In Vitro Low-Bortezomib Doses Induce Apoptosis and Independently Decrease the Activities of Glutathione S-Transferase and Glutathione Peroxidase in Multiple Myeloma, Taking into Account the GSTT1 and GSTM1 Gene Variants.

BACKGROUND: Multiple myeloma (MM) is a malignancy derived from plasma cells. Bortezomib affects the concentration of reduced glutathione (GSH) and the activity of glutathione enzymes. The aim of our study was to analyze deletion (null/present) variants of GSTT1 and GSTM1 genes and their association with the levels of glutathione and its enzymes in bortezomib-treated cell cultures derived from MM patients. MATERIALS AND METHODS: This study included 180 individuals (80 MM patients and 100 healthy blood donors) who were genotyped via multiplex PCR (for the GSTT1/GSTM1 genes). Under in vitro conditions, MM bone marrow cells were treated with bortezomib (1-4 nM) to determine apoptosis (via fluorescence microscopy), GSH concentration, and activity of glutathione enzymes (via ELISA). RESULTS: Bortezomib increased the number of apoptotic cells and decreased the activity of S-glutathione transferase (GST) and glutathione peroxidase (GPx). We found significant differences in GST activity between 1 nM (GSTT1-null vs. GSTT1-present), 2 nM (GSTT1-null vs. GSTT1-present), and 4 nM (GSTM1-null vs. GSTM1-present) bortezomib: 0.07 vs. 0.12, p = 0.02; 0.06 vs. 0.10, p = 0.02; and 0.03 vs. 0.08, p = 0.01, respectively. CONCLUSIONS: Bortezomib affects the activities of GST and GPx. GST activity was associated with GSTT1 and GSTM1 variants but only at some bortezomib doses.

Association of Chromosome 17 Aneuploidy, TP53 Deletion, Expression and Its rs1042522 Variant with Multiple Myeloma Risk and Response to Thalidomide/Bortezomib Treatment.

Multiple myeloma (MM) is a multifactorial genetic disorder caused by interactive effects of environmental and genetic factors. The proper locus of the TP53 gene (17p13.1) and its protein is essential in genomic stability. The most common variant of the TP53 gene-p.P72R (rs1042522)-shows functional variation. The aim of our study was a complex analysis of the TP53 p.P72R variant and TP53 gene expression in relation to chromosomal changes of the TP53 gene locus, as well as MM risk and outcome. Genomic DNA from 129 newly diagnosed MM patients was analyzed by methods of automated DNA sequencing (for TP53 variant analysis) and cIg-FISH (for chromosomal aberrations analysis). RNA was used in real-time PCR to determine the TP53 expression. In MM patients, the TP53 variant was not in Hardy-Weinberg equilibrium. The RR genotype was associated with lower MM risk (OR = 0.44, p = 0.004). A higher number of plasma cells was found in patients with RR genotype in comparison to those with PP + PR genotypes (36.74% vs. 28.30%, p = 0.02). A higher expression of the TP53 gene was observed in PP + PR genotypes vs. RR homozygote (p < 0.001), in smokers vs. non-smokers (p = 0.02). A positive Pearson's correlation was found between the TP53 expression level and the number of plasma cells (r = 0.26, p = 0.04). The presence of chromosome 17 aberrations with or without TP53 locus did not affect the MM risk and outcome. Similar results were observed in the case of TP53 gene expression and the p.P72R variant.

The Relationship of CCL5 and CCR1 Variants with Response Rate and Survival Taking into Account Thalidomide/Bortezomib Treatment in Patients with Multiple Myeloma.

(1) Background: Chemokines and chemokine receptors play an important role in tumor development. The aim of this study was to check the significance of CCL5 and CCR1 variants with response rate, survival, and the level of regulated on activation, normal T cells expressed and secreted (RANTES/CCL5) in multiple myeloma (MM) patients; (2) Methods: Genomic DNA from 101 newly diagnosed MM patients and 100 healthy blood donors were analyzed by Real-time PCR method (for CCL5 and CCR1 genotyping). In a subgroup of 70 MM patients, serum samples were collected to determine the level of RANTES; (3) Results: multivariate Cox regression showed increased risk of disease relapse or progression (HR = 4.77; p = 0.01) in MM patients with CG + CC genotypes of CCL5 rs2280788. In contrast, CT + TT genotypes of CCL5 rs2107538 were associated withdecreased risk of death (HR = 0.18; p = 0.028) and disease relapse or progression (HR = 0.26; p = 0.01). In MM patients with major genotypes of rs2280789, rs2280788, and rs2107538, higher survival rates were observed in response to treatment with thalidomide and bortezomib. Statistically significant lower RANTES levels were seen in minor genotypes and heterozygotes of CCL5 and CCR1 variants; (4) Conclusions: Major genotypes of CCL5 variants may be independent positive prognostic factors in MM.

Influence of Algae Supplementation on the Concentration of Glutathione and the Activity of Glutathione Enzymes in the Mice Liver and Kidney.

Algae are potential and natural source of long-chain polyunsaturated fatty acids like eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The diatom Pinnularia borealis accumulates high levels of EPA and may be considered as a source for commercial production of dietary supplements. In this study we asked the question whether diet supplementation with P. borealis may augment antioxidant defense and ameliorate risk factors for cardiovascular diseases. We fed mice (Mus musculus) with lyophilized diatom solutions of different concentrations (1%, 3%, and 5%) for 7 days. Then we measured glutathione content and the activity of glutathione redox system enzymes, total cholesterol and triacylglycerol concentrations, and malondialdehyde concentration in the liver and kidney. We found that cholesterol and triacylglycerol concentrations in the liver and kidneys were the lowest in mice who were fed with the highest concentration of Pinnularia borealis, suggesting protective properties of algae. Additionally, the lowest concentration of Pinnularia borealis was sufficient to improve antioxidant capacity. Our results suggest that P. borealis may be used as a source for dietary supplements rich in EPA, but the amount supplied to the organism should be limited.

ACE Insertion/Deletion Polymorphism (rs4646994) Is Associated With the Increased Risk of Multiple Myeloma.

Introduction: The insertion (I allele) deletion (D allele) polymorphism of ACE gene (rs4646994) may influence the etiopathogenesis of multiple myeloma (MM). ACE gene is expressed in bone marrow cells and encodes angiotensin converting enzyme (ACE). It converts angiotensin I to active peptide angiotensin II, which stimulates proliferation of hematopoietic stem cells. This suggests possible association of ACE I/D gene polymorphism with MM. The aim of our study was to check possible impact of this polymorphism on risk of development and outcome of MM, as well as, sensitivity to bortezomib in cell cultures derived from MM patients. Objects and Methods: Genomic DNA from 98 newly diagnosed MM patients and 100 healthy blood donors were analyzed by PCR method. Chromosomal aberrations were detected by use of cIg-FISH. In a subgroup of 40 MM patients nucleated bone marrow cells were treated with bortezomib in vitro. Results: The Hardy-Weinberg equilibrium test showed that genotypic frequencies diverged significantly from the equilibrium. The differences between I and D allele frequencies in control and study population were significant (p = 0.046). We observed the association between DD genotype and more than 2-fold risk of MM - OR = 2.69; p < 0.0001. We did not detect any significant differences among studied genotypes regarding clinical and laboratory parameters. Moreover, we did not observe the association between survival of MM patients and I/D genotypes. Bortezomib increased number of apoptotic and necrotic cells, but the only statistically significant differences were observed in the number of viable cells at 1 nM between ID and DD genotypes (p = 0.026). Conclusion: Presented results confirmed the significant relationship between ACE (I/D) polymorphism and risk of MM development. We did not observe the association of ACE I/D polymorphism with disease outcome and bortezomib in vitro sensitivity.

TP53 polymorphism in plasma cell myeloma.

INTRODUCTION: Significant and accessible predictive factors for bortezomib treatment in plasma cell myeloma (PCM) are still lacking. TP53 codon 72 polymorphism (P72R) results in proline (P) or arginine (R) at 72 amino acid position, which causes synthesis of proteins with distinct functions. The aims of our study were to: 1) analyze whether this polymorphism is associated with an increased risk of PCM; 2) study whether the P72R polymorphism affects overall survival (OS) among PCM patients; 3) assess the possible association of the P72R polymorphism with sensitivity to bortezomib in cell cultures derived from PCM patients. MATERIAL AND METHODS: Genomic DNA from newly diagnosed 59 patients (without IgVH gene rearrangements and TP53 deletions) and 50 healthy blood donors were analyzed by RFLP-PCR to identify TP53 polymorphism. Chromosomal aberrations were detected by use of cIg-FISH. The lymphocyte cell cultures from a subgroup of 40 PCM patients were treated with bortezomib (1, 2 and 4 nM). RESULTS: The P allele of the P72R polymorphism was more common than the R allele in PMC patients compared to controls (39% vs. 24%), and the difference was significant (p = 0.02). The PP and PR genotypes (in combina-tion) were more frequent among cases than in controls (65% vs. 42%, OR = 2.32, p = 0.04). At the cell culture level and 2 nM bortezomib concentration the PP genotype was associated with higher necrosis rates (10.5%) compared to the PR genotype (5.7%, p = 0.006) or the RR genotype (6.3%, p = 0.02); however, no effect of genotypes was observed at bortezomib concentrations of 1 and 4 nM. The shortest OS (12 months) was observed in patients with the PP genotype compared to patients with the PR or RR genotypes (20 months) (p = 0.04). CONCLUSIONS: The results suggest that P72R polymorphisms may be associated with an increased PCM risk and may affect OS of PCM patients. However, we saw no consistent results of the polymorphism effect on apoptosis and necrosis in cell cultures derived from PCM patients. Further studies are need in this regard.

Significance of Polymorphisms and Expression of Enzyme-Encoding Genes Related to Glutathione in Hematopoietic Cancers and Solid Tumors.

Antioxidant compounds such as glutathione and its enzymes have become the focus of attention of medical sciences. Glutathione, a specific tripeptide, is involved in many intercellular processes. The glutathione concentration is determined by the number of GAG repeats in gamma-glutamylcysteine synthetase. GAG polymorphisms are associated with an increased risk of schizophrenia, berylliosis, diabetes, lung cancer, and nasopharyngeal tumors. Cancer cells with high glutathione concentration are resistant to chemotherapy treatment. The oxidized form of glutathione is formed by glutathione peroxidases (GPXs). The changes in activity of GPX1, GPX2, and GPX3 isoforms may be associated with the development of cancers, for example, prostate cancer or even colon cancer. Detoxification of glutathione conjugates is possible due to activity of glutathione S-transferases (GSTs). Polymorphisms in GSTM1, GSTP1, and GSTO1 enzymes increase the risk of developing breast cancer and hepatocellular carcinoma. Gamma-glutamyl transpeptidases (GGTs) are responsible for glutathione degradation. Increased activity of GGT correlates with adverse prognosis in patients with breast cancer. Studies on genes encoding glutathione enzymes are continued in order to determine the correlation between DNA polymorphisms in cancer patients.

Antioxidants and oxidative stress in Helix pomatia snails during estivation.

Estivation enables land snails to survive a prolonged dryness but the return to active state imposes conditions of oxidative stress on internal organs due to a transient large increase in oxygen consumption, which augments mitochondrial production of reactive oxygen species. Therefore, activities of antioxidant enzymes, concentrations of reduced glutathione (GSH) and TBARS as an index of lipid peroxidation, were evaluated in Helix pomatia snails (i) during summer activity, (ii) during estivation, which was induced experimentally, (iii) at the start of arousal from estivation, and (iv) being aroused for 24 h. Estivation induced significant decreases in activity of catalase in the kidney and hepatopancreas and glutathione peroxidase in the kidney. Activity of glutathione reductase was unaffected by estivation/arousal cycle. Summer-active and estivating snails maintained high activity of glutathione transferase. Concentration of GSH was organ-dependent and was positively affected by estivation. Lack of increase in TBARS concentration during estivation/arousal cycle suggests that antioxidant defence system of H. pomatia snails is highly efficacious. In conclusion, permanent maintenance of relatively high activities of the antioxidant enzymes and the high concentration of GSH in H. pomatia snails indicate that they have well-developed strategy of defence against oxidative injury.

The influence of scopolamine and extreme temperatures on the lipid level in some mouse organs.

OBJECTIVES: The effect of scopolamine administration at dose 0,5 g/kg b.w. and high (+40 degrees C) and low (+4 degrees C) temperature on the level of triacyloglycerides, total lipids and cholesterol in the mouse liver, kidney and muscle of males and females. METHODS: The homogenates of the liver, kidney and muscle were taken for examination. The concentrations of triacyloglycerides, total lipids and cholesterol was estimated according to the Bio-La-Tests (Poland). RESULTS: The concentration of triacyloglycerides and total lipids in the liver, kidney and muscle increased of males and females after scopolamine injections and after exposure to high and low temperatures. CONCLUSION: Scopolamine may effect an increase in the rate of the lipid metabolism.

The effect of melatonin on glutathione and glutathione transferase and glutathione peroxidase activities in the mouse liver and kidney in vivo.

OBJECTIVES: The changes in reduced glutathione (GSH), activity of glutathione transferase (GST) and glutathione peroxidase (GSPx) after administration of exogenous melatonin (N-acetyl-5-methoxy-tryptamine) at a dose 20 mg/kg b.w. were investigated for five and ten days in the liver and kidney of male and female mice. METHODS: The liver and kidney were homogenized in 0.1 M phosphate buffer. Glutathione level and activity of studied glutathione enzymes were determined in the supernatants. RESULTS: Melatonin caused an increase in glutathione level, the activity of glutathione peroxidase as well as glutathione transferase in the examined organs. CONCLUSION: Melatonin may exert some effects on the activity of enzymes engaged in the metabolism of thiol compounds in the cell. Glutathione may be useful for monitoring melatonin's protective role on cell damage.