Juwita N. Rahmat

Lactobacillus Species is More Cytotoxic to Human Bladder Cancer Cells Than Mycobacterium Bovis (Bacillus Calmette-Guerin)

PURPOSE We determined if Lactobacillus species has growth inhibitory effects in human bladder cancer cell lines and how this effect compares with the known effects of Mycobacterium bovis, that is bacillus Calmette-Guerin (BCG). MATERIALS AND METHODS The growth of MGH and RT112 cells were determined by cell counts after 24, 48 and 72 hours of exposure to L. casei strain Shirota (Yakult, Singapore) or L. rhamnosus strain GG (National Collection of Industrial and Marine Bacteria, Ltd., Aberdeen, Scotland) (1 x 10 and 1 x 10 cfu) or BCG (1 x 10 cfu) in the presence and absence of streptomycin. Annexin-V was used to monitor the presence of pre-apoptotic cells. RESULTS L. rhamnosus GG inhibited MGH proliferation and it was cytotoxic to RT112 cells (p <0.05). L. casei Shirota was cytotoxic to the 2 cell lines (p <0.05). BCG had a similar cytotoxic effect in MGH cells as Lactobacillus species but was not as effective in RT112 cells. Streptomycin abrogated the cytotoxic effect of Lactobacillus species but not that of BCG. Cytotoxic activity was not found in Lactobacilli culture supernates but it was induced in the presence of mammalian cells. L. rhamnosus GG induced apoptosis in RT112 but not in MGH cells. No apoptotic cells were detected after treatment with L. casei Shirota. CONCLUSIONS Lactobacillus species induced cytotoxic effects in bladder cancer cells. Unlike BCG, it requires bacterial protein synthesis. Like BCG, L. casei Shirota induces cell death primarily via necrosis. The cytoxicity of these lactobacilli in bladder cancer cells raises the possibility of using this species of bacteria as intravesical agents for treating bladder cancer.

Lactobacillus rhamnosus GG induces tumor regression in mice bearing orthotopic bladder tumors

(Cancer Sci 2010; 101: 751–758)

Expression of chemokine/cytokine genes and immune cell recruitment following the instillation of Mycobacterium bovis, bacillus Calmette–Guérin or Lactobacillus rhamnosus strain GG in the healthy murine bladder

Mycobacterium bovis, bacillus Calmette–Guérin (BCG) is the current gold standard for bladder cancer therapy. In this study a profile of the gene expression changes that occur after BCG instillation in the bladders of healthy mice was produced and compared to the type of immune cells recruited into the bladder. A similar comparison was made for Lactobacillus rhamnosus strain GG (LGG) instillations in healthy mice to determine its potential in the immunotherapy of bladder cancer. Mice were given six weekly instillations and were killed after the fourth, fifth and sixth instillations of BCG or LGG. Their bladders were harvested for chemokine/cytokine messenger RNA analysis using an array as well as semi‐quantitative reverse transcription–polymerase chain reaction. In a second set of mice both the bladder and draining lymph nodes were harvested for the analysis of immune cells. BCG significantly upregulated genes for T helper type 1 (Th1) chemokines: Cxcl2, Cxcl9, Cxcl10, Xcl1; and increased the expression of Th1/Th2 chemokines: RANTES, Ccl6 and Ccl7; Th1 polarizing cytokines: Il1β and Tnfa; and Fcγr1 and iNOS as early as after four weekly instillations. Most of these genes remained highly expressed after 6 weeks. In contrast, LGG transiently induced Cxcl10, Il16, Fcεr1 and Il1r2. Despite these findings, LGG instillation induced the recruitment of natural killer cells into the bladder and draining lymph nodes, as was observed for BCG instillation.

Bacillus Calmette-Guérin induces cellular reactive oxygen species and lipid peroxidation in cancer cells.

OBJECTIVE To determine whether Bacillus Calmette-Guérin (BCG) and/or BCG-soluble factors could modulate cellular reactive oxygen species (ROS) in human bladder cancer cells and the impact this could have on response to therapy. METHODS The expression of α5β1 integrins on human bladder cancer cell lines and their ability to internalize BCG were determined. The effect of live and lyophilized BCG on cellular ROS, lipid peroxidation, and DNA damage was determined using H(2)DCF-DA, TBARS, and comet assays. The cytotoxic effects of live and lyophilized BCG on cancer cells were determined after 24 hours. ROS modulation by Antigen 85B and mycobacterial protein tyrosine phosphatases was monitored. RESULTS Live and lyophilized BCG were internalized to a similar extent, but live BCG increased cellular ROS, whereas lyophilized BCG reduced ROS. High ROS levels correlated with increased lipid peroxidation. The cytotoxic effect of BCG was independent of cellular ROS but dependent on internalization. Lyophilized BCG was more cytotoxic to bladder cancer cells than live BCG. BCG soluble factors such as Antigen85B could increase cellular ROS. Internalization of lyophilized BCG abrogated the ROS, and lipid peroxidation increase induced by BCG soluble factors. Both live and lyophilized BCG induced DNA damage but to different extents. CONCLUSION The end products of ROS, such as lipid peroxides and superoxide, could induce DNA damage, which could lead to mutations in cancer cells that select for their survival. Reducing BCG instillations may reduce the risk of mutational changes occurring in remnant cancer cells.

Lactobacillus rhamnosus GG Activation of Dendritic Cells and Neutrophils Depends on the Dose and Time of Exposure

This study evaluates the ability of Lactobacillus rhamnosus GG (LGG) to activate DC and neutrophils and modulate T cell activation and the impact of bacterial dose on these responses. Murine bone marrow derived DC or neutrophils were stimulated with LGG at ratios of 5 : 1, 10 : 1, and 100 : 1 (LGG : cells) and DC maturation (CD40, CD80, CD86, CD83, and MHC class II) and cytokine production (IL-10, TNF-α, and IL-12p70) were examined after 2 h and 18 h coculture and compared to the ability of BCG (the present immunotherapeutic agent for bladder cancer) to stimulate these cells. A 2 h exposure to 100 : 1 (high dose) or an 18 h exposure to 5 : 1 or 10 : 1 (low dose), LGG : cells, induced the highest production of IL-12 and upregulation of CD40, CD80, CD86, and MHC II on DC. In DCs stimulated with LGG activated neutrophils IL-12 production decreased with increasing dose. LGG induced 10-fold greater IL-12 production than BCG. T cell IFNγ and IL-2 production was significantly greater when stimulated with DC activated with low dose LGG. In conclusion, DC or DC activated with neutrophils exposed to low dose LGG induced greater Th1 polarization in T cells and this could potentially exert stronger antitumor effects. Thus the dose of LGG used for immunotherapy could determine treatment efficacy.

Extraction and quantification of biofilm bacteria: Method optimized for urinary catheters

Bacterial biofilms are responsible for the failure of many medical devices such as urinary catheters and are associated with many infectious and non-infectious complications. Preclinical and clinical evaluation of novel catheter coatings to prevent these infections needs to accurately quantify the bacterial load in the biofilm in vitro and ex vivo. There is currently no uniform gold standard for biofilm quantification for different surfaces and established biofilms. We have tried to establish a simple, accurate and reproducible method for extraction and measurement of biofilm bacteria on indwelling catheters, using a combination of vortexing and sonication. We demonstrate the usefulness of this method for catheters of different sizes – 3 Fr to 14 Fr – in vitro, in murine and porcine models, and indwelling in human clinical subjects. We also demonstrate consistent results with complex and polymicrobial biofilms. We believe that this standardized reproducible method will assist the assessment of biofilms in general and urological devices in particular in efforts to harness novel technologies to prevent healthcare associated infections.

Bacillus Calmette‑Guérin induces rapid gene expression changes in human bladder cancer cell lines that may modulate its survival

Bacillus Calmette-Guérin (BCG) immunotherapy is the standard therapy for non-muscle invasive bladder cancer. The aim of the present study was to identify genes that are induced in response to BCG immunotherapy, as these may be potential biomarkers for the response to clinical therapy. To model clinical therapy, human bladder cancer cell lines were incubated with BCG (live or lyophilized BCG Connaught) for 2 h. RNA was extracted and evaluated by Representational Differential Analysis (RDA) and oligo arrays. Gene expression was confirmed by reverse transcription polymerase chain reaction on fresh cell lines with differential abilities to internalize BCG. The effect of 2 major BCG soluble proteins, antigen 85B (Ag85B) and Mycobacterium protein tyrosine phosphatase A (MptpA) and BCG Tice® on gene expression was also determined. GAPDH and β-actin, which are normally used as control genes, were upregulated by BCG. Therefore, the ribosomal RNA gene ribosomal protein S27a was used to normalize gene expression. The genes likely to be induced by BCG internalization and soluble factors were: GSTT2, MGST2, CCL20, TNFα, CCNE1 and IL10RB. Those induced by BCG membrane interactions and/or soluble factors were: MGST1, CXCL6, IL12A, CSF2, IL1β and TOLLIP. MptpA decreased GSTT2 expression, and Ag85B increased TNFα expression. The two BCG strains significantly increased GSTT2, TNFα and TOLLIP levels in MGH cells. However, in J82 cells there was a BCG strain-dependent difference in TNFα expression. An important outcome of the present study was the determination that neither GAPDH nor β-actin were suitable control genes for the analysis of BCG-induced gene expression. BCG Connaught and Tice® induced similar expression levels of genes in bladder cancer cell lines. BCG soluble proteins modulated gene expression and therefore may affect therapeutic outcomes. The genes identified may be novel biomarkers of the response to BCG therapy.

MP65-05 IFNα MODULATES THE RESPONSE TO BCG IMMUNOTHERAPY IN BLADDER CANCER PATIENTS WITH SPECIFIC CTLA4 SINGLE NUCLEOTIDE POLYMORPHISMS

respect to the GSTT2B genotypes. The impact of BCG instillation (numbers) on recurrence was analyzed in a subset of patients for whom complete 10y follow-up data was available. Analysis was performed using SPSS 23.0 and p<0.05 was taken to be significant. RESULTS: GSTT2 was silenced in MGH cells (GSTT2B homozygous full length (GSTT2B)) and overexpressed in UMUC3 and U937 cells (GSTT2B homozygous deleted (GSTT2B)). A 2h exposure to BCG resulted in decreased ROS in GSTT2 silenced cells (p<0.05) and increased ROS in GSTT2 overexpressing cells (p<0.05). There was no difference in cellular cytotoxicity to BCG with respect to GSTT2 expression. However, intracellular BCG survival increased at 2 hours when GSTT2 was silenced (p<0.05) and decreased when GSTT2 was overexpressed (p<0.05). There was no significant difference between these groups at 24h. The majority of patients with complete 10y follow-up data, completed a 6+3 BCG schedule (n1⁄463) and n1⁄422 had less than 8 instillations. Patients with GSTT2B genotype (n1⁄46) who received 8 or less BCG instillations, were recurrence free (Likelihood ratio 1⁄4 0.040, p1⁄40.054). In the group that received at least 9 instillations of BCG, the GSTT2B was associated with earlier recurrence. CONCLUSIONS: GSTT2 expression decreases cellular ROS and BCG survival. GSTT2B was associated with lower likelihood of recurrence for patients who received 8 or less BCG instillations. In contrast patients with GSTT2B who received 9 or more instillations of BCG had earlier recurrences. Hence GSTT2B could be used as a marker for patients who will do well with less BCG therapy.

MP65-04 GSTT2 MODULATES PATIENT RESPONSE TO BCG IMMUNOTHERAPY

of BC patients. In addition, BCG induced expression of key immunomodulatory molecules in BC cells and within the EVs. This up-regulated expression of immuno-molecules in response to BCG supports the hypothesis that EVs have a role in activating the immune system during BCG immunotherapy. The immunologically active EVs detected in patients’ urine can be further explored as predictive biomarkers.