Selective Cytotoxic effect of Probiotic, Paraprobiotic and Postbiotics of L. casei strains against Colorectal Cancer Cells: Invitro studies

Abstract This study highlights the cytotoxic effect of three L. casei strains on colorectal cell lines in invitro conditions. Different concentrations of live, heat killed (HK) and cell free supernatant (CFS) of three L.casei strains were subjected to CaCo2 and MRC5 cell lines. The viability of the treated and untreated cells was determined after 72 hrs by MTT assay, and IC50 estimated. Apoptosis was evaluated by Annexin V-propidium iodide method using flow cytometry. The live, HK and CFS of the L. casei strains showed cytotoxic effects on colorectal cell lines with significant differences. The cytotoxicity effects of live cells on CaCo2 cells were significantly higher (p˂0.01) than the HK cells. A dose dependent response was observed, as higher concentrations resulted in enhanced cytotoxicity effects. Live L.casei 1296-2cells inhibited 91% of CaCo2 cell growth, with IC50 of less than 108 cfu/ml. MRS medium and concentrations of CFS at above 20% v/v, were cytotoxic to the normal cell lines. Flow cytometry analyses of L. casei 1296-2 indicated that cytotoxicity effects on CaCo2 cells is related to apoptotic induction. Invitro studies indicate that Live and CFS of L. casei 1296-2 might be promising candidate for the control of colorectal cancers

Dual effects of cell free supernatants from lactobacillus acidophilus and lactobacillus rhamnosus GG in regulation of MMP-9 by up-regulating TIMP-1 and down-regulating CD147 in PMA- differentiated THP-1 cells

Objective: recent studies have reported dysregulated expression of matrix metalloproteinases [MMPs], especially MMP-2, MMP-9, tissue inhibitor of metalloproteinase-1, -2 [TIMP-1, TIMP-2], and extracellular matrix metalloproteinase inducer [EMMPRIN/CD147] in activated macrophages of patients with inflammatory diseases. Therefore, MMP-2, MMP-9, and their regulators may represent a new target for treatment of inflammatory diseases. Probiotics, which are comprised of lactic acid bacteria, have the potential to modulate inflammatory responses. In this experimental study, we investigated the anti-inflammatory effects of cell-free supernatants [CFS] from Lactobacillus acidophilus [L.acidophilus] and L. rhamnosus GG [LGG] in phorbol myristate acetate [PMA]-differentiated THP-1 cells

Materials and Methods: in this experimental study, PMA-differentiated THP-1 cells were treated with CFS from L. acidophilus, LGG and uninoculated bacterial growth media [as a control]. The expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 mRNAs were determined using real-time quantitative reverse transcription polymerase chain reaction [RTPCR]. The levels of cellular surface expression of CD147 were assessed by flow cytometry, and the gelatinolytic activity of MMP-2 and MMP-9 were determined by zymography

Results: our results showed that CFS from both L. acidophilus and LGG significantly inhibited the gene expression of MMP-9 [P=0.0011 and P=0.0005, respectively], increased the expression of TIMP-1 [P<0.0001], decreased the cell surface expression of CD147 [P=0.0307 and P=0.0054, respectively], and inhibited the gelatinolytic activity of MMP-9 [P=0.0003 and P<0.0001, respectively] in PMA-differentiated THP-1 cells. Although, MMP-2 expression and activity and TIMP-2 expression remained unchanged

Conclusion: our results indicate that CFS from L. acidophilus and LGG possess anti-inflammatory properties and can modulate the inflammatory response

Effect of passage number and culture time on the expression and activity of insulin-degrading enzyme in Caco-2 cells

Background: Insulin-degrading enzyme [IDE] is a conserved zinc metallopeptidase. Here, we have evaluated the effect of passage number and culture time on IDE expression and activity in colorectal adenocarcinoma cell line [Caco-2]

Methods: Caco-2 cells were cultured with different passage ranges of 5-15, 25-35, 52-63 for 48, 72, and 120 hours. Subsequently, IDE expression and enzyme activity were assessed by Western blot analysis and fluorescent assay, respectively

Results: Our results confirmed that the amount of IDE was higher in cell extract compared to supernatant, and different passage numbers and culture times had small effect on IDE expression. However, when cells were cultured in the passage number range of 5-15 for 72 hours, the IDE activity was 35% higher compared to other passage numbers [p < 0.05]

Conclusion: The use of Caco-2 cells at passage number range of 5-15 and culture time of 72 hours provides proper conditions for IDE-related studies

Stem Cell Tracing Through MR Molecular Imaging

Stem cell therapy opens a new window in medicine to overcome several diseases that remain incurable. It appears such diseases as cardiovascular disorders, brain injury, multiple sclerosis, urinary system diseases, cartilage lesions and diabetes are curable with stem cell transplantation. However, some questions related to stem cell therapy have remained unanswered. Stem cell imaging allows approval of appropriated strategies such as selection of the type and dose of stem cell, and also mode of cell delivery before being tested in clinical trials. MRI as a non-invasive imaging modality provides proper conditions for this aim. So far, different contrast agents such as superparamagnetic or paramagnetic nanoparticles, ultrasmall superparamagnetic nanoparticles, fluorine, gadolinium and some types of reporter genes have been used for imaging of stem cells. The core subject of these studies is to investigate the survival and differentiation of stem cells, contrast agent's toxicity and long term following of transplanted cells. The promising results of in vivo and some clinical trial studies may raise hope for clinical stem cells imaging with MRI.

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

Lactobacilli modulate hypoxia-inducible factor [HIF]-1 regulatory pathway in triple negative breast cancer cell line

Objective: Hypoxia-Inducible Factor [HIF]-1 plays an essential role in the body's response to low oxygen concentrations and regulates expression of several genes implicated in homeostasis, vascularization, anaerobic metabolism as well as immunological responses. Increased levels of HIF-1alpha are associated with increased proliferation and more aggressive breast tumor development. Lactobacilli have been shown to exert anti-cancer effects on several malignancies including breast cancer. However, the exact mechanism of such effect is not clear yet. The aim of this study was to analyze the expression of selected genes from HIF pathway in a triple negative breast cancer cell line [expressing no estrogen and progesterone receptors as well as HER-2/Neu], MDA-MB-231, following treatment with two lactobacilli culture supernatants

Materials and Methods: In this experimental study, we analyzed the expression of HIF-1alpha, SLC2A1, VHL, HSP90, XBP1 and SHARP1 genes from HIF pathway in MDA-MB-231 cells, before and after treatment with Lactobacillus crispatus and Lactobacillus rhamnosus culture supernatants [LCS and LRS, respectively] by means of quantitative reverse-transcription polymerase chain reaction [qRT-PCR]

Results: Both LRS and LCS had cytotoxic effects on MDA-MB-231 cells, while the former type was more cytotoxic. LRS dramatically down-regulated expression levels of the HIF-1alpha, HSP90 and SLC2A1 in the MDA-MB-231 cells. LCS had similar effect on the expression of HSP90, to what was observed in the LRS treatment. The expression level of tumor suppressor genes VHL and SHARP1 were also decreased in LCS treated cells

Conclusion: Although both LCS and LRS had cytotoxic effects on the MDA-MB-231 cells, it is proposed that LRS could be more appropriate for pathway directed treatment modalities, as it did not decrease expression of tumor suppressor genes involved in HIF pathway. Down-regulation of HIF pathway mediated oncogenes by LRS suggests that the cytotoxic effects of this Lactobacillus may at least be partly caused by this mechanism. As previous studies have shown that inhibition of HIF-1alpha and HSP90 expressions have therapeutic impact on cancer treatment, the inhibitory effect of LRS on expression of these genes implies that this Lactobacillus can be used in treatment strategies

Homozygosity for a robertsonian translocation [13q;14q] in a phenotypically normal 44, XX female with a history of recurrent abortion and a normal pregnancy outcome

Background: Robertsonian translocations are structural chromosomal abnormalitiescaused by fusion of two acrocentric chromosomes. In carriers of such translocations,different modes of segregations would result in the formation of either balanced [alternatesegregation mode] or unbalanced [adjacent 1, adjacent 2, and 3:1 segregationmodes] gametes. In addition, there is an increased risk for imprinting disorders intheir offspring. Although it has been estimated that 1/1000 healthy persons carry aRobertsonian translocation, homozygosity for this type of structural chromosomalabnormality has been reported rarely. Most of reported cases are phenotypicallynormal but experience adverse pregnancy outcomes

Case Presentation: In this paper, a report was made on a normal female with a historyof 4 consecutive first trimester fetal losses and a normal son referred to Centerfor Comprehensive Genetics Services, Tehran, Iran, in summer 2015. Cytogeneticanalyses of proband and her infant showed 44,XX, der[13;14][q10;q10]x2 and 45,XY, der[13;14][q10;q10], respectively. Parents of proband have been shown to have45,XY,der[13q;14q] and 45,XX,der[13q;14q] karyotypes, respectively

Conclusion: The present report was in agreement with the few reports of homozygosityfor Robertsonian translocation which demonstrated normal phenotypes forsuch persons and possibility of giving birth to phenotypically normal heterozygotecarriers of Robertsonian translocations