Andrzej Klejewski

Analysis of MDR genes expression and cross-resistance in eight drug resistant ovarian cancer cell lines

BackgroundMultiple drug resistance (MDR) of cancer cells is the main reason of intrinsic or acquired insensitivity to chemotherapy in many cancers. In this study we used ovarian cancer model of acquired drug resistance to study development of MDR.We have developed eight drug resistant cell lines from A2780 ovarian cancer cell line: two cell lines resistant to each drug commonly used in ovarian cancer chemotherapy: cisplatin (CIS), paclitaxel (PAC), doxorubicin (DOX) and topotecan (TOP). A chemosensitivity assay - MTT was performed to assess drug cross-resistance. Quantitative real-time polymerase chain reaction and immunofluorescence were also performed to determine mRNA and protein expression of genes/proteins involved in drug resistance (P-gp, BCRP, MRP1, MRP2, MVP). Flow cytometry was used to determine the activity of drug transporters.ResultsWe could observe cross-resistance between PAC- and DOX-resistant cell lines. Additionally, both PAC-resistant cell lines were cross-resistant to TOP and both TOP-resistant cell lines were cross-resistant to DOX. We observed two different mechanisms of resistance to TOP related to P-gp and BCRP expression and activity. P-gp and BCRP were also involved in DOX resistance. Expression of MRP2 was increased in CIS-resistant cell lines and increased MVP expression was observed in CIS-, PAC- and TOP-, but not in DOX-resistant cell lines.ConclusionsEffectiveness of TOP and DOX in second line of chemotherapy in ovarian cancer can be limited because of their cross-resistance to PAC. Moreover, cross-resistance of PAC-resistant cell line to CIS suggests that such interaction between those drugs might also be probable in clinic.

Effect of Salvia miltiorrhiza root extract on brain acetylcholinesterase and butyrylcholinesterase activities, their mRNA levels and memory evaluation in rats

Salvia miltiorrhiza (Lamiaceae), one of the most important and popular plants of traditional medicine of Asia, is used for the prevention and treatment of cardiovascular diseases and in central nervous system disturbances. The main aim of this study was to assess the influence of subchronic (28-fold) administration of Salvia miltiorrhiza root extract (SE, 200mg/kg, p.o.) on behavioural activity and memory of rats and to evaluate the activities of cholinesterases (AChE and BuChE) and gene expression levels of AChE and BuChE as well as of beta-secretase (BACE1) in the hippocampus and frontal cortex in vivo. Huperzine A (HU, 0.5mg/kg b.w., p.o.) served as a positive control substance, whereas scopolamine (0.5mg/kg, i.p.) injection was used as a well-known model of memory impairment. The results showed that subchronic administration of SE led to an improvement of long-term memory of rats. Strong inhibition of AChE and BuChE mRNA transcription in the frontal cortex of rats treated with SE or HU was observed. The BACE1 transcript level was significantly decreased. AChE activity was statistically significantly inhibited in the frontal cortex and the hippocampus by SE (47% and 55%, respectively). Similar effects were observed in the case of HU. In summary, activity of SE provides evidence that the plant can be a source of drugs used in the treatment of Alzheimer disease.

The MAOA, COMT, MTHFR and ESR1 gene polymorphisms are associated with the risk of depression in menopausal women

OBJECTIVE The aim of the study was assessment of a possible relationship between the polymorphisms of the candidate genes participating in the etiology of some neurological and psychiatric disorders and the risk of depression in perimenopausal and postmenopausal women. METHODS A total of 167 (54 perimenopausal and 113 postmenopausal) Caucasian women from western Poland, aged 42-67, were recruited as the patient group in the study because of depressive symptoms, and another 321 healthy women (102 perimenopausal and 219 postmenopausal) served as the controls. All study participants were evaluated for climacteric and depressive disorders according to the Kupperman index and Hamilton rating scale for depression (HRSD), respectively. The following candidate genes were selected for the study: 5HTR2A, 5HTR1B, 5HTR2C, TPH1, TPH2, MAOA, COMT, NET, GABRB1, ESR1, MTHFR, MTR and MTHFD1. In each group the frequencies of the polymorphisms were determined using PCR-RFLP analysis. RESULTS After correcting for Bonferroni multiple tests, we found associations between the MAOA c.1460C>T (SNP 1137070), COMT c.472G>A (SNP 4680), MTHFR c.677C>T (SNP 1801133) and ESR1 454(-351) A>G (SNP 9340799) polymorphisms to mild and moderate depressive symptoms in menopausal women. In the perimenopausal and postmenopausal women, genotype association of the MAOA c.1460 CT and c.1460 CT+TT (OR=1.83; pcorr=0.009 and OR=1.85; pcorr=0.003, resp.), and of the MTHFR c.677 TT and c.677 CT+TT (OR=3.52; pcorr=0.00009 and OR=2.06; pcorr=0.0006, resp.), as well as of the COMT c.472 GA and COMT c.472 GA+AA genotypes (OR=2.23; pcorr=0.03 and OR=2.17; pcorr=0.027, resp.) in the postmenopausal women revealed significantly higher frequencies of these variants in depressed female patients than in controls, whereas the ESR1 454(-351) AG and 454(-351) AG+GG genotypes were associated with lower risk of depression in postmenopausal women (OR=0.48; pcorr=0.012, and OR=0.52; pcorr=0.015, resp.). CONCLUSIONS Our study substantiates the involvement of the MAOA and MTHFR polymorphisms in climacteric depression and offers evidence that the COMT and ESR1 genes may also play a role in the susceptibility to depressive mood in postmenopausal women.

Coexistence of ACE (I/D) and PAI-1 (4G/5G) gene variants in recurrent miscarriage in Polish population

OBJECTIVES Recurrent miscarriage (RM) is one of the most common obstetric complications. Numerous studies have suggested that genetic variants leading to an impaired balance between coagulation and fibrinolysis may contribute to elevated risk of pregnancy loss. The aim of the study was to investigate a possible association between angiotensin-converting enzyme (ACE, rs1799752) I/D and plasminogen activator inhibitor type 1 (PAI-1, rs1799768) 4G/5G polymorphisms with RM among Polish women. MATERIAL AND METHODS DNA was extracted from peripheral blood samples of 152 women with a history of ≥ 2 consecutive pregnancy losses before 22 weeks of gestation, and 180 healthy controls with at least 1 live birth at term and no history of pregnancy loss. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) were used to identify the polymorphisms. RESULTS No statistically significant differences were found in genotype and allele frequencies of the studied polymorphisms. The most relevant difference between the study group and controls was found for the ID genotype distribution of the ACE gene (52.6 vs. 46.7%, OR = 1.27, p = 0.28). The analysis of genotype coexistence revealed a higher incidence of the combination of the ACE II and the PAI-1 4G/4G genotypes in the control group (10.0 vs.5.9% in control group; p = 0.17). CONCLUSIONS The obtained results suggest no apparent association between the ACE I/D, PAI-1 4G/5G polymorphisms and increased RM susceptibility in the analyzed Polish population.

The RANKL/RANK/OPG signal trail: significance of genetic polymorphisms in the etiology of postmenopausal osteoporosis

OBJECTIVES Recent studies have demonstrated that disorders of bone metabolism, which is regulated by RANK/RANKL/OPG signaling pathway, are the cause of osteoporosis. The aim of the study was to investigate the distribution of genotypes of the RANK 575C>T and RANKL -643C>T polymorphisms and to analyze their relationship with bone parameters in postmenopausal women. MATERIAL AND METHODS A total of 310 postmenopausal Caucasian women (139 with osteoporosis, 107 with osteopenia, and 64 healthy postmenopausal controls) were included. Bone mineral density (BMD) at the lumbar region of the spine (L2-L4) was measured by dual energy X-ray absorptiometry (DXA). Genetic analysis was performed using the PCR-RFLP method. RESULTS Analysis of the frequency of genotypes and alleles of the RANK 575C>T and RANKL -643C>T polymorphisms did not show any statistically significant differences between the study groups (osteoporosis and osteopenia) and postmenopausal women with normal t-score value (ns). Notably, a significant association between the RANKL -643C>T polymorphism and body mass, such as BMI values in osteoporotic women (p<0.05), was observed. CONCLUSIONS Our results suggest lack of association between the 575C>T RANK polymorphism and the development of osteoporosis. The -643C>T RANKL polymorphism, through its significant influence on body weight and BMI value, may contribute to the development of osteoporosis in postmenopausal women.

Correlation between factor VII and PAI-1 genetic variants and recurrent miscarriage

BACKGROUND Polymorphisms which are presented below may be the cause of inherited thrombophilia and may result in pregnancy loss. The hypothesis is based on a number of cardiology studies which have confirmed the involvement of these polymorphisms in thrombotic incidents. OBJECTIVES To evaluate the role of polymorphisms of factor VII gene (Arg353Gln, -122T > C) and PAI-1 gene (-675 4G/5G) in the etiology of recurrent miscarriage. MATERIAL AND METHODS The study group included 152 women with a positive history of ≥ 2 consecutive pregnancy losses (114 and 38 women with 2 and ≥ 3 miscarriages, respectively), while 180 healthy women were recruited as controls. Genetic analysis was performed with the use of PCR/RFLP. RESULTS Lower frequency of Arg353/Gln353 was observed in women with 2 and ≥ 3 miscarriages as compared to controls (21.1% vs. 23.9% and 13.2% vs. 23.9%, respectively). The frequency of Gln353 was lower in women with ≥ 3 miscarriages as compared to controls (6.6% vs. 11.9%, p = ns). The frequency of -122TT was higher in women with ≥ 3 miscarriages as compared to controls (86.84% vs. 76.67%, p = ns), whereas -122TC was more frequent in controls (13.16% vs. 22.78% in controls, p = ns). The frequency of -122T was higher in patients with ≥ 3 abortions as compared to controls (93.42% vs. 88.06%, p = ns), and -122C was observed more frequently in controls (6.58% vs. 11.94% in controls, p = ns). There were no significant differences as far as the -675 4G/5G polymorphism was concerned. CONCLUSIONS The obtained results suggest a possible protective role of Gln353 and -122C alleles in recurrent miscarriage.

New and Old Genes Associated with Primary and Established Responses to Cisplatin and Topotecan Treatment in Ovarian Cancer Cell Lines

Low efficiency of chemotherapy in ovarian cancer results from the development of drug resistance. Cisplatin (CIS) and topotecan (TOP) are drugs used in chemotherapy of this cancer. We analyzed the development of CIS and TOP resistance in ovarian cancer cell lines. Incubation of drug sensitive cell lines (W1 and A2780) with cytostatic drugs was used to determine the primary response to CIS and TOP. Quantitative polymerase chain reaction (Q-PCR) was performed to measure the expression levels of the genes. We observed decreased expression of the MCTP1 gene in all resistant cell lines. We observed overexpression of the S100A3 and HERC5 genes in TOP-resistant cell lines. Increased expression of the S100A3 gene was also observed in CIS-resistant A2780 sublines. Overexpression of the C4orf18 gene was observed in CIS- and TOP-resistant A2780 sublines. A short time of exposure to CIS led to increased expression of the ABCC2 gene in the W1 and A2780 cell lines and increased expression of the C4orf18 gene in the A2780 cell line. A short time of exposure to TOP led to increased expression of the S100A3 and HERC5 genes in both sensitive cell lines, increased expression of the C4orf18 gene in the A2780 cell line and downregulation of the MCTP1 gene in the W1 cell line. Our results suggest that changes in expression of the MCTP1, S100A3 and C4orf18 genes may be related to both CIS and TOP resistance. Increased expression of the HERC5 gene seems to be important only in TOP resistance.

New and Old Genes Associated with Primary and Established Responses to Paclitaxel Treatment in Ovarian Cancer Cell Lines

Development of drug resistance is the main reason for low chemotherapy effectiveness in treating ovarian cancer. Paclitaxel (PAC) is a chemotherapeutic drug used in the treatment of this cancer. We analysed the development of PAC resistance in two ovarian cancer cell lines. Exposure of drug-sensitive cell lines (A2780 and W1) to PAC was used to determine the primary response. An established response was determined in PAC-resistant sublines of the A2780 and W1 cell lines. qRT-PCR was performed to measure the expression levels of specific genes. We observed decreased expression of the PCDH9, NSBP1, MCTP1 and SEMA3A genes in the PAC-resistant cell lines. Short-term exposure to PAC led to increased expression of the MDR1 and BCRP genes in the A2780 and W1 cell lines. In the A2780 cell line, we also observed increased expression of the C4orf18 gene and decreased expression of the PCDH9 and SEMA3A genes after PAC treatment. In the W1 cell line, short-term treatment with PAC upregulated the expression of the ALDH1A1 gene, a marker of Cancer stem cells (CSCs). Our results suggest that downregulation of the PCDH9, NSBP1, MCTP1 and SEMA3A genes and upregulation of the MDR1, BCRP, C4orf18 and ALDH1A1 genes may be related to PAC resistance.

The significance of lumican expression in ovarian cancer drug-resistant cell lines

Purpose The aim of the present study is to determine the expression of LUM in drug-resistant ovarian cancer cell lines. Methods Doxorubicin- (DOX), topotecan- (TOP) and vincristine- (VIN) resistant variants of the W1 ovarian cancer cell line were used in this study. We used quantitative real-time polymerase chain reactions to determine LUM mRNA levels. Protein expression was detected using Western blot and immunocytochemistry assays. Protein glycosylation was investigated using PGNase F digestion. Immunohistochemistry assays were used to determine protein expression in ovarian cancer patients. Results We observed increased expression of LUM in drug-resistant cell lines at both the mRNA and the protein level. The most abundant LUM expression was observed in TOP-resistant cell line. We observed LUM bands that corresponded to different molecular masses, and the most abundant LUM form was the secreted form, which had a mass of 50 kDa. Double immunofluorescence analysis showed co-expression of LUM and COL3A1 as well as the presence of extracellular COL3A1 in the TOP-resistant cell line. Finally, we detected the LUM protein in ovarian cancer tissue. Conclusion The expression of LUM in cytostatic-resistant cell lines suggests its role in drug resistance. The co-expression of LUM and COL3A1 indicates the significance of LUM in collagen fibre assembly. Expression in ovarian cancer tissue suggests that LUM can play a role in ovarian cancer pathogenesis in ways similar to other cancers.

Contribution of inherited thrombophilia to recurrent miscarriage in the Polish population

INTRODUCTION The aim of the study was to evaluate the contribution of genetic variants determining inherited thrombophilia to recurrent miscarriage (RM) in the Polish population. The following polymorphisms were analyzed: 1691G>A, 1328T>C of coagulation factor V, 20210G>A of coagulation factor II, R353Q (11496G>A) of coagulation factor VII, 667C>T, 1298A>C, 1793G>A of MTHFR. MATERIAL AND METHODS A total of 359 women with ≥ 2 subsequent recurrent miscarriages (303 < 13 weeks of gestation (w.g.) and 56 between 13-22 w.g.) and 400 healthy controls were included in the study. Frequency of the genetic polymor-phisms was determined with the PCR/RFLP method. RESULTS Higher frequency of the 20210GA genotype was found in the RM < 13 w.g. (2.97 vs. 1.50% in controls, OR = 2.01, ns) and the RM 13-22 w.g. (5.36 vs. 1.50% in controls, OR = 3.72, p = 0.09) subgroups. Statistically significantly higher frequency of the 11496GA genotype was noted in controls as compared to the RM 13-22 w.g. subgroup (10.71 vs. 23.00% in controls, OR = 0.40, p = 0.02). Statistically significantly higher frequency of the 1793GA genotype was observed in the RM < 13 w.g. subgroup as compared to controls (12.21 vs. 7.75% in controls, OR = 1.66, p = 0.03). No significant correlations were found as far as the rest of the analyzed polymorphisms are concerned. CONCLUSIONS The obtained results suggest that the 1793G>A MTHFR, R353Q (11496G>A) factor VII gene and the 20210G>A factor II gene polymorphisms play a role in the etiology of RM in the Polish population.