Abstract
OBJECTIVE There is a close correlation between interleukin-6 (IL-6) and malignant proliferation of gliomas. We investigated the effect of bradykinin on the expression of IL-6 in C6 glioma cells.
METHODS Semi-quantitive RT-PCR and radioimmunoassay were used to examine the effect of bradykinin on the expression of IL-6 in C6 glioma cells and on the level of IL-6 in the culture medium.
RESULTS Using semi-quantitive RT-PCR, the expression of IL-6 mRNA was examined in C6 glioma cells at 0, 5, 10,15, 30, 60 min following addition of 1 μmol/L bradykinin. There was no statistical difference in expression of IL-6 mRNA between the treatment and control groups (P>0.05) and IL-6 was not detected in the cell culture medium.
CONCLUSION Within an hour, IL-6 expression in C6 glioma cells is not induced by bradykinin, suggesting that its clinical application may be useful as a potential therapeutic agent for tumors of the central nervous system.
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INTRODUCTION
Gliomas are brain tumors with a high incidence and poor prognosis[1]. In the past, several studies have detected the expression of IL-6 in human gliomas,glioma cell lines and normal astrocytes[2,3], and abnormal expression of IL-6 has been suggested to be closely correlated with glioma pathogenesis, progression and the malignant glioma degree[4]. While bradykinin at a low dose may increase the permeability of blood tumor barrier, which would favor the delivery of antitumor agents to tumor tissue, and thus enforce the effects of chemotherapy[5,6], nevertheless, as an inflammatory mediator, bradykinin also has a potent stimulatory effect on the secretion of IL-6 in many types of cells such as pulmonary cells [7]. It is possible that bradykinin would stimulate the secretion of IL-6 in glioma cells when it opened the blood tumor barrier, enhancing the potential risk of using bradykinin for glioma therapy. Therefore, we tested the effect of bradykinin on the expression of IL-6 in C6 glioma cells and in the culture medium.
MATERIALS AND METHODS
Cell line and culture
The C6 glioma cell strain was provided by our laboratory, and cultured in Dulbecco’s modified Eagle’s medium (DMEM; high glucose) containing 10% fetal bovine serum in a humidified atmosphere of 5% CO2. When the cultures became confluent, the cells were treated with 0.25% trypsin. All experiments were conducted when the cultures had become confluent.
RT-PCR analysis of IL-6mRNA
Cultured C6 glioma cells were treated with 1 μmol/L bradykinin for 0~60 min, after which total RNA was extracted from the cells, according to the manufacturer’s instructions for the RNAout (Tiandz). Then 1 ug of total RNA was converted to first-strand cDNA, and the resulting cDNA was subjected to PCR analysis in accordance with the manufacturer’s instructions (TaKaRa) using 30 cycles. Each cycle consisted of 30 s at 94°C for denaturation, 30 s at 55°C for annealing, and 45 s at 72°C for extension. The PCR products were stained with ethidium bromide after agarose gel electrophoresis and photographed with GAPDH serving as the control mRNA. Integrated density values (IDV) of these mRNA levels were normalized with respect to GAPDH gene expression with software (FluorChen V2.0). The actual sequences of specific primers are summarized as following: (1) IL-6: sense primer CTA CGA AGA ACT GGC AAT ATG, antisense primer AAA CCA TCT GGC TAG GTA AGA, 207 bp; (2) GAPDH: sense primer AAG GGC TCA TGA CCA CAG TCC, antisense primer ACC CTG TTG CTG TAG CCA TCC, 461 bp.
Radioimmunoassay analysis of IL-6
Cultured C6 glioma cells were treated with 1 μmol/L bradykinin for 0~60 min, then the culture media were collected, and radioimmunoassay analysis was performed using an IL-6RIA kit according to the manufacturer’s instructions.
Statistical analysis
The data are presented as the mean±SD. Student’s t tests were calculated for two group comparisons with SPSS 13.0 statistical software. Statistical significance was assumed if P value was <0.05.
RESULTS
RT-PCR analysis of IL-6 mRNA
As shown in Fig.1 and Table1, the expression of IL-6 mRNA was detected in untreated C6 glioma cells. However, there was no statistical difference in the groups treated with bradykinin for 5~60 min compared with the control group, P>0.05.
RT-PCR analysis for IL-6 mRNA in cultured C6 glioma cells treated with 1 μmol/L bradykinin for the indicated times. GAPDH served as the control mRNA. Lane M, marker.
Expression of IL-6 mRNA in glioma cells treated with bradykinin.
Radioimmunoassay analysis of IL-6
IL-6 bioactivity was not observed in the culture media of the C6 glioma cells treated with 1 μmol/L bradykinin for 0~60 min.
DISCUSSION
Van et al.[8] have demonstrated that IL-6 is produced and released by human glioblastomas. In their report, IL-6 bioactivity was also detected in 19 glioblastoma cell lines, and that the expression of IL-6 could be observed in the cell culture media. Our study demonstrated that IL-6 mRNA was detected in C6 glioma cells without any exogenous stimulus, which is in accordance with previous reports. Even though our radioimmunoassay showed that IL-6 bioactivity was not observed in the C6 glioma cell culture media, we believe this is not a contradiction with previous reports, as our studies employed fresh C6 glioma cell cultures treated with bradykinin for only 0~60 min. Treatment with bradykinin for 5~60 min was too short a duration for IL-6 to be detected in the media.
Autocrine production of IL-6 has been implicated with the genesis and development of human gliomas in various studies[9,10]. Goswami et al.[11] have demonstrated the coexpression of IL-6 and IL-6R in the glioblastoma cell line U87MG as confirmed by immunofluorescence staining. Moreover, both specific monoclonal antibodies against IL-6 and IL-6R and antisense oligonucleotide to IL-6 mRNA inhibited the growth of U87MG cells in culture. Furthermore, studies had revealed that elevated levels of IL-6 and IL-6R in gliomas have a correlation with the pathological grade. Highly malignant tumors such as glioblastoma multiforme, anaplastic astrocytoma, medulloblastoma had a remarkably higher positive expression rate and expression intensity. IL-6 and IL-6R to a certain extent are currently considered to contribute to the malignant proliferation of gliomas. IL-Iβ also can stimulate the secretion of IL-6 in glioma cells[12,13], while bradykinin may stimulate many types of cells to secret IL-6, TNFα and IL-1 β. Further studies are required to determine whether bradykinin stimulates IL-6 secretion by glioma cells and if bradykinin is involved in the autocrine regulation of IL-6 in glioma cells.
Bradykinin or its analog, RMP-7 can selectively open the blood brain barrier in tumor tissue while having no effect in normal tissue. Matsukado et al.[14] have verified that intracarotid infusion of bradykinin or RMP-7 can selectively increase transport of carboplatin into brain tumors and result in higher survival in rats with gliomas. Chemotherapy plays an important role in the treatment of gliomas. Studies have shown that actinotheraphy in combination with chemotherapy is highly efficient in contrast to actinotheraphy alone, especially, in a relapse following surgery. The combination of bradykinin and antitumor agents might be able to solve the blood-brain problem. Unfortunately, bradykinin has some vasoactive side effects, so it is important to determine if the application of bradykinin in clinical therapy would result in potential danger for the patients.
Our results showed that the expression levels of IL-6 in glioma cells remained unchanged after bradykinin treatment for 5~60 min, suggesting that IL-6 levels in glioma cells might not be influenced by bradykinin. These results lend evidence for the future clinical use of bradykinin. However the effect of bradykinin beyond 60 min of treatment is unkown.
Other studies have demonstrated that the maximal effect of bradykinin to open the blood tumor barrier occurred at about 15 min after intracarotid infusion, followed by a reduction in the effect, indicating a rapid and transient action[15]. Since the administration of bradykinin would be less than 30 min, effects on IL-6 secretion beyond 60 min should not be of importance.
Footnotes
This work was supported by grants from the National Natural Science Foundation of China (No.30670723, No.30600060, No.30400145 and No.30570650), and Natural Science Foundation of Liaoning Province (No.20052102).
- Received December 24, 2006.
- Accepted February 8, 2007.
- Copyright © 2007 by Tianjin Medical University Cancer Institute & Hospital and Springer
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