Glutathione-S-Transferase Theta 2 (GSTT2) Modulates the Response to Bacillus Calmette-Guérin Immunotherapy in Bladder Cancer Patients.

Glutathione-S-transferases (GST) enzymes detoxify xenobiotics and are implicated in response to anticancer therapy. This study evaluated the association of GST theta 1 (GSTT1), GSTT2, and GSTT2B with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) response in non-muscle-invasive bladder cancer treatment. In vitro assessments of GSTT2 knockout (KO) effects were performed using cell lines and dendritic cells (DCs) from GSTT2KO mice. Deletion of GSTT2B, GSTT1, and single-nucleotide polymorphisms in the promoter region of GSTT2 was analysed in patients (n = 205) and healthy controls (n = 150). Silencing GSTT2 expression in MGH cells (GSTT2BFL/FL) resulted in increased BCG survival (p < 0.05) and decreased cellular reactive oxygen species. In our population, there are 24.2% with GSTT2BDel/Del and 24.5% with GSTT2BFL/FL. With ≤ 8 instillations of BCG therapy (n = 51), 12.5% of GSTT2BDel/Del and 53.8% of GSTT2BFL/FL patients had a recurrence (p = 0.041). With ≥9 instillations (n = 153), the disease recurred in 45.5% of GSTT2BDel/Del and 50% of GSTT2BFL/FL. GSTT2FL/FL patients had an increased likelihood of recurrence post-BCG therapy (HR 5.5 [1.87-16.69] p < 0.002). DCs from GSTT2KO mice produced three-fold more IL6 than wild-type DCs, indicating a robust inflammatory response. To summarise, GSTT2BDel/Del patients respond better to less BCG therapy and could be candidates for a reduced surveillance regimen.

Tumor-targeting albumin nanoparticles as an efficacious drug delivery system and potential diagnostic tool in non-muscle-invasive bladder cancer therapy.

Current intravesical chemotherapy for non-muscle invasive bladder cancer (NMIBC) has limited efficacy due to loss of the instilled agent from urine voiding and the agent's lack of specificity for the tumors. We developed a nanocarrier (txCD47-HNP, ∼100 nm) based on human serum albumin conjugated with a peptide that targets the cluster of differentiation 47 receptor overexpressed on bladder cancer (BC) cells. The IC50 of gemcitabine elaidate (GEM) loaded in the txCD47-HNP was almost an order of magnitude lower than that of free GEM. In a mouse orthotopic BC model, GEM loaded in txCD47-HNP effectively reduced the tumor burden. Tumor cells in BC patients' urine can also be targeted by fluorescence-labeled txCD47-HNP resulting in >83 % of the cells exhibiting fluorescence. Thus, txCD47-HNP can potentially be a theranostic agent in NMIBC management by serving as a targeted drug delivery vehicle as well as an alternative to urine cytology.

Predictive biomarkers of response to bacillus Calmette-Guérin immunotherapy and bacillus Calmette-Guérin failure for non-muscle invasive bladder cancer.

Within the heterogeneous population of patients with bacillus Calmette-Guérin failure, there are clear differences in prognosis and therapy with regard to the timeline when bacillus Calmette-Guérin failure occurred. There are a variety of classifications which include bacillus Calmette-Guérin refractory disease, relapsing, unresponsive, and intolerant. Further profiling of these patients may help to shed light on other forms of therapy that are less radical. We hereby summarize the different biomarkers that predicts for response to bacillus Calmette-Guérin immunotherapy and bacillus Calmette-Guérin failure for non-muscle invasive bladder cancer.

Polymorphism in autophagy gene ATG2B is not associated with bladder cancer recurrence after intravesical Bacillus Calmette-Guerin (BCG) immunotherapy in Asian patients.

OBJECTIVES: Optimal patient stratification is critical in the era of personalized medicine. Germline polymorphisms play an important role in the treatment response of various human diseases, including bladder cancer. Intravesical BCG therapy is widely-used for bladder cancer. However, tumor recurrence and progression are very common. Stratification based on germline polymorphisms may contribute to circumvent this clinical challenge. Autophagy pathway plays an important role in the nonspecific protective effects of BCG. Patients that carry C allele of rs3759601 in autophagy gene ATG2B showed increased risk of recurrence and progression in European population. We thus sought to analyze rs3759601 and its relevance in BCG response in Asian NMIBC patients. METHOD: Functional impact of rs3759601 ATG2B (p.Gln1383Glu) was analyzed by bioinformatics programs including NCBI Conserved Domain Search, Clustal Omega, Polyphen and SIFT. NMIBC patients who received intravesical BCG at multiple hospitals in Singapore from 1995 to 2016 were included. These patients were genotyped for rs3759601 using high resolution melt analysis. The rs3759601 polymorphism was studied in correlation with the bladder cancer recurrence rate and disease progression rate in our cohort. Statistical analysis was conducted using Kaplan-Meier plots and the Chi-squared analysis. RESULTS: In total, 307 individules were included in the study including 161 NMIBC patients and 146 healthy controls, predominately Chinese. The rs3759601 genotype distributions in our NMIBC patients were (GG 72.1%; GC 27.9%; CC 0%), which were distinct from the Dutch report (GG 32.8%; GC 47.4% and CC 19.8%, Buffen K et al, 2014). Consistently, the C allele frequencies of rs3759601 are 0.171 in our controls and 0.177 in East Asians from 1,000 Genome, but 0.406 in Europeans from 1,000 Genome. In silico analysis suggested rs3759601 ATG2B (p.Gln1383Glu) alteration is unlikely to be functionally deleterious. Statistical analysis revealed no significant association between ATG2B rs3759601 C allele and risk of bladder cancer recurrence (P= 0.353, GC vs. GG: hazard ratio [HR]= 1.324), or cancer progression (P= 0.454, GC vs. GG: HR = 0.658). CONCLUSION: In contrast to European NMIBC patients, ATG2B rs3759601 C allele is much less common in Asians and it not associated with BCG response in Asian NMIBC patients.

Expression of major HDL-associated antioxidant PON-1 is gender dependent and regulated during inflammation.

Paraoxonase 1, an HDL-associated enzyme that confers antioxidant activity on HDL, and its activity in serum have been correlated with protection against atherosclerosis, an oxidative disease. However, serum PON-1 activity is highly variable and its regulation is complex, involving both genetic and environmental factors. It is influenced by gender and inflammation, two important factors in atherosclerosis. Serum PON-1 activity has been shown to be lower in male mice and is decreased in male Syrian hamster during inflammation. Here we show that male mice had lower hepatic PON-1 mRNA that increased by 170% after castration. Our data also suggested that this effect was testes but not plasma testosterone dependent. Ovariectomy had no effect on PON-1 mRNA in female mice. LPS caused hepatic PON-1 mRNA to decrease further in male mice, and to increase moderately in female mice. Anti-inflammatory dexamethasone enhanced PON-1 mRNA level by 2-fold in male and female LPS-treated mice, and increased PON-1 expression by 8-fold in Hepa cell, a mouse hepatoma cell line. Therefore, antioxidant PON-1 is regulated at the mRNA level in a gender-specific manner by proinflammatory LPS and anti-inflammatory dexamethasone.

Gonadal effects on plasma ACE activity in mice.

Angiotensin-converting enzyme (ACE) regulates blood pressure and is an important target in the management of hypertension. Hypertension is a gender biased disease. Plasma ACE activity is significantly higher in male mice (309 U/l) than female mice (237 U/l) and is reduced significantly upon gonadectomy to 224 and 209 U/l, respectively. Although, the gonads influence plasma ACE activity in both male and female mice, the effect is more pronounced in male mice. Plasma ACE is derived from the cleavage of tissue ACE and lung has the highest concentration of tissue ACE. However, lung ACE activity is not gender dimorphic but increases significantly upon gonadectomy in both male and female. ACE mRNA level in the lung is not influenced by gender or gondaectomy. Therefore, the gonads affect plasma ACE activity by influencing cleavage of tissue ACE to plasma ACE and/or decrease stability of plasma ACE in gonadectomized mice is mediated.

NRAMP1 and hGPX1 gene polymorphism and response to bacillus Calmette-Guérin therapy for bladder cancer.

BACKGROUND: The natural resistance-associated macrophage protein 1 (NRAMP1) gene is associated with susceptibility to Mycobacterium tuberculosis in humans and to bacillus Calmette-Guérin (BCG) in mice. The detoxification enzyme, human glutathione peroxidase 1 (hGPX1), is associated with recurrence of bladder cancer (BCa). OBJECTIVE: To determine whether NRAMP1 and hGPX1 gene polymorphisms correlate with response to BCG immunotherapy for non-muscle-invasive BCa (NMIBC). DESIGN, SETTING, AND PARTICIPANTS: DNA was obtained from the peripheral blood of 99 NMIBC patients who were prospectively randomized to receive postresection intravesical BCG (81 mg [n=50] or 27 mg [n=19]) or BCG (27 mg) with interferon alpha (IFN-α; n=30). The median follow-up time was 60 mo. INTERVENTION: Intravesical BCG or BCG-IFN-α. MEASUREMENTS: Restriction fragment length polymorphism (RFLP) analysis was performed to identify polymorphisms in the NRAMP1 promoter region (GT repeat number) and at position 543 (aspartate [D] and/or asparagine [N] expression) within the NRAMP1 protein (D543N) and position 198 (proline and/or leucine expression) within the hGPX1 protein (Pro198Leu). Data were analyzed using χ(2) analysis, multivariate analysis, and Kaplan-Meier curves. RESULTS AND LIMITATIONS: On univariate analysis, the NRAMP1 D543N G:G genotype had decreased cancer-specific survival (CSS; p=0.036). The hGPX1 CT genotype (Pro-Leu) had decreased recurrence time (p=0.03) after BCG therapy. On multivariate analysis, patients with the NRAMP1 D543N G:G genotype and allele 3 (GT)n polymorphism had decreased recurrence time (p=0.014 and p=0.03) after BCG therapy. The limitation of this study was its small sample size. CONCLUSIONS: Polymorphisms of the NRAMP1 and hGPX1 genes may be associated with recurrence of BCa after BCG immunotherapy.

AFP(+), ESC-derived cells engraft and differentiate into hepatocytes in vivo.

A major problem in gene therapy and tissue replacement is accessibility of tissue-specific stem cells. One solution is to isolate tissue-specific stem cells from differentiating embryonic stem (ES) cells. Here, we show that liver progenitor cells can be purified from differentiated ES cells using alpha-fetoprotein (AFP) as a marker. By knocking the green fluorescent protein (GFP) gene into the AFP locus of ES cells and differentiating the modified ES cells in vitro, a subpopulation of GFP(+) and AFP-expressing cells was generated. When transplanted into partially hepatectomized lacZ-positive ROSA26 mice, GFP(+) cells engrafted and differentiated into lacZ-negative and albumin-positive hepatocytes. Differentiation into hepatocytes also occurred after transplantation of GFP(+) cells in apolipoprotein-E- (ApoE) or haptoglobin-deficient mice as demonstrated by the presence of ApoE-positive hepatocytes and ApoE mRNA in the liver of ApoE-deficient mice or by haptoglobin in the serum and haptoglobin mRNA in the liver of haptoglobin-deficient mice. This study describes the first isolation of ES-cell-derived liver progenitor cells that are viable mediators of liver-specific functions in vivo.