Characterization of Effective Native Lactic Acid Bacteria as Potential Oral Probiotics on Growth Inhibition of Streptococcus mutans

Background and Objective: Probiotics' effects on harmful oral bacteria have been verifed. As antibiotic resistance becomes a major problem, searching for novel potential species is important. The objective of this study was to select novel safe strains of lactic acid bacteria with potentials as oral probiotics. Furthermore, ability of these strains to suppress growth and attachment of Streptococcus mutans as the most important cariogenic bacteria in tooth decay was investigated. Material and Methods: Initial identification tests, including Gram staining and catalase and oxidase tests, were carried out on 22 strains of lactic acid bacteria isolated from Iranian traditional dairy products. Safety of the strains was assessed using hemolysis test and antibiotic resistance assessment. Strains were then assessed for probiotic characteristics such inhibition of Streptococcus mutans growth, tolerance to lysozyme enzymes and ability of adhesion as well as ability of decreasing Streptococcus mutans adhesion. Selected strains were identified using16S rRNA molecular method. Results and Conclusion: Of all strains, four strains with the optimal probiotic characteristics were selected. These included one Lactobacillus brevis, one Lactobacillus casei and two Lactobacillus paraceasei. These four strains showed strong antimicrobial characteristics against Streptococcus mutans, were resistant to oral lysozyme enzymes and included high adhesion abilities to polystyrene wells. Furthermore, they decreased Streptococcus mutans attachment; thus, biofilm formation by this bacterium was prevented. These strains were recognized as safe strains since they were approved in assessments of antibiotic susceptibility and hemolytic activity. Therefore, these four strains are suggested as oral probiotics. Conflict of interest: The authors declare no conflict of interest

The detergent Triton X-100 induces a death pattern in human carcinoma cell lines that resembles cytotoxic lymphocyte-induced apoptosis

AbstractThe detergent Triton X-100 (TX100) was used with the intention to establish a model for necrotic cell death. However TX100 was found to induce apoptotic and necrotic death in prostate and colon cancer cell lines. Apoptosis was characterized by the typical morphological features and internucleosomal DNA fragmentation. The rapid onset within 60 min and the lack of inhibition by cycloheximide indicated that apoptosis induced by TX100 was not dependent on protein synthesis. Removal of extracellular calcium blocked internucleosomal DNA fragmentation. This pattern of cell death shows a striking similarity to the effect of cytotoxic lymphocytes on their target cells

Ex vivo assessment of chemotherapy-induced apoptosis and associated molecular changes in patient tumor samples

BACKGROUND: There are inherent conceptual problems in investigating the pharmacodynamics of cancer drugs in vivo. One of the few possible approaches is serial biopsies in patients. However, this type of research is severely limited by methodological and ethical constraints. MATERIALS AND METHODS: A modified 3-dimensional tissue culture technique was used to culture human tumor samples, which had been collected during routine cancer operations. Twenty tumor samples of patients with non-small cell lung cancer (NSCLC) were cultured ex vivo for 120 h and treated with mitomycin C, taxotere and cisplatin. The cytotoxic activity of the anticancer agents was quantified by assessing the metabolic activity of treated tumor cultures and various assays of apoptosis and gene expression were performed. RESULTS: The proliferative activity of the tissue was maintained in culture as assessed by Ki-67 staining. Mitomycin C, cisplatin and taxotere reduced the metabolic activity of the tumor tissue cultures by 51%, 29% and 20%, respectively, at 120 h. The decrease in metabolic activity corresponded to the induction of apoptosis as demonstrated by the typical morphological changes, such as chromatin condensation and nuclear fragmentation. In addition, activated caspase-3 could be verified in apoptotic cells by immunohistochemistry. To verify functional aspects of apoptosis, the induction of chemotherapy-induced cell death was inhibited with the caspase inhibitor z-VAD.fmk. RNA was extracted from the tissue cultures after 120 h of ex vivo drug treatment and was of sufficient quality to allow quantitative PCR. CONCLUSION: The 3-dimensional ex vivo culture technique is a useful method to assess the molecular effects of pharmacological interventions in human cancer samples in vitro. This culture technique could become an important tool for drug development and for the prediction of in vivo drug efficacy

Novel functional profiling approach combining reverse phase protein microarrays and human 3-D ex vivo tissue cultures: expression of apoptosis-related proteins in human colon cancer

Cancer is caused by a complex pattern of molecular perturbations. To understand the biology of cancer, it is thus important to look at the activation state of key proteins and signaling networks. The limited amount of available sample material from patients and the complexity of protein expression patterns make the use of traditional protein analysis methods particularly difficult. In addition, the only approach that is currently available for performing functional studies is the use of serial biopsies, which is limited by ethical constraints and patient acceptance. The goal of this work was to establish a 3-D ex vivo culture technique in combination with reverse-phase protein microarrays (RPPM) as a novel experimental tool for use in cancer research. The RPPM platform allows the parallel profiling of large numbers of protein analytes to determine their relative abundance and activation level. Cancer tissue and the respective corresponding normal tissue controls from patients with colorectal cancer were cultured ex vivo. At various time points, the cultured samples were processed into lysates and analyzed on RPPM to assess the expression of carcinoembryonic antigen (CEA) and 24 proteins involved in the regulation of apoptosis. The methodology displayed good robustness and low system noise. As a proof of concept, CEA expression was significantly higher in tumor compared with normal tissue (p<0.0001). The caspase 9 expression signal was lower in tumor tissue than in normal tissue (p<0.001). Cleaved Caspase 8 (p=0.014), Bad (p=0.007), Bim (p=0.007), p73 (p=0.005), PARP (p<0.001), and cleaved PARP (p=0.007) were differentially expressed in normal liver and normal colon tissue. We demonstrate here the feasibility of using RPPM technology with 3-D ex vivo cultured samples. This approach is useful for investigating complex patterns of protein expression and modification over time. It should allow functional proteomics in patient samples with various applications such as pharmacodynamic analyses in drug development