1. Introduction 
Glioblastoma Multiforme (GBM) is an aggressive neurological malignancy with non-specific signs and poor prognosis (Yin et al., 2018). Pro-inflammatory cytokines, like Interleukin-6 (IL-6) released from glial cells may contribute to the progression of GBM (Chen et al., 2016; Matias et al., 2018; Wang et al., 2016). Despite immunostaining similarities between glial cells and glioblastoma, studies highlighted the role of astrocytes and oligodendrocytes in tumorigenesis (Krcek, Matschke, Theis, Adamietz, Buhler, & Theiss, 2017; Yin et al., 2018). The etiology of glioblastoma remains unknown; however, the upregulation of FBXL18 in glioma tissues which induces the phosphorylation of Forkhead Box O (FOXO3), was recently reported (Zhang, Yang, Ou, Xia, Zhi, & Cui, 2017). One of the target genes for the stimulated FOXO transcription factors is Matrix Metalloproteinases (MMPs) (Diebold, Petry, Burger, Hess, & Gorlach, 2011).
MMPs are metalloproteinases that are calcium-dependent zinc-containing endopeptidases that can digest various Extracellular Matrix (ECM) macromolecules that play a role in the GBM invasion (Wang, Tong, Jiang, & Yang, 2017). Typically, MMPs are critical mediators of ECM remodeling and regulate normal development and tissue repair. Several studies highlighted the high expression of MMPs and tumor progression in an experimental model and cancer patients (Blaes et al., 2018; Ko et al., 2016). Recently, MMP-9 has been considered a glioma grades biomarker due to its capability to increase capillary permeability (Fan et al., 2018; Li et al., 2016). Thus, MM inhibitors might help control glioblastoma invasiveness.
In malignant tumors, voltage-gated potassium (Kv) channels are highly expressed and involved in the progression of various cancer types (Aissaoui et al., 2018; Martinez et al., 2015). Furthermore, 4-Aminopyridine (4-AP) is a potent inhibitor of the voltage-dependent potassium channel. In vitro studies revealed that it can be improved action potential conduction in poorly myelinated nerve fiber (Rabadi, Kreymborg, & Vincent, 2013). Moreover, 4-AP enhanced apoptotic properties in various cancer cells and passed through the blood-brain barrier (Hassan et al., 2018; Luo et al., 2018; Wang et al., 2014).
The high-grade glioma comprised a cell collection of astrocytoma and others; therefore, using the U-373 MG glioblastoma astrocytoma cells should be an appropriate model to resemble its aggressive behavior. Moreover, these cells can produce pro-inflammatory cytokines. In this study, the cultured U-373 MG was treated by 4-AP, and its cytotoxic and anti-invasive effect was evaluated. Moreover, the impact of 4-AP in the repression of IL-6, IL-1, and Tumor necrosis factor (TNF) levels in the samples was qualitatively determined by ELISA. Reduced cell viability, IL-6 secretion, and MMP-9 activity in an experimental model of GBM might be assumed 4-AP as adjuvant therapy in cancer.

2. Methods
Cell culture
The U-373 MG (human glioblastoma astrocytoma cell line) was obtained from the National Cell Bank of Iran (NCBI), Pasteur Institute of Iran, Tehran, Iran. U-373 MG was seeded at an initial density of 2x104cells/well in 96-well tissue culture plates containing RPMI-1640 medium with 5% fetal calf serum, penicillin at 100 U/mL, and streptomycin at 100 µg/mL (Gibco BRL, Life Technologies, NY, USA). In this experiment, U-373 MG cells were cultured in the presence of 50ng/mL Lipopolysaccharide (LPS) to stimulate and then were pretreated for 4 h with 4-AP at selected concentrations. 

Cytotoxicity analysis 
Triplicate, twofold dilutions of 4-AP (Merck, Frankfurter, Germany) preparations at concentrations of 0.1–10 mM were added to cultured cells. After overnight incubation under 5% CO2, 37oC, and saturated humidity, cells were subjected to a colorimetric assay using 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis, MO, USA) assay. MTT (5mg/mL PBS) was added to cell culture, incubated for 4 h at 37 °C, then solubilized with acidic isopropanol. The absorbance was measured on a SPECTRA max PLUS384 reader (Molecular Devices Corp., Sunnyvale, CA, USA) at 570 nm. Based on MTT results, cytotoxicity as the percentage of the viable cell at different concentrations of samples and IC50 as the dose at which 50% of the cell death was calculated. Moreover, viability was evaluated after removing 4-AP from the cell medium. 

Zymoanalysis
This technique detected MMP-9 in corresponding supernatants of cells conditioned-media with some modifications (Khorramizadeh et al., 2005). Briefly, the aliquots of conditioned media were subjected to electrophoresis in a polyacrylamide gel containing gelatin (2mg/mL) (Sigma-Aldrich, St. Louis, MO, USA) for 3 hours (80 volts). The gel was washed (2.5% Triton X100), incubated at 37oC overnight (0.1 M Tris HCl, pH 7.4), and afterward stained (0.5% Coomassie Blue). After de-staining, using a UVI Pro Gel Documentation system (GDS-8000 System), proteolysis bands were quantitatively evaluated and expressed as the “relative expression” of gelatinolytic activity. 

The quantification of cytokine levels
The levels of IL-1, IL-6, and TNF in the samples were assessed using the ELISA kits (R&D Systems, Minneapolis, USA). The cytokine in the samples is bound to an immobilized antibody as directed by the manufacturer, followed by incubation with an enzyme-linked monoclonal antibody specific to the cytokine provided. After washing, a substrate was applied, and the color changed in proportion to the cytokine quantity. The response was stopped, and the absorption was measured with an ELx800brand ELISA system at 450 nm (BioTek Instruments, Inc., Winooski, VT). The effects of the examined agent on cell proliferation, cytokine secretion, and gelatinase inhibition were analyzed using the Student’s t-test in SPSS. The obtained data were presented as Mean±SD with n=3 for in vitro experiments. Besides, the differences were considered significant at P<0.05.

3. Results
4-AP decreased the proliferation of human glioma cell lines 
The efficacy of 4-Ap therapy is presented on the U-373 MG cell line at various doses (0.1-10μM) in Figure 1.

The administration of 4-AP at various concentrations indicates no noticeable low-dose cytotoxicity. The results of Student’s t-test indicated that mean % viability value at 0.1 μM of 4-Ap was significantly higher than one at 2 μM (P<0.05), 4 μM, and 10μM of it (P<0.001). The mean % viability value at 2 μM of 4-Ap was significantly different to one at 4 μM (P<0.05) and 10 μM of 4-Ap (P<0.001). The difference in mean % viability value between concentrations 4 μM and 10 μM of 4-Ap was also significant (P<0.001). Thus, the biocompatibility and tolerability of U-373 MG against higher amounts of 4-AP were significantly less (P<0.001).The calculated median lethal dose (LD50) for 4-AP was at concentration of 0.48µM according to equation y= -4.5x+10.3 x2 + 94.8. The inhibitory effect of 4-Ap on cell proliferation at 48 hours is similar to 24 hours in a concentration below 5 µM (data not presented). Moreover, cell culture changed after 24 hours of drug intervention showed a static effect on cell proliferation at doses over 5 µM after two overnight.

4-AP inhibited matrix metalloproteinase activity in human glioma cell lines
The dose-response study of 4-Ap on MMP-9 activity is depicted in Figure 2.

Comparison of the densitometric analysis of gelatinase activity at different doses demonstrated that the inhibitory efficacy of 4-Ap at all doses was statistically significant compared with non-treated cells (P<0.05). However, the mean % gelatinase activity at 0.1 μM of 4-Ap was not significantly higher than one at 2 μM, 4 μM, and 10μM of 4-Ap. 

4-AP diminished cytokine secretion in human glioma cell lines
Our cytokine assay for IL-1 didn’t show any statistical differences between treated and non-treated cells (P=0.05). The mean differences between non-treated and treated with 0.1µM were 13.3+ 1.24 vs. 12.8+0.8, respectively. As per Figure 3, the IL-1 level for 4-Ap at a unique concentration of 10µM was statistically significant, compared with non-treated cells (5.5+0.5 vs. 13.3+1.24, P<0.05).

Using the ELISA method for measuring TNF, we determined that 4-Ap-treated cells produced little TNF than non-treated cells (122.5+4.9 pg/mL). The TNF decrement due to 4-AP was not statistically significant (Figure 4).

Furthermore, administrating 4-AP suggested a significant reduction of IL-6 secretion compared with the control group (P<0.001) (Figure 5).

The mean differences of IL-6 between non-treated (320+8.0 pg/mL) and treated with 0.1µM, 2 µM, 4 µM, and 10 µM (209+5.1, 155+8.7, 113+3.0 & 14+1.2), respectively.

4. Discussion
Immune system dysregulation is among the critical mechanisms in the pathophysiology of numerous tumors (Voutsadakis, 2018a, 2018b). Moreover, selective and non-selective ion channels are necessary for the physiopathology of several cancers, including GBM (Morelli et al., 2016). This study attempted to clarify the influence of 4-AP, a broad-spectrum blocker of potassium channels, on MMP-9 activity, various types of cytokines, and its cytotoxic effects on the experimental cell model U373-MG. 
Cytotoxicity test for treated cells revealed that 4-AP could exert its toxicity at 2uM up to 10µM. Furthermore, our data and other investigations showed that this agent elicited more minor cytotoxic characteristics at doses below Its LD50 (Wang et al., 2014). Moreover, after removing 4-Ap in cell culture, the cell proliferates, suggesting potassium channel blocking should be considered a probable mechanism for 4-Ap inhibitory function. The mechanism by which potassium gate inhibitors, such as 4-AP, exert antitumor effects are not entirely defined but are postulated to involve up-regulation of phosphatase and tensin homolog, modulation of protein kinase B signaling pathway, and apoptosis enhancement (Hassan et al., 2018; Luo et al., 2018; Wang et al., 2014). 
The degradation of ECM, primarily by MMPs, is one of the fundamental stages of tumor metastasis (Shariftabrizi et al., 2005; Zhong et al., 2018). The upregulation of collagen XVI can induce MMP-9 expression by certain glioma cell lines, like U-373 MG; thus, the inhibition of MMPs can prevent tumor metastasis (Bedal, Grassel, Oefner, Reinders, Reichert, & Bauer, 2014; Senner, Ratzinger, Mertsch, Grassel, & Paulus, 2008). Few records are available on the impact of voltage-dependent potassium channels in regulating the action and biosynthesis of MMPs. Some studies reported no TLR4 expression on astrocytes and oligodendroglial cells; however, others demonstrated its presence on gliomal cell lines like U-373 MG (Che et al., 2017). LPS stimulation activated TLR 4 signaling, which facilities immune evasion of gliomal cells through MMP secretion. As per zymography results, there was a significant reduction of MMP-9 activity in these LPS induced cells; it might indicate various signaling pathways blockage. Our finding revealed that the tested drug inhibited MMP activity in a dose-response fashion (Figure 2). Therefore, this agent’s ability to inhibit matrix metalloproteinase could be valuable to prevent tumor invasion and metastasis. 
Among different cell subpopulations in the brain, astrocytes play their role as an Antigen-Presenting Cell (APC). These cells produce a collection of cytokines such as IL-6 and TNF in response to provocation. Evidence signified that the inflammatory response that adjusts cytokines pattern occurs in the tumor microenvironment (Roato & Ferracini, 2018; Worzfeld et al., 2017). The effect of 4-AP in modulation cytokine secretion was examined. According to our finding among several cytokines, IL-6 was significantly reduced in astrocytoma cells by treatment with 4-AP compared with the control group. Our data agreed with experiments that demonstrated that GBM cells are up-regulated, and secreted IL-6 and anti-IL6 directed therapies are potential utility for anti-cancer treatments (Trikha, Corringham, Klein, & Rossi, 2003; West et al., 2018). 
The strategy for GBM treatment is chemotherapy, radiotherapy, and surgery. Because of cellular complexity, undefined pathology, and the short survival rate of patients with GBM, new promising medication was considered. The meaningful MMP and IL-6 inhibition and the capability to pass through the blood-brain barrier with mild side effects might be recommended 4-AP for chemoprevention of GBM patients. However, the clinical consequence of these findings must be further studied. 

5. Conclusion
In summary, our study provides evidence that repression in the expression of MMP and cytokine secretion is an important aspect in glioblastoma. Targeting K+ channel induces cell viability reduction, IL-6 and MMP-9 activity decrement in an in vitro model of glioblastoma. However, more studies are required to confirm 4-AP significant role as chemopreventive agent aginst glioblastoma.

Ethical Considerations
Compliance with ethical guidelines
The research protocol was approved by the local Ethics Committee of the Tehran University of Medical Sciences.

Funding
The study was supported by Tehran University of Medical Sciences (Grant No.: 6890.0).

Authors' contributions
All authors equally contributed to preparing this article.

Conflict of interest
The authors declared no conflict of interest.

Acknowledgments
We are grateful to the Tehran University of Medical Sciences for providing us financial support to implement this work.


Refrences
Aissaoui, D., Mlayah-Bellalouna, S., Jebali, J., Abdelkafi-Koubaa, Z., Souid, S., & Moslah, W., et al. (2018). Functional role of Kv1.1 and Kv1.3 channels in the neoplastic progression steps of three cancer cell lines, elucidated by scorpion peptides. International Journal of Biological Macromolecules, 111, 1146-55. [DOI:10.1016/j.ijbiomac.2018.01.144] [PMID]
Bedal, K. B., Grassel, S., Oefner, P. J., Reinders, J., Reichert, T. E., & Bauer, R. (2014). Collagen XVI induces expression of MMP9 via modulation of AP-1 transcription factors and facilitates invasion of oral squamous cell carcinoma. PloS One, 9(1), e86777. [DOI:10.1371/journal.pone.0086777] [PMID] [PMCID]
Blaes, J., Thome, C. M., Pfenning, P. N., Rubmann, P., Sahm, F., & Wick, A., et al. (2018). Inhibition of CD95/CD95L (FAS/FASLG) signaling with APG101 prevents invasion and enhances radiation therapy for glioblastoma. Molecular Cancer Research, 16(5), 767-76. [DOI:10.1158/1541-7786.MCR-17-0563] [PMID]
Che, F., Yin, J., Quan, Y., Xie, X., Heng, X., & Du, Y., et al. (2017). TLR4 interaction with LPS in glioma CD133+ cancer stem cells induces cell proliferation, resistance to chemotherapy and evasion from cytotoxic T lymphocyte-induced cytolysis. Oncotarget, 8(32), 53495-507. [DOI:10.18632/oncotarget.18586] [PMID] [PMCID]
Chen, H. Y., Lin, L. T., Wang, M. L., Lee, S. H., Tsai, M. L., & Tsai, C. C., et al. (2016). Musashi-1 regulates AKT-derived IL-6 autocrinal/paracrinal malignancy and chemoresistance in glioblastoma. Oncotarget, 7(27), 42485-501. [DOI:10.18632/oncotarget.9890] [PMID] [PMCID]
Diebold, I., Petry, A., Burger, M., Hess, J., & Gorlach, A. (2011). NOX4 mediates activation of FoxO3a and matrix metalloproteinase-2 expression by urotensin-II. Molecular Biology of the Cell, 22(22), 4424-34. [DOI:10.1091/mbc.E10-12-0971] [PMID] [PMCID]
Fan, X., Qi, C., Liu, X., Wang, Y., Liu, S., & Li, S., et al. (2018). Regional specificity of matrix metalloproteinase-9 expression in the brain: Voxel-level mapping in primary glioblastomas. Clinical Radiology, 73(3), 283-9. [DOI:10.1016/j.crad.2017.10.013] [PMID]
Hassan, S., Ejaz, S. A., Saeed, A., Shehzad, M., Ullah Khan, S., & Lecka, J., et al. (2018). 4-Aminopyridine based amide derivatives as dual inhibitors of tissue non-specific alkaline phosphatase and ecto-5’-nucleotidase with potential anticancer activity. Bioorganic Chemistry, 76, 237-48. [DOI:10.1016/j.bioorg.2017.11.013] [PMID]
Khorramizadeh, M. R., Aalizadeh, N., Pezeshki, M., Ghahary, A., Zeraati, H., & Berahmeh, A., et al. (2005). Determination of gelatinase A using a modified indirect hemagglutination assay in human prostate cancer screening and assessment of its correlation with prostate-specific antigen parameters. International Journal of Urology, 12(7). [DOI:10.1111/j.1442-2042.2005.01094.x] [PMID]
Ko, J. H., Ho Baek, S., Nam, D., Chung, W. S., Lee, S. G., & Lee, J., et al. (2016). 3-Formylchromone inhibits proliferation and induces apoptosis of multiple myeloma cells by abrogating STAT3 signaling through the induction of PIAS3. Immunopharmacology and Immunotoxicology, 38(5), 334-43. [DOI:10.1080/08923973.2016.1203928] [PMID]
Krcek, R., Matschke, V., Theis, V., Adamietz, I. A., Buhler, H., & Theiss, C. (2017). Vascular endothelial growth factor, irradiation, and axitinib have diverse effects on motility and proliferation of glioblastoma multiforme cells. Frontiers in Oncology, 7, 182. [DOI:10.3389/fonc.2017.00182] [PMID] [PMCID]
Li, Q., Chen, B., Cai, J., Sun, Y., Wang, G., & Li, Y., et al. (2016). Comparative analysis of matrix metalloproteinase family members reveals that mmp9 predicts survival and response to temozolomide in patients with primary glioblastoma. PloS One, 11(3), e0151815. [DOI:10.1371/journal.pone.0151815] [PMID] [PMCID]
Luo, Z., Wang, J., Li, C., Qiu, Y., Huang, J., & Huang, Y., et al. (2018). Upregulation of phosphatase and tensin homolog is essential for the effect of 4-aminopyridine on A549/CDDP cells. Molecular Medicine Reports, 17(4), 5996-6001. [DOI:10.3892/mmr.2018.8585] [PMID]
Martinez, R., Stuhmer, W., Martin, S., Schell, J., Reichmann, A., & Rohde, V., et al. (2015). Analysis of the expression of Kv10.1 potassium channel in patients with brain metastases and glioblastoma multiforme: Impact on survival. BMC Cancer, 15, 839. [DOI:10.1186/s12885-015-1848-y] [PMID] [PMCID]
Matias, D., Balca-Silva, J., da Graca, G. C., Wanjiru, C. M., Macharia, L. W., & Nascimento, C. P., et al. (2018). Microglia/astrocytes-glioblastoma crosstalk: Crucial molecular mechanisms and microenvironmental factors. Frontiers in Cellular Neuroscience, 12, 235. [DOI:10.3389/fncel.2018.00235] [PMID] [PMCID]
Morelli, M. B., Nabissi, M., Amantini, C., Tomassoni, D., Rossi, F., & Cardinali, C., et al. (2016). Overexpression of transient receptor potential mucolipin-2 ion channels in gliomas: Role in tumor growth and progression. Oncotarget, 7(28), 43654-68. [DOI:10.18632/oncotarget.9661] [PMID] [PMCID]
Rabadi, M. H., Kreymborg, K., & Vincent, A. S. (2013). Sustained-release fampridine (4-aminopyridine) in multiple sclerosis: And impact on motor function. Drugs in R&D, 13(3), 175-81. [DOI:10.1007/s40268-013-0020-x] [PMID] [PMCID]
Roato, I., & Ferracini, R. (2018). Cancer stem cells, bone and tumor microenvironment: Key players in bone metastases. Cancers, 10(2). [DOI:10.3390/cancers10020056] [PMID] [PMCID]
Senner, V., Ratzinger, S., Mertsch, S., Grassel, S., & Paulus, W. (2008). Collagen XVI expression is upregulated in glioblastomas and promotes tumor cell adhesion. FEBS Letters, 582(23-24), 3293-300. [DOI:10.1016/j.febslet.2008.09.017] [PMID]
Shariftabrizi, A., Khorramizadeh, M. R., Saadat, F., Alimoghadam, K., Safavifar, F., & Ebrahimkhani, M. R. (2005). Concomitant reduction of matrix metalloproteinase-2 secretion and intracellular reactive oxygen species following anti-sense inhibition of telomerase activity in PC-3 prostate carcinoma cells. Molecular and Cellular Biochemistry, 273(1-2), 109–16. [DOI:10.1007/s11010-005-8158-7] [PMID]
Trikha, M., Corringham, R., Klein, B., & Rossi, J. F. (2003). Targeted anti-interleukin-6 monoclonal antibody therapy for cancer: A review of the rationale and clinical evidence. Clinical Cancer Research, 9(13), 4653-4665. [PMID] [PMCID]
Voutsadakis, I. A. (2018). Expression and function of immune ligand-receptor pairs in NK cells and cancer stem cells: Therapeutic implications. Cellular Oncology (Dordrecht), 41(2), 107-21. [DOI:10.1007/s13402-018-0373-9] [PMID]
Voutsadakis, I. A. (2018). Immune blockade inhibitors and the radiation abscopal effect in gastrointestinal cancers. World Journal of Gastrointestinal Oncology, 10(9), 221-7. [DOI:10.4251/wjgo.v10.i9.221] [PMID] [PMCID]
Wang, C., Tong, X., Jiang, X., & Yang, F. (2017). Effect of matrix metalloproteinase-mediated matrix degradation on glioblastoma cell behavior in 3D PEG-based hydrogels. Journal of Biomedical Materials Research. Part A, 105(3), 770-8. [DOI:10.1002/jbm.a.35947] [PMID] [PMCID]
Wang, W., Fan, Y., Wang, S., Wang, L., He, W., & Zhang, Q., et al. (2014). Effects of voltage-gated K+ channel on cell proliferation in multiple myeloma. The Scientific World Journal, 2014, 785140. [DOI:10.1155/2014/785140] [PMID] [PMCID]
Wang, Y., Chen, X., Tang, G., Liu, D., Peng, G., & Ma, W., et al. (2016, December). AS-IL6 promotes glioma cell invasion by inducing H3K27Ac enrichment at the IL6 promoter and activating IL6 transcription. FEBS Letters, 590(24), 4586-93. [DOI:10.1002/1873-3468.12485] [PMID]
West, A. J., Tsui, V., Stylli, S. S., Nguyen, H. P. T., Morokoff, A. P., & Kaye, A. H., et al. (2018). The role of interleukin-6-STAT3 signalling in glioblastoma. Oncology Letters, 16(4), 4095-4104. [DOI:10.3892/ol.2018.9227] [PMID] [PMCID]
Worzfeld, T., Pogge von Strandmann, E., Huber, M., Adhikary, T., Wagner, U., & Reinartz, S., et al. (2017). The unique molecular and cellular microenvironment of ovarian cancer. Frontiers in Oncology, 7, 24. [DOI:10.3389/fonc.2017.00024] [PMID] [PMCID]
Yin, X. F., Zhang, Q., Chen, Z. Y., Wang, H. F., Li, X., & Wang, H. X., et al. (2018). NLRP3 in human glioma is correlated with increased WHO grade, and regulates cellular proliferation, apoptosis and metastasis via epithelial-mesenchymal transition and the PTEN/AKT signaling pathway. International Journal of Oncology, 53(3), 973-86. [DOI:10.3892/ijo.2018.4480] [PMID] [PMCID]
Zhang, J., Yang, Z., Ou, J., Xia, X., Zhi, F., & Cui, J. (2017). The F-box protein FBXL18 promotes glioma progression by promoting K63-linked ubiquitination of Akt. FEBS Letters, 591(1), 145-54. [DOI:10.1002/1873-3468.12521] [PMID]
Zhong, Y., Lu, Y. T., Sun, Y., Shi, Z. H., Li, N. G., & Tang, Y. P., et al. (2018). Recent opportunities in matrix metalloproteinase inhibitor drug design for cancer. Expert Opinion on Drug Discovery, 13(1), 75-87. [DOI:10.1080/17460441.2018.1398732] [PMID]