Dual anti-metastatic and anti-proliferative activity assessment of two probiotics on Hela and HT-29 cell lines

Objective: Lactobacilli are a group of probiotics with beneficial effects on prevention of cancer. However, there is scant data in relation with the impacts of probiotics in late-stage cancer progration, especially metastasis. The present original work was aimed to evaluate the anti-metastatic and anti-proliferative activity of lactobacillus rhamnosus supernatant [LRS] and lactobacillus crispatus supernatant [LCS] on the human cervical and colon adenocarcinoma cell lines [HeLa and HT-29, respectively]

Materials and Methods: In this experimental study, the anti-proliferative activities of LRS and LCS were determined through MTT assay. MRC-5 was used as a normal cell line. Expression analysis of CASP3, MMP2, MMP9, TIMP1 and TIMP2 genes was performed by quantitative reverse transcriptase-polymerase chain reaction [qRT-PCR], following the cell synchronization

Results: Supernatants of these two lactobacilli had cytotoxic effect on HeLa, however LRS treatment was only effective on HT-29 cell line. In addition, LRS had no side-effect on normal cells. It was shown that CASP3 gene expression has been reduced after treatment with supernatants of two studied lactobacilli. According to our study, LRS and LCS are efficacious in the prevention of metastasis potency in HeLa cells with decreased expression of MMP2, MMP9 and increased expression of their inhibitors. In the case of HT-29 cells, only LRS showed this effect

Conclusion: Herein, we have demonstrated two probiotics which have anti-metastatic effects on malignant cells and they can be administrated to postpone late-stage of cancer disease. LRS and LCS are effective on HeLa cell lines while only the effect of LRS is significant on HT-29, through cytotoxic and anti-metastatic mechanisms. Further assessments are required to evaluate our results on the other cancer cell lines, in advance to use these probiotics in other extensive trial studies

Mycoplasma infection in pyospermic infertile and healthy fertile men

Infections are one of the correctable causes of infertility with low cost and cost effective treatment. The 50% of infertile cases is related to men in some way, and 30% of them are absolutely related to them. Mycoplasmas are the smallest microorganisms with capability of DNA replication. Present study is planned to compare the mycoplasma infection in infertile men and men with established fertility. 45 Semen samples were collected from case and control persons who referred to Royan Infertility and Fertility Institute between 2004 and 2005 and stored in -20 degree°C until time of test. DNA was extracted from semen using phenol chloroform. PCR reaction was done by mycoplasma specific primers. Mycoplasma genitalium gene was amplified in 6 [40%] cases from 15 infertile semen samples and 11 [36.6%] from 30 control semen samples. Probability of genital infection, at least, in these studies group, is very lower than other communities' reports

Two matrix metalloproteinase inhibitors from scrophularia striata boiss

Many species belonging to the Scrophularia genus have been used since ancient times as folk remedies for many medical conditions such as scrofulas, scabies, tumors, eczema, psoriasis, inflammations. The aim of this study was to characterize the matrix metalloproteinases [MMPs] inhibitor compounds of the Scrophularia striata extract by bio-guide fractionation. The aerial parts of S. striata were collected and different extracts were sequentially prepared with increasingly polar solvents. The MMPs inhibitory activity of the crude extract and its fractions were evaluated by the Zymoanalysis method. The pure compounds were purified from the active fraction by chromatography methods. Chemical structures were deduced by nuclear magnetic resonance and mass spectrometry. Two active compounds [acteoside and nepitrin] were identified by bio-guide fractionation. The inhibitory effects of nepitrin and acteoside at 20 Micro g/ml were about 56 and 18 percent, respectivly. The inhibitory effects of acteoside at 80 Micro g/ml were increased to about 73 percent. In summary, the results suggest that nepitrin effectively inhibited MMPs inhibitory activity at low concentrations, whereas acteoside showed inhibition at high concentrations

Down regulation of sidB gene by use of RNA interference in aspergillus nidulans

Introduction of the RNA interference [RNAi] machinery has guided the researchers to discover the function of essential vital or virulence factor genes in the microorganisms such as fungi. In the filamentous fungus Aspergillus nidulans, the gene sidB plays an essential role in septation, conidiation and vegetative hyphal growth. In the present study, we benefited from the RNAi strategy for down-regulating a vital gene, sidB, in the fungus A. nidulans. The 21-nucleotide small interfering RNA [siRNA] was designed based on the cDNA sequence of the sidB gene in A. nidulans. Transfection was performed through taking up siRNA from medium by 6 hour-germinated spores. To evaluate the morphologic effects of siRNA on the fungus, germ tube elongation was followed. Moreover, total RNA was extracted and quantitative changes in expression of the sidB gene were analyzed by measuring the cognate sidB mRNA level by use of a quantitative real-time RT-PCR assay. Compared to untreated-siRNA samples, a significant inhibition in germ tube elongation was observed in the presence of 25 nM of siRNA [42 VS 21 microM]. In addition, at the concentration of 25 nM, a considerable decrease in sidB gene expression was revealed. Usage of RNAi as a kind of post-transcriptional gene silencing methods is a promising approach for designing new antifungal agents and discovering new drug delivery systems

Down-regulation of the ALS3 gene as a consequent effect of RNA-mediated silencing of the EFG1gene in candida albicans

The most important virulence factor which plays a central role in Candida albicans pathogenesis is the ability of this yeast to alternate between unicellular yeast and filamentous hyphal forms. Efg1 protein is thought to be the main positive regulating transcription factor, which is responsible for regulating hyphal-specific gene expression under most conditions. ALS3 is one of the Efg1-associated genes encoding a multi-functional adhesive polypeptide, which mediates adherence to diverse host substrates. In this study, the EFG1 gene was knocked down by using synthetic siRNA in C. albicans and the regulation in ALS3 as one of the Efg1-dependent genes was investigated. The 19-nucleotide siRNA was designed based on cDNA sequence of EFG1 gene in C. albicans. Transfection was performed using modified- plyethylen glycol/LiAc method. To quantify the level of EFG1 and the hyphal-specific ALS3 gene expression, the cognate EFG1 and ALS3 mRNA were measured in C. albicans by quantitative real-time RT-PCR. Fluorescent microscopy pictures indicated that transfection was performed successfully. Also, according to relative expression software tool, expression of EFG1 gene was decreased significantly with 500 nM siRNA as well as 1 micro M siRNA [P<0.05]. However, more significant downregulations were observed in the expression of ALS3 in both concentrations of 500 nM and 1 micro M siRNA [P<0.05]. In conclusion, we demonstrated the down-regulation of ALS3 gene as a consequent of applying EFG1-specific siRNA in C. albicans. This may lead us to design anti-fungal-specific agents in order to face with C. albicans-associated infections

Angiotensin II differentially induces matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 production and disturbs MMP/TIMP balance

Angiotensin II, the main component of the rennin-angiotensin system, is associated with cardiovascular diseases such as hypertension, vascular remodeling and inflammation. Remodeling process results from dysregulation of Matrix Metalloproteinases [MMPs] and their tissue inhibitors [TIMPs]. MMPs are considered as important target genes for angiotensin II. The aim of this study was to determine the effects of angiotensin II on MMP-9 and TIMP-1 production and MMP/TIMP balance in a monocytic cell type. Human monocytic U-937 cells were cultured and treated with 100 nM angiotensin II. Supernatants were analyzed for MMP-9 and TIMP-1 using ELISA and zymography methods. Real-time PCR was utilized to evaluate relative MMP-9 and TIMP-1 genes expression following treatments. Cytotoxicity potentials of treatments were determined by assaying lactate dehydrogenase leakage from the cells. Stimulation of the monocytic cells with angiotensin II significantly increased MMP-9 and TIMP-1 secretion as measured by ELISA [p<0.05]. It also augmented gelatinolytic activity of MMP-9 in the conditioned media as much as 49% [p<0.05]. Incubation of the cells with angiotensin II for 12 hr increased MMP-9 and TIMP-1 gene expression 2.7 and 1.8 folds, respectively [p<0.05]. Angiotensin II treatments did not establish significant cytotoxic effects. In summary, our data provide further evidences that monocytic MMP-9 is a major effector of angiotensin II. It is induced more efficiently than TIMP-1 by angiotensin II that leads to MMP/TIMP imbalance. Our data also reveal by the pivotal participation of these cells in pathological cardiovascular remodeling mediated by angiotensin II

Intermitted feeding attenuates clinical course of experimental autoimmune encephalomyelitis in C57BL/6 mice

Multiple Sclerosis [MS] is an automimmune inflammatory, demyelinating disease of human central nervous sytem. Experimantal Autoimmune Encephalomyelitis [EAE] is the commonly used animal model of MS. Calorie restriction has been found to reduce inflammation and autoimmune responses and promote neruoprotection. In this study we evaluated the effects of intermittent feeding protocol of the calorie restriction in a mouse model of EAE. Fifty four female mice [C57BL/6] were used in this study. The animals were divided into two dietary groups: ad libitum [AL] [n=25] with access to food on alternate days. After 8 weeks, EAE was induced in animals by immunization with MOG antigen [hooke labs, Lawrence, MA, USA] subcutaneously. AL and IF groups were then further divided into two groups each: AA [ad libitum until the end of study] [n=16] and Al [subjected to intermittent feeding regimen after immunization day] [n=13]. The IF group was divided into II [continued intermittent feeding regimen until the end of study] [n=13] and IA [changed to AL regimen after immunization day][n=12]. All the animals were behaviorally monitored for 35 days after immunization and observed daily for the signs and severity of disease with EAE scoring scale [0.5] and cumulative disease index [CDI] score. Intermittent feeding significantly reduced the incidence of EAE in IF groups [Al 0%, II 18.5%, IA 22.2%, p<0.05]. In addition, intermittent feeding significantly delayed the onset of EAE in Al group [p<0.05] and also, intermittent feeding significantly reduced the severity of disease in II and IA groups [AA vs. II, p<0.05 and AA vs. IA p<0.05] groups. The CDI was also significantly reduced in intermittent feeding fed groups [Al, II and to compared to AA group [p<0.05, <0.01, <0.05 respectively]]. Intermittent feeding regimen protocol of the calorie restriction significantly suppressed EAE incidence, induction, and severity. The results of this study suggest possible role of intermittent feeding in the treatment of Multiple Sclerosis patients

Green synthesis of small silver nanoparticles using geraniol and its cytotoxicity against fibrosarcoma-Wehi 164

Many reports have been published about the biogenesis of silver nanoparticles using several plant extracts such as Pelargonium graveolens [P.graveolens-geranium] and Azadirachta indica [neem] but the capacity of their natural reducing constituents to form silver nanoparticles has not yet been studied. In this research the synthesis of silver nanoparticles using geraniol has been investigated. We successfully synthesized uniformly dispersed silver nanoparticles with a uniform size and shape in the range of 1 to 10 nm with an average size of 6 nm. Also the cytotoxicity of the prepared silver nanoparticles was investigated using a cancer cell line [Fibrosarcoma-Wehi 164]. The cytotoxicity analysis of the sample shows a direct dose-response relationship; cytotoxicity increased at higher concentrations. At concentration of 1 pg/ml, silver nanoparticles was able to inhibit the cell line's growth by less than 30%. Conversly, the presence of 5 pg/m/of silver nanoparticlse significantly inhibited the cell line's growth [> 60%]. The concentration necessary to produce 50% cell death was 2.6 microg/m/for this silver nanoparticles preapared with geraniol

Expression of HLA-G in the skin of patients with pemphighus vulgaris

Studies on HLA-G, a nonpolymorphic antigen of non-classical HLA class I, is of basic and clinical significance. In the present study, the expression of HLA-G proteins in the human skin tissue sections of normal and autoimmune pemphigus vulgaris [PV] individuals were investigated using monoclonal antibodies. The antibodies recognized both membrane-bound and soluble isoforms of HLA-G. RT-PCR was performed to assess the patterns of HLA-G mRNA transcripts in the epidermal cells of PV and normal subjects. HLA-G expression could only be detected at transcriptional level in normal skin tissues. However cells derived from PV subjects expressed detectable HLA-G molecules at both transcriptional and translational levels. In addition, the RT-PCR patterns of HLA-G amplification revealed a reduction in HLA-G2 and an increase in HLA-G1 transcripts in epidermal cells of PV patients as compared to normal cells. These observations further support suggestions in the literature regarding the role of HLA-G in induction of tolerance in autoimmune individuals

Suppression of telomerase activity by pyrimethamine: implication to cancer

Although pyrimethamine [Tindurin[TM]] 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. 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. Cytotoxicity analysis of pyrimethamine revealed a dosedependent 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. 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

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 micro g/ml and 1, 5, 10 micro g/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 micro g/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 micro g/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

Study of correlation between prostate specific antigen and matrix metallloproteinase-2 activity in benign and malignant prostate hyperplasia

There are emerging data on novel tumor markers such as matrix metalloproteinase-2 [MMP-2 or gelatinase-A], which play a key role in tissue invasion and metastasis. We designed an investigation to assess the usefulness of MMP-2 activity as compared to prostate specific antigen [PSA] in cancer staging process. We have analyzed the circulating form of MMP-2 in serum samples of patients suffering from either benign prostate hyperplasia [BPH, n = 54] or prostate cancer [PC, n = 26], and prostatitis [n = 4] as compared to control normal individual [n = 26], respectively. Protein-content adjusted samples were separated by gelatin-embedded polyacrylamide gel electrophoresis then were subjected to densitometric analysis. Total PSA [tPSA] and free PSA [fPSA] were quantified using a standard ELISA technique. Correlation coefficient [r] between tPSA and MMP-2 activity in patient group was +0.938 and in controls group was 0.799 [P<0.01]. In addition, [r] between tPSA and MMP-2 activity in PC patients was 0.940 and in BPH patients was 0.962 [P<0.01]. [r] between fPSA and MMP-2 activity in PC patients was 0.913, in BPH patients was 0.644, and in prostatitis patients was -0.994 [P<0.01]. These results demonstrate that MMP-2, compared to PSA, might be considered as a better tumor marker in monitoring and screening patients with PC

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. Nontreated cells were used as controls. The production of IFN-alpha and IFN-beta 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-alpha production in cell culture media, whereas, adding the BV to K562 cell line could significantly increase the production level of IFN-beta 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

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 ofMMPs 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 gelatinaselike 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 betweenACD cells and normal fibroblasts both in time-course and MMP-2 activity per cell fashions, 163.7 +/- 16.21 for meanACD fibroblasts vs. 130 +/- 9.09 for normal cells respectively. These data suggest that fibroblasts overproliferated in the process ofACD. Moreover, simultaneous overexpression of MMPs observed inACD fibroblasts vs. normal strains, is indicative of altered fibroblast functionality in the process of allergic contact dermatitis. 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

Rt-PCR cloning and expression of complementary DNA for human tumor necrosis factor alpha

U-937, a monocytic cell line was induced with Phorbol Myristate Acetate [PMA] for human tumor necrosis factor alpha [hTNF- alpha] production. An optimized RT-PCR was employed for construction of hTNF- alpha complementary DNA [cDNA]. The resulted fragment was verified by restriction digestion mapping with PvuII.The verified fragment was cloned in pUC18 plasmid and transformants were pelletted onto LB agar medium containing ampicillin, X-gal and IPTG. The resulting white colonies were verified by PCR and cultured in LB medium containing ampicillin and IPTG. The biological activity and the quantity of hTNF- alpha expression was assessed by an ELISA method using a monoclonal anti hTNF- alpha antibody together with a bioassay utilizing L-929 line as sensitive cells