The Repression of Matrix Metalloproteinases and Cytokine Secretion in Glioblastoma by Targeting K+ Channel.

Introduction: Glioblastoma is an aggressive human brain malignancy with poorly understood pathogenesis. Voltage-gated potassium (Kv) channels and Matrix Metalloproteinases (MMPs) are highly expressed in malignant tumors and involved in the progression and metastasis of glioblastoma. This study aimed to determine whether a voltage-dependent potassium channel blocker could modulate astrocytes as a cell involved in the immunopathogenesis of glioblastoma. Methods: The cytotoxic effect of 4-Aminopyridine (4-AP) at different doses in the cell model of glioblastoma was measured by MTT assay. The ELISA technique and gelatin zymography were used to assess cytokine levels and MMP-9 after 4-AP treatment. Results: Cytotoxicity analysis data indicated that cell viability reduced by increasing 4-AP level and cell growth decreased gradually by removing 4-AP from the cell medium. 4-AP inhibits the secretion of IL-6 and IL-1 (P<0.05). MMP9 activity significantly inhibits with increased 4-AP dose, compared to non-treated cells. Conclusion: The reduction of cell viability, IL-6 secretion, and MMP-9 activity in an in vitro model of glioblastoma might be assumed 4-AP as an agent for chemoprevention of cancer. Highlights: 4-Aminopyridine, as a K channel blocker, inhibits the secretion of IL-1.A voltage-gated potassium channel inhibits the secretion of IL-6.MMP9 activity, as a tumor metastasis marker, significantly decreased by 4-AP. Plain Language Summary: Glioblastoma is the most common primary malignant of the brain, which remains mainly untreatable. A group of enzymes -matrix metalloproteinases- can digest various extracellular matrix macromolecules. They express at a high level and play a role in the glioblastoma invasion. Besides, several substances are secreted by multiple cells and affect cancer metastasis. Among them, cytokines, like interleukin-6, released from glial cells, may contribute to glioblastoma progression. The present study determined whether an agent as a potassium channel blocker could modulate the immunopathogenesis of glioblastoma. We realized the cytotoxic effect of potassium channel blocker at different doses in the U-373 MG glioblastoma astrocytoma cells. Our chosen agent inhibits the secretion of both interleukin and matrix metalloproteinases activity. Overall, we suggest potassium channel blocker as an agent for cancer chemoprevention.

Targeted Intracellular Heat Transfer in Cancer Therapy: Assessment of Asparagine-laminated Gold Nanoparticles in Cell Model of T cell Leukemia.

BACKGROUND: High temperatures have destructive effects on cancer cells by damaging proteins and structures within cells. Gold nanoparticles (AuNPs) can act as drug delivery vehicles, especially for cancer therapy. Due to the selective intake of asparagine molecules into malignant cells, AuNPs were coated with asparagine; and CCRF-CEM human T-cell leukemia cells were treated with the new combination, Asn-AuNPs, at 39 °C. METHODS: The co-authors from a number of collaborative labs located at Tehran University of Medical Sciences, Tehran, Iran, have initiated the idea and preliminary design of this study in 2011. Hydroxyl surfaced AuNPs were preliminary prepared by tannin free ethanol extract of black tea leaves. These biogenic AuNPs were further capped with asparagines to form asparagine-gold nanoparticle conjugates (Asn-AuNP conjugates). Then CCRF-CEM human T-cell leukemia cells were separately treated with different concentrations of AuNPs and Asn-AuNP conjugates (3, 30, 300 µg/mL). MTT assay and zymography analysis were carried out, and the apoptotic and necrotic effects of Asn-AuNPs were determined in comparison with AuNPs, using flow cytometry assay. RESULTS: Asn-AuNP conjugates at 300 µg/mL significantly inhibited MMPs at 39 °C, compared to AuNPs. In terms of cytotoxicity, a remarkable decrease was observed in the percentage of viable cells treated with Asn-AuNP conjugates, rather than AuNPs. Moreover, the AuNPs and Asn-AuNP conjugates enhanced the level of apoptosis at almost similar rates. CONCLUSION: AuNPs are coated with asparagine molecules and the temperature is slightly increased by 2 °C, the apoptosis is not only enhanced among cells but also shifts to necrosis in higher concentrations of Asn-AuNP conjugates. More investigations should be carried out to explain the exact mechanism underlying the necrotic effects of Asn-AuNPs.

Effect of toll-like receptor 3 agonists on the functionality and metastatic properties of breast cancer cell model.

There exists compelling evidence that Toll-like receptor 3 (TLR3) agonists can directly affect human cancer cells. The aim of this study was to investigate anti-cancer effects of TLR3 agonist in human breast cell line. We assessed potential effects of poly (A:U) on human breast cell line (MDA-MB-231) on a dose-response and time-course basis. Human breast cell line MDA-MB-231 was treated with different concentrations of poly (A:U) and lipopolysaccharide (LPS). Then, the following assays were performed on the treated cells: dose-response and time-course cytotoxicity using colorimetric method; matrix metalloproteinase-2 (MMP-2) activity using gelatin zymography method; apoptosis using annexin-v flowcytometry method; and relative expression of TLR3 and MMP-2 mRNA using reverse transcriptase polymerase chain reaction (RT-PCR) method. Following treatments, dose- response and time-course cytotoxicity using a colorimetric method, (MMP-2) activity (using gelatin zymography), apoptosis (using annexin-v flowcytometry method) assays and expression of TLR3 and MMP-2 genes (using PCR method) were performed. Cytotoxicity and flowcytometry analysis of poly (A:U) showed that poly (A:U) do not have any cytotoxic and apoptotic effects in different concentrations used. MMP-2 activity analysis showed significant decrease in higher concentrations (50 and 100 µg/ ml) between treated and untreated cells. Moreover, poly A:U treated cells demonstrated decreased expression of MMP-2 gene in higher concentrations. Collectively, our data indicated that human breast cancer cell line (MDA-MB-231) was highly responsive to poly (A:U). The antimetastatic effect of direct poly (A:U) and TLR3 interactions in MDA-MB-231 cells could provide new approaches in malignant tumor therapeutic strategy.

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.

BACKGROUND: Prostate cancer is the most common malignancy affecting men and is a major cause of cancer death. There are increasing data on novel tumor markers, such as gelatinase A, which play a key role in tissue invasion and metastasis. OBJECTIVES: We designed a study to evaluate total gelatinase A content using a simple and applicable Indirect hemagglutination (IHA) test in harmony with gelatinase A activity in serum samples as compared with prostate-specifc antigen (PSA) parameters. METHODS: In this study, we analysed the circulating form of gelatinase A (MMP-2) in patients suffering from either benign prostate hyperplasia (n=54) or prostate cancer (n=26) versus normal individuals as control (n=26). The gelatinolytic activity was determined by zymography and total MMP-2 content was measured by a novel IHA method. Total PSA and free PSA were quantified using a standard ELISA technique. RESULTS: Correlation of densitometric analysis of gelatinase A activity and IHA titer is significant at the 0.01 level (P<0.01, rho=0.916). Correlation of PSA and IHA titer is significant at the 0.01 level (P<0.01, rho=0.746). Correlation of free PSA and IHA titer is significant at the 0.01 level (P<0.01, rho=0.749). Borderline of IHA titer in patients with prostate cancer was 512+/-1 tube titer, in benign prostate hyperplasia patients was 128+/-1 tube titer and the titer in normal individuals was 8+/-1 tube titer. CONCLUSIONS: These results demonstrate that assessment of gelatinase A might be a promising procedure for monitoring and screening patients with prostate cancer.

In vitro assessment of bee venom effects on matrix metalloproteinase activity and interferon production.

Controversial immunomodulatory properties of bee venom (BV) have provided an appropriate field for more investigation. The aim of present research was to verify the effects of honeybee venom on matrix metalloproteinase activity and interferon production as well as cell proliferation in monocyte and fibroblast cell lines.The monocyte and fibroblast cell lines (K562, HT-1080, WEHI-164) were used in order to assess proliferative response, interferon-1 production and matrix metalloproteinase-2 (MMP-2) activity. Australian BV (ABV) and Iranian BV (IBV) preparations at concentrations of 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, and 1microg/ml were added to each overnight cultured cells. In time course study, cells were treated each with ABV and IBV. In all cases supernatants were collected 24 hours after treatment. A sample of the each medium was used for zymography and interferons assay. Non-treated cells were used as controls.The production of IFN-a and IFN-b in supernatant of cell cultures was assessed using enzyme linked immunoassay procedure. MMP-2 activity, as an inflammatory index, was evaluated using zymoanalysis method.The results of this study showed that, there were no significant difference between two sources of honey bee venoms when they were added to an identical cell line, whereas, the responses of various cell lines against bee venom were different. The increasing amounts of bee venom to human monocyte cell line (K562) revealed a significant increase in proliferative response. Our findings showed that the bee venom had no influence on IFN-a production in cell culture media, whereas, adding the BV to K562 cell line could significantly increase the production level of IFN-b only on day 8 post-treatment. In addition the effect of bee venom on MMP-2 activity in both cell culture media, WEHI-164 and K562 was similar. The stimulatory effect of bee venom on MMP-2 activity occurred at low doses. In contrast, its inhibitory effect was seen at high concentrations.It is concluded that, honeybee venom affects on MMP-2 activity and interferon beta production as well as cell proliferation in a time and dose-dependent manner.