Effect of lactobacillus crispatus and lactobacillus rhamnosus culture supernatants on expression of autophagy genes and hpv e6 and e7 oncogenes in the hela cell line

Objective: The aim of this study was to clarify the mechanism by which lactobacilli exert their cytotoxic effects on cervical cancer cells. In addition, we aimed to evaluate the effect of lactobacilli on the expression of human papilloma virus [HPV] onco-genes

Materials and Methods: In this experimental study, using quantitative real-time polymerase chain reaction [PCR], we analyzed the expression of CASP3 and three autophagy genes [ATG14, BECN1 and alpha 2 catalytic subunit of AMPK [PRKAA2]] along with HPV18 E6 and E7 genes in HeLa cells before and after treatment with Lactobacillus crispatus and Lactobacillus rhamnosus culture supernatants

Results: The expression of CASP3 and autophagy genes in HeLa cells was decreased after treatment with lactobacilli culture supernatants. However, this de-crease was not significant for PRKAA2 when compared with controls. In addition, expression of HPV E6 was significantly decreased after treatment with lactobacilli culture supernatants

Conclusion: Lactobacilli culture supernatants can decrease expression of ATG14 and BECN1 as well as the HPV E6 oncogene. It has been demonstrated that the main changes occurring during cervical carcinogenesis in cell machinery can be reversed by suppression of HPV oncogenes. Therefore, downregulation of HPV E6 by lactobacilli may have therapeutic potential for cervical cancer. As the role of autophagy in cancer is complicated, further work is required to clarify the link between downregulation of autophagy genes and antiproliferative effects exerted by lactobacilli

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

Upregulation of RHOXF2 and ODF4 expression in Breast Cancer tissues

Objective: During the past decade, the importance of biomarker discovery has been highlighted in many aspects of cancer research. Biomarkers may have a role in early detection of cancer, prognosis and survival evaluation as well as drug response. Cancer-testis antigens [CTAs] have gained attention as cancer biomarkers because of their expression in a wide variety of tumors and restricted expression in testis. The aim of this study was to find putative biomarkers for breast cancer

Materials and Methods: In this applied-descriptive study, the expression of 4 CTAs, namely acrosin binding protein [ACRBP], outer dense fiber 4 [ODF4], Rhox homeobox family member 2 [RHOXF2] and spermatogenesis associated 19 [SPATA19] were analyzed at the transcript level in two breast cancer lines [MCF-7 and MDA-MB-231], 40 invasive ductal carcinoma samples and their adjacent normal tissues as well as 10 fibroadenoma samples by means of quantitative real-time reverse transcription polymerase chain reaction [RT-PCR]

Results: All four genes were expressed in both cell lines. Expression of ODF4 and RHOXF2 was detected in 62.5% and 60% of breast cancer tissues but in 22.5 and 17.5% of normal tissues examined respectively. The expression of both RHOXF2 and ODF4 was upregulated in cancerous tissues compared with their normal adjacent tissues by 3.31- and 2.96-fold respectively. The expression of both genes was correlated with HER2/neu overexpression. RHOXF2 expression but not ODF4 was correlated with higher stages of tumors. However, no significant association was seen between expression patterns and estrogen and progesterone receptors status

Conclusion: ODF4 and RHOXF2 are proposed as putative breast cancer biomarkers at the transcript level. However, their expression at protein level should be evaluated in future studies

Zinc and low-dose of cadmium protect sertoli cells against toxic-dose of cadmium: the role of metallothionein

The impact of cadmium [Cd] on male infertility may be related to the interaction with metal-binding proteins known as metallothioneins [Mts]. Trace elements like zinc [Zn] have protective effects on testicular damage induced by Cd. We determined the effect of Zn and low-dose Cd pre-treatment on the expression of Mt1 and Mt2 genes on testicular Sertoli cells. The cultured TM4 mouse sertoli cells were treated with 50 micro M ZnSO4 [Zn pre-treated group; ZnPG], 2 micro M CdCl2 [Cd pre-treated group; CdPG], or distilled water [DW pre-treated group; DWPG]. After 18 hour, all of these groups were exposed to 100 micro M CdCl2 for different periods of time [1, 2, 3, and 6 hours]. There was also a control group for all three groups, which was treated only with distilled water [without Cd or Zn pre-treatment]. Cellular viability, Zn and Cd concentrations and gene expression were assessed by MTT, atomic absorption spectrometry and real time PCR methods, respectively. The expression of Mt1 and Mt2 genes in ZnPG, CdPG, and DWPG was greater than the control group [p=0.02 and p=0.01, respectively]. Cd concentrations in CdPG and DWPG were greater than the control group [p=0.00]. Expression of both genes in ZnPG and CdPG increased after 3 hours of treatment and Cd concentration decreased simultaneously, which was more obvious in ZnPG. Zn and short term low-dose Cd pre-treatment might reduce the adverse effects of Cd by increasing expression of Mts genes in Sertoli cells. The protective effect of Zn was stronger than Cd

Generation of a uracil auxotroph strain of the probiotic yeast Saccharomyces boulardii as a host for the recombinant protein production

Saccharomyces boulardii [S. boulardii] is the best known probiotic yeast. The genetic engineering of this probiotic strain requires the availability of appropriate mutants to accept various gene constructs carrying different selection markers. As the auxotrophy selection markers are under focus, we have generated a ura3 auxotroph mutant of S. boulardii for use in further genetic manipulations. Classical UV mutagenesis was used for the generation of auxotroph mutants. The mutants were selected in the presence of 5-FOA [5-Fluoroorotic acid], uracil and uridine. Uracil auxotrophy phenotype was confirmed by the ability of mutants to grow in the presence of uracil and the lack of growth in the absence of this compound. To test whether the uracil auxotrophy phenotype is due to the inactivation of URA3, the mutants were transformed with a plasmid carrying the gene. An in vitro assay was used for the analysis of acid and bile resistance capacity of these mutants. Three mutants were found to be ura3 auxotroph as they were able to grow only in the presence of uracil. When the URA3 gene was added, these mutants were able to grow normally in the absence of uracil. Further in vitro analysis showed that the acid and bile resistance capacity of one of these mutants is intact and similar to the wild type. A uracil auxotroph mutant of the probiotic yeast, S. boulardii, was generated and characterized. This auxotroph strain may have potential applications in the production and delivery of the recombinant pharmaceutics into the intestinal lumen

Real-Time RT-PCR on SAG1 and BAG1 Gene Expression during Stage Conversion in Immunosuppressed Mice Infected with Toxoplasma gondii Tehran Strain

Toxoplasmic encephalitis is caused by reactivation of bradyzoites to rapidly dividing tachyzoites of the apicomplexan parasite Toxoplasma gondii in immunocompromised hosts. Diagnosis of this life-threatening disease is problematic, because it is difficult to discriminate between these 2 stages. Toxoplasma PCR assays using gDNA as a template have been unable to discriminate between an increase or decrease in SAG1 and BAG1 expression between the active tachyzoite stage and the latent bradyzoite stage. In the present study, real-time RT-PCR assay was used to detect the expression of bradyzoite (BAG1)- and tachyzoite-specific genes (SAG1) during bradyzoite/tachyzoite stage conversion in mice infected with T. gondii Tehran strain after dexamethasone sodium phosphate (DXM) administration. The conversion reaction was observed in the lungs and brain tissues of experimental mice, indicated by SAG1 expression at day 6 after DXM administration, and continued until day 14. Bradyzoites were also detected in both organs throughout the study; however, it decreased at day 14 significantly. It is suggested that during the reactivation period, bradyzoites not only escape from the cysts and reinvade neighboring cells as tachyzoites, but also converted to new bradyzoites. In summary, the real-time RT-PCR assay provided a reliable, fast, and quantitative way of detecting T. gondii reactivation in an animal model. Thus, this method may be useful for diagnosing stage conversion in clinical specimens of immunocompromised patients (HIV or transplant patients) for early identification of tachyzoite-bradyzoite stage conversion.

Production and Evaluation of Toxoplasma gondii Recombinant GRA7 for Serodiagnosis of Human Infections

The precise diagnosis of the acute toxoplasmosis in pregnant women and immunocompromsied patients has critical importance. Most of the commercially available assays use the whole Toxoplasma soluble extract as the antigen. However, the assays currently available for the detection of specific anti-Toxoplasma antibodies may vary in their abilities to detect serum immunoglobulins, due to the lack of a purified standardized antigen. The aim of this study was production and evaluation of the usefulness of the recombinant Toxoplasma gondii GRA7 antigen for the serodiagnosis of Toxoplasma gondii IgM and IgG by ELISA. A total of 70 T. gondii IgM positive sera, 74 T. gondii IgG positive sera, and 60 sera from subjects who were not infected with T. gondii were examined. These sera were shown different absorbance values in ELISA test. To control the specificity of the rGRA7 other parasitic diseases, for example, echinococcosis, malaria, leishmaniasis, fascioliasis, and strongyloidiasis were tested of which none showed positive results. Sensitivity and specificity of the generated recombinant IgG ELISA in comparison with commercial ELISA (com ELISA) were 89% and 90%, and the sensitivity and specificity of the generated recombinant IgM ELISA were 96% and 90%, respectively. The results obtained here show that this antigen is useful for diagnostic purposes.

Expression of testis-specific genes, TEX101 and ODF4, in chronic myeloid leukemia and evaluation of TEX101 immunogenicity

Cancer-testis [CT] antigens are a group of antigens with a restricted expression in normal tissues, except testis, and they have aberrant expression in different tumors. This pattern of expression has made them promising targets for immunotherapy and cancer detection. Our aim was to find new members of this group that might be useful as markers in the detection of cancer and immunotherapy. A descriptive study conducted in referral centers of Tehran University of Medical Science from january 2008 to January 2009. We analyzed the expression of two testis-specific genes named ODF4 [outer dense fiber of sperm tails 4] and TEX101 [testis expressed 101] in 20 chronic myeloid leukemia [CML] and 20 normal samples by reverse transcription-polymerase chain reaction and sequencing. Immunogenicity of TEX101 was evaluated by means of enzyme-linked immunosorbent assay. These two genes were expressed in 30% of CML patients but not in any of the healthy donors. Humoral response against TEX101 was not detected in any samples. TEX101 and ODF4 are CT genes useful for detection of CML. Unlike many CT genes, overexpression of TEX101 was not shown to induce immunologic responses in these samples. According to the previous studies, overexpression of TEX101 leads to suppression of cancer invasion and metastasis; thus, the induction of the expression of TEX101 in cancer by epigenetic mechanisms may be a treatment strategy

role of Ile3434Thr XRCC7 gene polymorphism in differentiated thyroid cancer risk in an Iranian population

The aim of this study was to understand any association between differentiated thyroid carcinoma [DTC] and Ile3434Thr XRCC7 gene polymorphism [GenBank accession number: rs7830743]. DTC is the most prevalent thyroid neoplasm, which includes papillary and follicular cell carcinoma. XRCC7 gene encodes a protein that functions in non-homologous end joining DNA repair pathway. Non-synonymous polymorphisms in this gene may alter DNA repair capacity of the cell and change the risk of developing cancers. DTC patients [n = 173] and cancer free individuals [n = 204] were enrolled in a case-control study. The Ile3434Thr polymorphic alleles were discriminated by using amplification refractory mutation system-PCR method. The frequencies of this single nucleotide polymorphism in case and control groups were compared. Also, risk ratio for developing DTC in dichotomized genotypes was estimated by multivariate logistic regression analysis. Dichotomized genotypes into those with and without the 3434Thr allele showed that this allele was associated with DTC [OR [odd ratio]: 1.89, 95% confidence interval [CI] = 1.29-2.79, P<0.001]. Also, TC genotype was significantly associated with increased risk of DTC [OR: 2.42, 95% CI = 1.55-3.81, P = 0.0001] in individuals carrying this genotype. Allele 3434Thr in XRCC7 gene might be associated with differentiated thyroid cancer risk. Further studies with larger samples are needed to verify these initial findings

[Cloning and evaluation of expression of a novel overlapping region of NS3 gene of Hepatitis C virus by expressing vector]

In this project, our aim was to construct a novel expressing vector harboring a new sequence from overlapping region of NS3 gene of HCV from infected Iranian patient. The partial NS3 [pNS3] gene was amplified by Nested-RT-PCR method using sera of HCV infected patients harboring genotype 1a. After purification and cloning the pNS3 into TA-cloning vector, the best colony was selected based on Blue/White screening and colony-PCR following by confirmation with sequencing and restriction digestion with BglII. The sequenced gene was compared with other reference sequences using alignment softwares. The resultant pNS3 gene subcloned into the expression vector, IRES vector, followed by selection the suitable clones by 2 different colony-PCRs. The gene expression was evaluated using GFP detection, RT-PCR and western blotting techniques after transfection of the IRES-pNS3 vector into the 293 cell line. After pNS3 sequence amplification by RT-PCR, sequencing results showed high homology among the sequences with other reference sequences. This result also showed that it belonged to genotype 1 of HCV. Colony-PCR showed the insertion of gene into expressing vector with the right orientation. GFP expression, RT-PCR and western blotting confirmed transfection of vector, expression of pNS3 gene and production of its protein in 293 cells respectively. This novel expressing vector harboring partial region of NS3 gene in compare to full NS3 gene maybe more useful in immune induction by antigen presenting cells due to absence of genes responsible for protease activity of the protein in the setting of HCV vaccine