Józef Drzewoski

Metformin in cancer prevention and therapy

The prevalence of diabetes is dramatically increasing worldwide. The results of numerous epidemiological studies indicate that diabetic population is not only at increased risk of cardiovascular complications, but also at substantially higher risk of many forms of malignancies. The use of metformin, the most commonly prescribed drug for type 2 diabetes, was repeatedly associated with the decreased risk of the occurrence of various types of cancers, especially of pancreas and colon and hepatocellular carcinoma. This observation was also confirmed by the results of numerous meta-analyses. There are however, several unanswered questions regarding the exact mechanism of the anticancer effect of metformin as well as its activity against various types of cancer both in diabetic and nondiabetic populations. In the present work we discuss the proposed mechanism(s) of anticancer effect of metformin and preclinical and clinical data suggesting its anticancer effect in different populations.

Polymorphisms of the promoter regions of matrix metalloproteinases genes MMP-1 and MMP-9 in breast cancer

SummaryPurposeMatrix metalloproteinases play a crucial role in the cancer invasion and metastasis, angiogenesis and tumorigenicity. A single guanine insertion – the 1G/2G polymorphism in the promoter of the matrix metalloproteinase 1 (MMP-1) gene creates a binding site for the transcription factor AP-1 and thus may affect the transcription level of MMP-1. The C→T substitution at the polymorphic site of the MMP-9 gene promoter results in a higher transcription activity of the T-allelic promoter trough the loss of binding site for a repressor protein. The aim of this work was to investigate the influence of 1G/2G and C→T polymorphisms on the MMP-1 and MMP-9 level and therefore on the occurrence and progression of breast cancer.Experimental designWe investigated the distribution of genotypes and frequency of alleles of the 1G/2G and C→T polymorphisms for 270 patients with breast cancer and 300 healthy women served as control. The genotypes were determined by RFLP-PCR. Additionally, we estimated the level of MMP-1 and MMP-9 antigens in tumor samples and normal breast tissue using ELISA.ResultsThe levels of MMP-1 in tumor samples of node positive patients ware significantly higher than in samples of node negative patients (p<0.05). Increased level of MMP-9 correlates with Bloom-Richardson grading III (p<0.05), increased tumor size (p<0.05) and absence of estrogen and progesterone receptors (p<0.01). Additionally, both MMP-1 and MMP-9 levels were higher in tumor than in the normal breast tissue. We showed the higher risk of metastasis development in lymph node for the 2G/2G genotype (OR=2.14; CI 95% 1.24;3.69) and the 2G allele carriers (OR=1.68; CI 95% 1.19;2.39). We found correlation between the T allele (OR=2.61; CI 95% 1.33;4.87), 2G (OR=2.58; CI 95% 1.35;4.91) and malignance.ConclusionThe results suggest that MMP-1 is responsible for the local invasion and MMP-9 is associated with the malignance and the growth of the tumor. We suggest that the 2G allele of the 1G/2G MMP-1 gene polymorphism may be associated with the lymph node metastasis in patients with breast cancer and therefore it can be considered as a progression marker in this disease.

In vitro genotoxicity of ethanol and acetaldehyde in human lymphocytes and the gastrointestinal tract mucosa cells

The influence of ethanol and acetaldehyde on DNA in human lymphocytes, gastric mucosa (GM) and colonic mucosa (CM) was investigated by using the comet assay. All kinds of cells were exposed to ethanol and acetaldehyde in two regimens: the cells were incubated with either chemical and analysed or they were exposed first to ethanol, washed and then exposed to acetaldehyde and analysed. Lymphocytes were exposed to ethanol at final concentrations of 30 mM and acetaldehyde at 3 mM. GM cells were incubated with ethanol at 1 M and acetaldehyde at 100 mM. CM cells were exposed to ethanol at 10 mM and acetaldehyde at 100 mM. In combined exposure, the cells were subsequently exposed to ethanol and acetaldehyde at all combination of the concentrations of the agents. Ethanol caused DNA strand breaks, which were repaired during 4 hr, except when this agent was applied in GM cells at a concentration of 1 M. A dose-dependent decrease in the tail moment of all types of acetaldehyde-treated cells was observed. Similar results were obtained when a recognized DNA crosslinking agent, formaldehyde, was used. These results suggest that acetaldehyde may form crosslinks with DNA. These crosslinks were poorly repaired. CM cells showed the highest sensitivity of all cell types to ethanol than lymphocytes and GM cells. There were no differences in the sensitivity to acetaldehyde of all the cell types. Our results clearly indicate that ethanol and acetaldehyde can contribute to cancers of the digestive tract.

Polymorphisms of the BRCA2 and RAD51 genes in breast cancer.

SummaryWe performed a case-control study (150 cases and 150 controls) to test the association between three polymorphisms in BRCA2 and RAD51 genes and breast cancer risk. Genotypes were determined in DNA from blood cells by PCR–RFLP. Cancer occurrence was strongly associated with the BRCA2 Met/1915Thr homozygous polymorphic variants, whereas heterozygous variant was associated with significant reduction in breast cancer risk. Gene-gene interaction between the BRCA2-Met1915Thr Thr/Thr and BRCA2-Met784Val Met/Met homozygous variants increased the risk. Therefore, the Met1915Thr polymorphism in the BRCA2 gene may be considered as an independent marker of breast cancer.

Free radical scavengers can differentially modulate the genotoxicity of amsacrine in normal and cancer cells.

Amsacrine is an acridine derivative drug applied in haematological malignancies. It targets topoisomerase II enhancing the formation of a cleavable DNA-enzyme complex and leading to DNA fragmentation in dividing cancer cells. Little is known about other modes of the interaction of amsacrine with DNA, by which it could affect also normal cells. Using the alkaline comet assay, we showed that amsacrine at concentrations from the range 0.01 to 10 microM induced DNA damage in normal human lymphocytes, human promyelocytic leukemia HL-60 cells lacking the p53 gene and murine pro-B lymphoid cells BaF3 expressing BCR/ABL oncogene measured as the increase in percentage tail DNA. The effect was dose-dependent. Treated cells were able to recover within a 120-min incubation. Amifostine at 14 mM decreased the level of DNA damage in normal lymphocytes, had no effect on the HL-60 cells and potentiated the DNA-damaging effect of the drug in BCR/ABL-transformed cells. Vitamin C at 10 and 50 microM diminished the extent of DNA damage in normal lymphocytes, but had no effect in cancer cells. Pre-treatment of the cells with the nitrone spin trap, N-tert-butyl-alpha-phenylnitrone or ebselen, which mimics glutathione peroxidase, reduced the extent of DNA damage evoked by amsacrine in all types of cells. The cells exposed to amsacrine and treated with endonuclease III and 3-methyladenine-DNA glycosylase II, the enzymes recognizing oxidized and alkylated bases, respectively, displayed greater extent of DNA damage than those not treated with these enzymes. The results obtained suggest that free radicals may be involved in the formation of DNA lesions induced by amsacrine. The drug can also methylate DNA bases. Our results indicate that the induction of secondary malignancies should be taken into account as diverse side effects of amsacrine. Amifostine may potentate DNA-damage effect of amsacrine in cancer cells and decrease this effect in normal cells and Vitamin C can be considered as a protective agent against DNA damage in normal cells.

DNA damage and repair in human lymphocytes and gastric mucosa cells exposed to chromium and curcumin.

Human population can be considered as a subject of combined exposure to chemicals. Hexavalent chromium is a well-known mutagen and carcinogen. Curcumin, a popular spice and pigment, is reported to have antineoplastic properties. The single cell gel electrophoresis (Comet assay) is a sensitive technique that allows detecting double- and single-strand DNA breaks caused by a broad spectrum of mutagens. In the present work the ability of curcumin to reduce DNA damage induced by chromium in human lymphocytes and gastric mucosa (GM) cells was investigated by using the comet assay. Chromium at 500 microM evoked DNA damage measured as significant (P < 0.001), about a two-fold increase in comet tail moment of both lymphocytes and GM cells. Curcumin at 10, 25, and 50 microM also damaged DNA of both types of cells in a dose-dependent manner: the increase in the tail moment reached about twenty times of the control value (P < 0.001). The combined action of chromium at 500 microM and curcumin at 50 microM resulted in the significant (P < 0.001) increase in the comet tail moment of both types of cells. In each case, treated cells were able to recover within 60 min. Our study clearly demonstrates that curcumin does not inhibit DNA damaging action of hexavalent chromium in human lymphocytes and GM cells. Moreover, curcumin itself can damage DNA of these cells and the total effect of chromium and curcumin is additive. Further studies are needed to establish the role of interaction of curcumin with DNA in carcinogenesis.

Determination by liquid chromatography of free and total cysteine in human urine in the form of its S-quinolinium derivative

A reversed-phase high-performance liquid chromatographic method for the determination of free and total cysteine in urine is described. The method involves reductive conversion of cysteine dimer and cysteine mixed disulphides to their reduced counterpart with the use of tri-n-butylphosphine, ultraviolet-labeling with 2-chloro-1-methylquinolinium tetrafluoroborate, and liquid chromatographic separation with isocratic conditions. In developing this method the following parameters were investigated and optimized: the time, pH and reagent excess in the derivatization step, and mobile phase buffer concentration, pH, organic modifier and column temperature in the separation step. The method provides quantitative information on free and total cysteine based on assays with derivatization before and after reduction with tri-n-butylphosphine. The calibration graph, obtained with the use of normal urine spiked with growing amounts of cystine, was linear over the concentration range covering most experimental and clinical cases. The assay has a low pmol detection and quantitation limits, low imprecision and high recovery. The method was validated for urine samples received from several donors. Cystine was chosen as a primary calibrator for these assays.

In vitro effect of gliclazide on DNA damage and repair in patients with type 2 diabetes mellitus (T2DM).

Type 2 diabetes mellitus is associated with elevated level of oxidative stress, which is one of the most important factors responsible for the development of chronic complications of this disease. Moreover, it was shown that diabetic patients had increased level of oxidative DNA damage and decreased effectiveness of DNA repair. These changes may be associated with increased risk of cancer in T2DM patients, since DNA damage and DNA repair play a pivotal role in malignant transformation. It was found that gliclazide, an oral hypoglycemic drug with antioxidant properties, diminished DNA damage induced by free radicals. Therefore, the aim of the present study was to evaluate the in vitro impact of gliclazide on: (i) endogenous basal and oxidative DNA damage, (ii) DNA damage induced by hydrogen peroxide and (iii) the efficacy of DNA repair of such damage. DNA damage and DNA repair in peripheral blood lymphocytes of 30 T2DM patients and 30 non-diabetic individuals were evaluated by alkaline single cell electrophoresis (comet) assay. The extent of oxidative DNA damage was assessed by DNA repair enzymes: endonuclease III and formamidopyrimidine-DNA glycosylase. The endogenous basal and oxidative DNA damages were higher in lymphocytes of T2DM patients compared to non-diabetic subjects and gliclazide decreased the level of such damage. The drug significantly decreased the level of DNA damage induced by hydrogen peroxide in both groups. Gliclazide increased the effectiveness of DNA repair in lymphocytes of T2DM patients (93.4% (with gliclazide) vs 79.9% (without gliclazide); P< or =0.001) and non-diabetic subjects (95.1% (with gliclazide) vs 90.5% (without gliclazide); P< or =0.001). These results suggest that gliclazide may protect against the oxidative stress-related chronic diabetes complications, including cancer, by decreasing the level of DNA damage induced by reactive oxygen species.

DNA damage and repair in human lymphocytes exposed to three anticancer platinum drugs

Cisplatin is a widely used anticancer drug, but its application is limited due to severe side effects. To reduce these effects, many other platinum drugs have been synthesized. In the present work comparative analysis of the toxicity of cisplatin, oxoplatin, and a conjugate (NH(3))(2)Pt(SeO(3)) (Se-Pt) in terms of cell viability, DNA binding, and DNA damage and repair in human lymphocytes was performed using the Trypan blue exclusion test, atomic absorption spectroscopy, and the comet assay, respectively. Cisplatin and oxoplatin did not cause a significant change in the viability of the lymphocytes even at the highest used concentration (750 microM), but the conjugate dramatically diminished viability at 100 microM only about 60% of the lymphocytes were viable (P < 0.05), and at 750 microM, less than 20% (P < 0.001). Se-Pt bound to isolated DNA was about 100 times weaker than the remaining two compounds; the binding of cisplatin was about 30% stronger than oxoplatin. Cisplatin and oxoplatin formed crosslinks with DNA in lymphocytes, whereas the conjugate induced DNA strand breaks. The lesions evoked by cisplatin and oxoplatin were slowly removed, but damage induced by Se-Pt was not repaired after 5 h even at a drug concentration of 10 microM. Severe cytotoxic and genotoxic effects exerted by Se-Pt in normal human lymphocytes preclude its intravenous application in cancer therapy. Teratogenesis Carcinog. Mutagen. 20:119-131, 2000.

Application of 2-halopyridinium salts as ultraviolet derivatization reagents and solid-phase extraction for determination of captopril in human plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic method was developed for the determination of captopril in human plasma. 1-Benzyl-2-chloropyridinium bromide (BCPB) was used as a precolumn derivatizing reagent. The mercapto group of captopril was arylated by the reagent to generate a stable UV-sensitive product. The derivative was solid-phase extracted (SPE), separated on a C18 column using reversed-phase ion-paring chromatography and monitored by a spectrophotometric detector at 314 nm. The method enabled sensitive determination of captopril and its disulphides in human plasma in patients after oral administration. Disulphides of captopril with captopril itself and with endogenous thiol compounds are reduced with triphenylphosphine to form captopril, followed by derivatization with the same reagent. The quantification limit was 10 ng/ml. Calibration curves were prepared for human plasma samples spiked with captopril and captopril disulphide. The calibration curves were linear in the range of 10 to 500 ng/ml for captopril and 10 to 1000 ng/ml for captopril disulphide.