor cells drug resistance (Shahar et al., 2012; Triscott et al., 2012).
It has been demonstrated that mda-7/IL-24 could inhibit growth and selectively induce apoptosis in tumor cells while only minimally affecting normal cells (Emdad et al., 2007). In this study, we investigated the effects of mda-7/IL-24 gene in glioma cell lines in order to understand the anti-tumor effect of this gene, to find a pathway that can reverse drug resistance in tumor, and to provide basic information about gene therapy that can be applied in the clinical setting.
We hypothesized that down-regulation of mda-7/IL-24 may be one of the mechanisms through which tumor cells develop drug resistance. Therefore, in this study, we constructed a stable mda-7/IL-24 over-expressing glioma cell line by plasmid transfection, and confirmed it by Western blotting. The subsequent cytotoxicity results showed that the glioma cell lines stably over-expressing mda-7/IL-24 had increased sensitivity, which was evidenced by a decrease in the IC50 value, and the mda-7/IL-24 expression was positively correlated with the sensitivity to cisplatin. The higher the expression of the mda-7/IL-24 gene showing positive correlation, the higher the sensitivity was to the drug. In order to further elucidate the molecular mechanisms of increased drug sensitivity after elevated mda-7/IL-24 expression levels, we detected the rate of apoptosis in tumor cells by flow cytometry, and found that mda-7/IL-24 could significantly enhance the apoptosis rate of tumor cells.
P-gp is an ATP-dependent transporter protein, which can antiport many types of molecules and compounds out of cells. Rh-123, a specific substrate of P-gp, is a common fluorescent substance which can indicate the function of P-gp and reflect the ability of drug accumulation. Glutathione is an antioxidative oligopeptide which could detoxify anti-cancer drugs and may confer drug resistance. Our results showed that the Rh-123 content was significantly enhanced whereas, expressions of cell surface P-gp and intracellular glutathione were significantly reduced in the mda-7/IL-24 over-expressing groups. These results showed that mda-7/IL-24 was associated with improved cisplatin sensitivity and intracellular drug accumulation.
Bcl-2 protein family plays an important role in regulating the cellular program of apoptosis. Higher levels of Bcl-2 expression have been demonstrated to be closely related to drug resistance in many tumors. Bcl-2 can inhibit apoptosis through interactions with other proteins. It has been confirmed that a variety of proteins can interact with Bcl-2, thereby preventing the activation of caspase proteases in the cytoplasm and inhibiting cell apoptosis (Banerjee et al., 2012; Wang et al., 2012). Caspase-3 and -8 play an irreplaceable role in cell apoptosis. The main substrate of caspase-3 and -8 is poly (ADP-ribose) polymerase (PARP), an enzyme related to the DNA repair and maintenance of genomic stability. Activation of caspase-3 and -8 can induce apoptosis (Li et al., 2012; Kim et al., 2012). Unlike the other anti-apoptotic genes, expression of survivin is highly specific in tumor cells. Although survivin is not expressed in normal adult human tissues, it is expressed in various human cancers. It has been shown that survivin expression is closely associated with the degree of malignancy and prognosis of patients with cancers (Lu et al., 2013.
Our research showed that Bcl-2 and survivin expression levels were significantly decreased in mda-7/IL-24 over-expressing cells, indicating that mda-7/IL-24 is a negative regulator of Bcl-2 and survivin. Decreased expressions of Bcl-2 and survivin resulting from mda-7/IL-24 up-regulation could be one of the major mechanisms of increased drug sensitivity of tumor cells. Real time-PCR further confirmed that down-regulation of Bcl-2 and survivin was achieved by increased gene transcription levels of Bcl-2 and survivin. With the down-regulation of Bcl-2 and survivin, expressions of caspase-3 and –8 were elevated in cells, thus promoting apoptosis.
The Akt signaling pathway has been shown to be closely associated with tumor resistance. In our study, the results of Western blotting showed that Akt phosphorylation levels were elevated in mda-7/IL-24 over-expressing cells, suggesting that the activation of Akt is also involved in the regulation of the sensitivity of tumor cells to cisplatin. This regulation was related to the mda-7/IL-24 expression levels.
In summary, this study shows that mda-7/IL-24 gene silencing could enhance the sensitivity of tumor cells to cisplatin, and the main possible mechanisms involved are the down-regulation of Bcl-2 and survivin expression levels and enhancement of the Akt signaling pathway. Therefore, mda-7/IL-24 can be used as a promising predictive biomarker and potential target for chemotherapy in glioma.