Gut micobiota alteration by Lactobacillus rhamnosus reduces pro-inflammatory cytokines and glucose level in the adult model of Zebrafish.

OBJECTIVE: Type 2 diabetes mellitus (T2DM) is still a challenge for physicians to manage patient's circumstances. It is assumed that alterations in the normal flora may be involved in the pathogenesis of T2DM through inducing chronic inflammation. To investigate the effect of Lactobacillus rhamnosus as a common probiotic on T2DM, we induced an experimental model of T2DM in adult male Zebrafish by gradient hyper-glucose accumulation methodology. RESULTS: In this trial 3-month old male adult Zebrafish were divided in to four groups including two control groups and T2DM induced groups with or without probiotic treatment. After 5 days of acclimation, T2DM was induced by a gradient hyper-glucose accumulation methodology. Diabetic fishes had statistically abnormal blood glucose and pro-inflammatory cytokine levels compared to control group (p = 0.0001). These results suggest that probiotic intervention decreased the blood glucose level in the T2DM-P group by decreasing pro-inflammatory cytokines responsible for signaling in T2DM therapeutic modalities.

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.

Transcranial Magnetic Stimulation for the Treatment of Concussion: A Systematic Review.

BACKGROUND: Post-concussive symptoms (PCSs) are common, disabling, and challenging to manage. Evolving models of concussion pathophysiology suggest evidence of brain network dysfunction that may be amenable to neuromodulation. Repetitive transcranial magnetic stimulation (rTMS) has emerged as a potential novel treatment option for PCSs. OBJECTIVES: To systematically review rTMS trials for the treatment of symptoms following concussion/mild traumatic brain injury (mTBI). MATERIALS AND METHODS: We conducted a systematic review of Pubmed/Medline, Embase, and PsychINFO databases were searched up to May 19, 2020. Studies were included if they were prospective rTMS treatment studies of patients with mTBI/concussion. Variables including patient demographics, study design, rTMS protocol parameters, primary outcome measures, and efficacy data were extracted and qualitatively synthesized. rTMS methodology and study quality were also evaluated. RESULTS: Of the 342 studies identified, 11 met eligibility criteria and were included for synthesis. Forty-one percent of patients were female and age ranged from 18 to 65 (average age = 38.5 years). Post-concussive depression (seven studies) and headache (four studies) were the most commonly investigated symptoms. The majority of trials were sham-controlled with randomized control trial (RCT) designs, but all were small pilot samples (n < 30). Methodological heterogeneity and a low number of identified trials precluded quantitative meta-analysis. Regarding rTMS for post-concussive depression, positive results were found in two out of four studies with depression as a primary outcome, and all three studies that assessed depression as a secondary outcome. All four rTMS studies for post-concussive headache reported positive results. CONCLUSIONS: rTMS for the treatment of concussion/mTBI shows promising preliminary results for post-concussive depression and headache, symptoms that otherwise have limited effective treatment options. More studies with larger sample sizes are needed to further establish potential efficacy.

Augmented cAMP Signaling by Co-Administration of Resveratrol and Curcumin: A Cellular Biosensor Kinetic Assessment.

BACKGROUND: Curcumin and resveratrol are two polyphenolic compounds extensively investigated for their medicinal effects on inflammatory signaling. However, there is a paucity of information on the Adenosine-3', 5'-cyclic monophosphate (cAMP) kinetics following administration of curcumin and resveratrol in biological systems. In this study, kinetic modulation of cAMP as a target detection messenger in pro-inflammatory pathways was assessed by co-administration of curcumin and resveratrol using a cellular sensor model. METHODS: To evaluate their putative activity, curcumin and resveratrol compounds were administered alone or in combination on the media culture of cAMP EPAC (exchange protein directly activated by cAMP) bioluminescence resonance energy transfer (BRET) biosensor. The study was performed at the following two centers at Tehran University of Medical Sciences (TUMS): 1- Biotechnology Research Center, and, 2- Endocrinology and Metabolism Research Institute (EMRI) in 2017. Time course kinetic of cAMP response signals were plotted. Forskolin and IBMX were used to stabilize the cAMP signals. RESULTS: When we treated HEK-293T biosensor cells at 10uM concentration, curcumin and resveratrol upregulated cAMP signaling. Co-administration of resveratrol and curcumin revealed an augmented cAMP level, as compared to treatments with the compounds alone. CONCLUSION: Co-administration of curcumin and resveratrol leverage cAMP kinetic response in a time-course manner. The presented methodology can be readily adopted for drug development and novel biopharmaceutical functional analyses.

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.

Silencing the wild-type and mutant K-ras increases the resistance to 5-flurouracil in HCT-116 as a colorectal cancer cell line.

Colon cancer is the second to third common cancer worldwide. Several efforts have been made to reveal the pathways responsible for drug resistance in this type of cancer. We aimed to investigate the effect of silencing both mutant and wild-type Kristen Rous sarcoma (k-ras) on the response of human colorectal tumor 116 (HCT-116) as a colon cancer cell line to the cytotoxic effect of 5-flurouracil (5-FU). One oligonucelotide against mutant k-ras (12th codon, namely 207) and two against wild-type k-ras (namely 535 and 689) were cloned into pSilencer neo2.1. The linearized vectors besides the negative control plasmid were stably transfected into HCT-116. The proliferation rates of these cells in different concentrations of 5-FU and the apoptosis rates of the cells after treatment with lethal doses of 5-FU were studied. Moreover, the cell cycle in these cells was also analyzed by staining the cells with propidium iodide. Stably transfected cells were named HCT207ks, HCT535ks, HCT689ks, and HCT-Sc (transfected with the negative control plasmid). Decreased expression of k-ras in HCT207ks, HCT535ks, and to a lesser extent in HCT689ks was proved by quantitative real-time PCR. Although in HCT207ks the cells were mostly in G0/G1 and G2/M phases, in HCT535ks and HCT689ks, the cells in the S phase were higher in comparison with nontransfected HCT-116. Lethal doses of 5-FU in HCT-116 and HCT-Sc were 2.5-3 and 3-3.5 µmol/l, whereas in HCT207ks, HCT535ks, and HCT689ks, they were 35-40, 37.5-40, and 22.5-25 µmol/l. In conclusion, silencing mutant and wild-type k-ras would increase the resistance of HCT-116 cell line as a model of colorectal cancer to 5-FU. The degree of resistance was related directly to the k-ras mRNA level. Therefore, both mutant and wild-type k-ras may play a role in sensitizing colorectal cancer cells to 5-FU as a common chemotherapeutic drug.

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.

Interaction of CpG-oligodeoxynucleotides with Toll like receptor 9 induces apoptosis and modulates metaloproteinase-2 activity in human intestinal epithelium.

Recent reports have indicated different effects of immunostimulatory sequences containing CpG-Oligodeoxynucleotides (ODN) on various immune cells. However, the exact role of CpG-ODN in the human gut is unclear. In the present study, we assessed potential effects of CpG-ODN on non lymphoid cell (intestinal epithelial cell line HT-29) on a dose-response and time-course basis. Intestinal epithelial cell line HT-29 was treated with CpG-ODN (CpG 2006) and lipopolysaccharide (LPS) at 5, 10, 25, 50 microg/ ml and 1, 5, 10 microg/ ml concentrations, respectively. Following treatments, dose- response and time-course cytotoxicity using a colorimetric method, Metaloproteinase-2 (MMP-2) activity (using gelatin zymography) and apoptosis (using annexin-v flowcytometry method) assays were performed. Chloroquine treatment was also used for its inhibitory effect on endosomal acidification process to verify specific CpG-ODN and Toll Like Receptor 9 (TLR9) interactions. Cytotoxicity analysis of CpG-ODN showed that CpG-ODN increased significantly the proliferation of CpG-ODN treated cells, as compared to untreated cells, at concentrations of 10-25 microg/ml (p < 0.05). Overall MMP-2 activity analysis showed significant differences between treated and untreated cells. However, minimal changes were observed when MMP-2 activity was assessed per cell. Moreover, CpG-ODN treated cells demonstrated an increasing apoptosis rate of 0.8 %, 6.46 % and 14.21% at concentrations of 5, 10, 25 microg/ml, respectively. Collectively, our data indicated that intestinal epithelial cell line HT-29 is highly responsive to CpG effect in vitro and exhibits modified activities. The direct CpG-ODN and TLR-9 interactions in HT-29 cells could provide new approaches in malignant tumor therapeutic strategies.

Suppression of telomerase activity by pyrimethamine: implication to cancer.

BACKGROUND: Although pyrimethamine (Tindurin) appears to be effective in the prevention and treatment of some infectious diseases, very little information exists on its unpredictable properties. We design this study to evaluate its anti-tumoral effect on a model of cell line. METHODS: The cytotoxic influence of Pyrimethamine on prostate cell line was investigated using an in vitro colometric assay. The potential modulatory effects on metastasis, apoptosis, and immortality characteristics of cells were assessed with gelatin zymography, terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay and telomeric repeat amplification protocol, respectively. RESULTS: Cytotoxicity analysis of pyrimethamine revealed a dose-dependent fashion. An apoptotic influence of pyrimethamine was also confirmed by data obtained from TUNEL assay. Dose-dependent inhibitory effect on matrix metalloproteinases (MMP) was seen in pyrimethamine. A potent inhibitory effect of pyrimethamine was also established by data achieved from TRAPeze telomerase detection kit. CONCLUSIONS: Collectively, as induction of apoptosis together with MMP and telomerase inhibition could be indicative of cancer treatment, pyrimethamine might be considered as a chemopreventative agent in cancer.

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.

BACKGROUND: The level of activity of the telomerase has been shown to correlate with the degree of invasiveness in several tumor types. In addition, cellular redox state is believed to regulate the secretion of matrix metalloproteinase-2 (MMP-2). AIMS: To determine the effect of anti-sense telomerase treatment of prostate cancer cells on MMP-2 activity, and the reactive oxygen and nitrogen species (two effectors of cellular redox state). METHODS: Anti-sense oligonucleotide against RNA component of human telomerase (hTR) was introduced into the cells using Fugene-6 transfection reagent. The activity of telomerase was assessed using Telomere Repeat Amplification Protocol (TRAP assay). Activity of matrix metalloproteinase-2 (MMP-2) was determined by zymography. Levels of intracellular reactive oxygen species (ROS) and nitric oxide metabolites were measured by dichlorofluorescein diacetate (DCFH-DA) staining and Griess reagent, respectively. The level of apoptosis was determined using TUNEL assay. RESULTS: TRAP assay showed more than 90% inhibition of telomerase activity after 72 h of transfection. Pro-MMP-2 activity was decreased down to 50% of the control levels. Intracellular reactive oxygen species were also significantly decreased. Neither apoptosis rate nor the level of nitric oxide metabolites was significantly different between anti-sense treated and control cells. CONCLUSIONS: Concomitant reduction of the pro-MMP-2 secretion and ROS in PC-3 cells following hTR inhibition suggests that over-activity of telomerase in cancer cells might increase the level of matrix metalloproteinase-2 and thus, be directly involved in the invasion process through enhancement of intracellular oxidative stress.

Dermal Wound Fibroblasts and Matrix Metaloproteinases (MMPs): Their Possible Role in Allergic Contact Dermatitis.

This study was conducted to examine if allergic contact dermatitis (ACD) alters the expression of MMPs in human dermal fibroblasts. Fibroblasts are the primary source for MMP and matrix production in skin. MMPs are known to involve in a number of physiological and pathological processes. Some published data indicated a gelatinase-like activity in acute and chronic phases of allergic contact dermatitis. However, no exact source of gelatinase activity was demonstrated. Moreover, little is known about the role of MMPs in immune responses.To study and predict the pathophysiological effects of (MMP-2) in allergic contact dermatitic (ACD) patients, we established an in vitro tissue culture survey based on fibroblast explanted from ACD wounds and normal tissues respectively. We also employed a precise proliferation assay [i.e. MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to analyze and compare three ACD vs. three normal cell strains. Parallel to MTT assay, we assessed the activity as well as the kinetics of gelatinase (MMP-2) in conditioned media using a zymogeraphy analysis. There was a significant difference in proliferation capacity between mean ACD fibroblast strains vs. mean normal cells, particularly in days 6 to 8 post explantation, 492.5+/-6.6 vs. 361.75+/-8.25 respectively. Zymoanalyses indicated significant differences between ACD cells and normal fibroblasts both in time-course and MMP-2 activity per cell fashions, 163.7+/-16.21 for mean ACD fibroblasts vs. 130+/-9.09 for normal cells respectively. These data suggest that fibroblasts overproliferated in the process of ACD. Moreover, simultaneous overexpression of MMPs observed in ACD fibroblasts vs. normal strains, is indicative of altered fibroblast functionality in the process of allergic contactdermatitis. The activity per cell analysis showed that MMP-2 expression in ACD fibroblasts is independent of cell number, suggesting that either intra- or inter-cellular control signals are also altered and that ACD fibroblasts exhibit hyper-responsiveness to mitogenic or fibrogenic stimulants. Altogether, these data address the chronocity and non-healing tendency of ACD wounds. However, more studies are required to examine possible MMPs inhibition and differential expression of mytogenic, fibrogenic and antifibrogenic cytokines in ACD wound beds. In particular, MMP-2 is postulated to be an aim for further gene therapy protocols.