[Importance of expression of DNA repair proteins in non-small-cell lung cancer]. / Význam stanovovania expresie DNA reparacných mechanizmov u nemalobunkového karcinómu plúc.

BACKGROUND: Proteins XRCC1 and ERCC1 are involved in DNA repair. XRCC1 plays a role in DNA base excision repair and ERCC1 in nucleotide excision repair pathway. Higher expression profile of both proteins in cancer cells may contribute to development of drug resistance. ERCC1 is involved in removal of platinum adducts and might be a potential predictive and prognostic marker in NSCLC (non-small-cell lung cancer) treated with a cisplatin-based regimen. The purpose of study was determination of XRCC1 and ERCC1 levels and their correlation with basic clini-copathological parameters in NSCLC. PATIENTS AND METHODS: In this study, 107 tumor samples diagnosed as NSCLC were immunohistochemically examined for expression of XRCC1 and ERCC1 proteins. Our results were compared to basic clinicopathological parameters: type of tumor, tumor grade and stage of disease. For statistical analysis, the chi-square test was used. RESULTS: In squamous cell carcinoma and large cell carcinoma samples, the XRCC1 protein level was twofold higher (60% of positive samples) than in adenocarcinoma samples (35.5% of positive samples). We have found statistical correlation between XRCC1 protein expression and type of tumor (p = 0.0306). On the other hand, the statistical importance between the protein level versus grade and stage was not found. In the case of the ERCC1 protein, we observed the highest protein level in adenocarcinoma (64.5%) and squamous cell carcinoma (62.5%) samples. Next, we determined a significant difference in content of XRCC1 versus ERCC1 (35.5% vs 64.5%) in adenocarcinoma samples. Statistical chi-square test did not reveal any correlation between ERCC1 status and clinicopathological parameters. CONCLUSION: According to our results, XRCC1 represents an important mechanism of DNA repair in squamous cell and large cell carcinomas. Besides that, expression of XRCC1 was in correlation with type of tumor. In patients with adenocarcinoma and squamous cell carcinoma, we could assume increased resistance to platinum-based therapy because of high expectation of ERCC1 protein expression. However, its levels did not correlate with monitored clinicopathological parameters. The ERCC1 protein will be possibly an independent prognostic factor in NSCLC. To prove a true survival benefit of patients with expression of ERCC1, prospective validation of ERCC1 before clinical implication is needed in the future.

Cytogenetic abnormalities predict treatment-free interval and response to therapy in previously untreated chronic lymphocytic leukemia patients.

We evaluated the prognostic impact of chromosomal abnormalities as detected by interphase fluorescence in situ hybridization (iFISH) in 86 chronic lymphocytic leukemia (CLL) patients. Overall, 39 of 86 (45%) patients displayed one (35%) or more (10%) chromosomal abnormalities, del13q (31%) being more frequently detected than trisomy 12 (19%) followed by del11q (17%), del17p (6%) and del6q (5%). Significant differences in the treatment free intervals (TFIs) were observed among individual cytogenetic subgroups (p=0.027) with the shortest mean TFIs in subgroups with del17p, del11q and trisomy 12 (10, 12 and 14 months, respectively) as compared to subgroups with normal cytogenetics (38 months) and del13q (68 months). Poor response to therapy was observed in subgroups with del11q (p=0.044) and trisomy 12 (p=0.047) while patients with normal cytogenetics had good response (p=0.003). Furthermore, del17p and del11q were associated with highest tumor burden and disease activity as reflected by corresponding laboratory data.

Antiangogenic effect of selected phytochemicals.

Angiogenesis, the formation of new blood vessels from a preexisting vascular network is considered a key step in tumour growth, invasion, and metastasis. Recent studies show that several natural compounds inhibit angiogenesis and nowadays numerous bioactive plant compounds are tested for their antiangiogenic potential. This review examines current knowledge regarding the antiangiogenic potential of several phytochemicals, including polyphenols resveratrol and curcumin as well as miscellaneous compounds from garlic, Hypericum perforatum, Panax ginseng, Coptis chinensis and Rheum palmatum.

MDR1 (C3435T) polymorphism: relation to the risk of breast cancer and therapeutic outcome.

P-glycoprotein (PGP), the product of the MDR1 gene, is a transmembrane active efflux pump for a variety of carcinogens and cytostatics. It has been suggested that MDR1 polymorphisms contribute to the variability in cancer risk and therapeutic outcome. We examined the relevance of C3435T polymorphism in relation to breast cancer susceptibility, clinical and pathological characteristics of breast carcinoma, the therapeutic response and hematologic toxicities after anthracycline-based chemotherapy. A significant association between allele frequencies and histological type, stage and histological grade was observed (P=0.024, 0.014, 0.006, respectively, chi(2)-test or Fisher's exact test). We also found significantly higher (P=0.019, chi(2)-test) T allele frequency in breast cancer patients (n=221) than in controls (n=113). A significantly enhanced therapeutic outcome after neoadjuvant therapy (n=38; P=0.021, Fisher's exact test) and longer time to progression after anthracycline-based chemotherapy (n=102; P=0.049, log-rank test) were observed in CC homozygotes. However, no significant association between hematologic toxicities and C3435T polymorphism was detectable.

In vitro antiproliferative and antiangiogenic effects of Flavin7.

Flavin7 (F7) is a nutritional supplement often taken by cancer patients in Central Europe during chemo- and radiation therapy. In this study, investigation of the antiproliferative and antiangiogenic activities of this supplement were performed. Flavin7 showed antiproliferative activity in Jurkat as well as in HeLa cells. It significantly reduced the growth of both cancer cell lines at the doses of 200 microg/ml to 20 microg/ml (p<0.001 and p<0.01, respectively). In F7-treated Jurkat cells we found a significant increase in the fraction of cells with sub-G(0)/G(1) DNA content, which is considered to be a marker of apoptotic cell death. Apoptosis was also confirmed by annexin V staining and DNA fragmentation. Furthermore, F7 at the doses of 100 microg/ml to 4 microg/ml inhibited endothelial cell migration and capillary tube formation what indicates its potential antiangiogenic properties. Flavin7 also inhibited the activity of matrix metalloproteinases (MMPs), preferentially MMP-9, at the doses of 100 microg/ml to 4 microg/ml. Our data suggest that F7 possesses marked antiproliferative and antiangiogenic properties in vitro. Further research is needed to elucidate also its in vivo activities.

Antiangiogenic effects of flavonoids and chalcones.

Angiogenesis, the development of new blood vessels from the existing vasculature, is essential in normal developmental processes. Uncontrolled angiogenesis is a major contributor to a number of disease states such as inflammatory disorders, obesity, asthma, diabetes, cirrhosis, multiple sclerosis, endometriosis, AIDS, bacterial infections and autoimmune disease. It is also considered a key step in tumour growth, invasion, and metastasis. Angiogenesis is required for proper nourishment and removal of metabolic wastes from tumour sites. Therefore, modulation of angiogenesis is considered as therapeutic strategies of great importance for human health. Numerous bioactive plant compounds are recently tested for their antiangiogenic potential. Among the most frequently studied are polyphenols present in fruits and vegetables. Plant polyphenols inhibit angiogenesis and metastasis through regulation of multiple signalling pathways. Specifically, flavonoids and chalcones regulate expression of VEGF, matrix metalloproteinases (MMPs), EGFR and inhibit NFkappaB, PI3-K/Akt, ERK1/2 signalling pathways, thereby causing strong antiangiogenic effects. This review focuses on the antiangiogenic properties of flavonoids and chalcones and examines underlying mechanisms.

Expression of MDR proteins in breast cancer and its correlation with some clinical and pathological parameters.

The aim of this work was to determine the expression of the multidrug resistance (MDR) proteins, namely MDR1 (P-glycoprotein), MRP1 (multidrug resistance-related protein) and LRP (lung resistance-related protein), in 87 samples of breast carcinoma. Detection of these proteins was provided by using indirect enzymatic immunohistochemistry. Our findings were compared with the other clinical and pathological parameters: expression of Her2/neu, estrogen receptor status (ER), progesteron receptor status (PR), histological grade and regional lymph node status. For statistical analysis, non-parametric two sided Mann-Whitney-U test was used. Majority of breast carcinoma specimens show positivity for these proteins. The MDR1 and MRP1 signal was found in the cytoplasm of cancer cells. The expression of LRP was detected in the cytoplasm close to the nuclear membrane. The samples were positive for MDR1 protein in 57%, for MRP1 in 84% and for LRP in 79%. Comparing our results with other clinical and pathological parameters, negative correlation between ER, PR and MDR1 expressions and histological grading status was found. No associations were observed between the MRP1 and LRP proteins and histological grading, as well as between the expression of three MDR proteins and the other clinically relevant parameters. In conclusion, high frequency of expression of MDR proteins in breast carcinoma cells suggests, that these proteins might be an important factor of drug resistance in breast carcinoma. Nevertheless, the negative correlation between the histological grade of malignancy of tumor and the expression of ER, PR and MDR1 indicates possible influence of progressive tumor cell de-differentiation. However, this finding has to be confirmed in additional evaluations.

Protective effect of selected flavonoids on in vitro daunorubicin-induced cardiotoxicity.

Flavonoids are an ubiquitous group of polyphenolic substances with varied chemical structures present in foods of plant origin and act as free radical scavenging and chelating agents with a variety of biological activities. Using a model of spontaneously beating, cultured adult rat cardiomyocytes, this study examined the cardioprotective role of quercetin, naringenin, pycnogenol and a model antioxidant, trolox, against daunorubicin-induced toxicity. Cardiomyocyte protection was assessed by MTT test and extracellular lactate dehydrogenase detection. Protection of cardiomyocytes was concentration/dose dependent for quercetin > naringenin > pycnogenol > trolox. Quercetin (10(-4)-10(-6) mol/L) after 24 h of co-incubation with daunorubicin significantly increased the cardiomyocyte survival in all tested concentrations (p < 0.001). The cytoprotective effect of naringenin (10(-4)-10(-6) mol/L) was similar to those of quercetin (p < 0.001 and p < 0.01, respectively). Pycnogenol was the least effective of the flavonoids studied. On the other hand, all tested flavonoids had significantly better protective effects than trolox. The leakage of lactate dehydrogenase induced by daunorubicin was also prevented by the studied compounds and was in accordance with their cytoprotective activity.

Photoinduced antitumour effect of hypericin can be enhanced by fractionated dosing.

The in vivo antitumour activity of the natural photosensitizer hypericin was evaluated. C3H/DiSn mice were inoculated with fibrosarcoma G5:1:13 cells. When the tumour reached a volume of 40-80mm3 the mice were intraperitoneally injected with hypericin, either in a single dose (5mg/kg; 1 or 6h before laser irradiation) or two fractionated doses (2.5 mg/kg; 6 and 1 h before irradiation with laser light; 532 nm, 70mW/cm2, 168 J/cm2). All tumours in control groups treated with hypericin alone as well as those irradiated with laser light alone had similar growth rates and none of these tumours regressed spontaneously. Complete remission of tumour in photodynamic therapy (PDT)-treated groups was similar (14-17% single dose vs. 33% fractionated dose), but the fractionated schedule of hypericin dosing was found to be more efficient than the single dose, measured by survival assay (p < 0.05). Our experimental model showed that fractionated administration of hypericin can produce a better therapeutic response than single administration.

Diazepam enhances hypericin-induced photocytotoxicity and apoptosis in human glioblastoma cells.

Glioblastoma multiforme (GBM) is neoplasm which is resistant to all currently used treatment modalities including surgery, radiation therapy and chemotherapy. Photodynamic therapy (PDT) has been suggested as a novel therapeutical approach to the treatment of malignant gliomas. Here, we attempted to enhance hypericin-induced photocytotoxicity and apoptosis by diazepam, a non-selective ligand of peripheral benzodiazepine receptors (PBR) which seem to play an important role in apoptosis regulation. For the study, we used U-87 MG and U373 MG glioma cell lines and primary cultures of GBM cells prepared from peroperatively obtained tumor specimens. The patients included 7 histologically confirmed GBMs. Colorimetric MTT assay was employed to study the photocytotoxic effects of hypericin and diazepam. Flow cytometry was used to detect apoptosis and assess the proapoptotic effects of diazepam. We found that hypericin upon photoactivation exerts strong cytotoxic effects against U-87 MG and U373 MG cells as well as primary GBM cell cultures. No cytotoxic effect of hypericin was observed under dark conditions. Diazepam inhibited cell growth in U-87 MG cells and primary cultures whereas proliferation of U373 MG cells remained unaffected. When hypericin was combined with diazepam, photocytotoxicity was increased in U-87 MG cells and primary cultures unlike U373 MG cells. Flow cytometric analysis revealed photoactivated hypericin-induced apoptosis in both cell lines. Apoptosis was significantly enhanced by diazepam in U-87 MG cells. However, no such effect was observed in U373 MG cells. In the present study, we showed that photocytotoxic effect of hypericin in glioma cells can be potentiated by diazepam. This effect is underlied by the ability of diazepam to facilitate hypericin-induced apoptosis. This work provides support to performe clinical studies involving diazepam in the antiglioma treatment regimens as an apoptosis-modulating agent.

Cruciferous phytoalexins: antiproliferative effects in T-Jurkat leukemic cells.

We tested antiproliferative activity of selected cruciferous phytoalexins including brassinin, 1-methoxybrassinin, (+/-)-spirobrassin, (+/-)-1-methoxyspirobrassinin and (+/-)-1-methoxyspirobrassinol, in leukemic Jurkat cell. The most effective of the tested phytoalexins was 1-methoxybrassinin with IC(50) 10 micromol l(-1). However, significant effect of all phytoalexines was also determined at concentration 1 micromol l(-1). In 1-methoxybrassinin-treated Jurkat cells, we found significant increase in the fraction of cells with a sub-G(0)/G(1) DNA content, which is considered to be a marker of cell death by apoptosis. Apoptosis was also confirmed by the annexin V staining. In summary, 1-methoxybrassinin exerted potent antiproliferative activity probably due to cell cycle arrest and apoptosis induction.

Expression of the multidrug resistance-associated protein 1 (MRP1) and the lung resistance-related protein (LRP) in human lung cancer.

Fifty lung cancer samples (41 non-small cell lung cancer-NSCLC and 9 small cell lung cancer-SCLC) were immunohistochemically analyzed for lung resistance-related protein (LRP) and multidrug resistance-associated protein 1 (MRP1) expressions which were then correlated with histopathological subtype of the tumor. To detect these proteins, monoclonal antibodies LRP-56 and MRPm6 were used. NSCLC samples were divided into two groups, adenocarcinomas (17 samples) and squamous cell carcinomas (24 samples). Four categories of LRP and MRP1 quantity were distinguished: +++ = high level--90--100% of positive cells, ++ = lower level--10--90% of positive cells, + = low level--up to 10% of positive cells, - = negative cells--0% of positive cells. Within the NSCLC group the most samples (36/41) had the similar level of LRP and MRP1. Significantly higher expression of both proteins was observed in the adenocarcinomas in comparison with squamous cell carcinomas. The lowest positive staining for LRP and MRP1 proteins has been found in SCLC. It is suggested that our finding can confirm the overall empirical clinical knowledge about much higher chemosensitivity of untreated SCLC comparing to NSCLC.

Effect of quercetin on daunorubicin-induced heart mitochondria changes in rats.

Cancer therapy with daunorubicin is limited by its cardiotoxicity. It has been suggested that daunorubicin-induced free radical generation can be involved. The precise molecular mechanism of daunorubicin-induced cardiotoxicity is still not well understood but it is believed that mitochondria play an important role in this process. It has been reported that flavonoids with antioxidant properties may prevent anthracycline-induced cardiotoxicity. In this work, we investigated the effects of daunorubicin and quercetin on mitochondrial enzyme activities such as ATPase, glutathione peroxidase (GPx) and glutathione reductase (GR). Moreover, we also studied the changes of outer mitochondrial membrane using synchronous fluorescence spectra. The activity of ATPase and GR were significantly increased after daunorubicin application. Pretreatment with quercetin significantly alleviated this increase. On the other hand, GPx activity was significantly decreased and quercetin prevented this decrease. Treatment with quercetin alone had no significant effect on the enzyme activity studied. Quercetin also completely prevented daunorubicin-induced changes in fluorescence of the outer mitochondrial membrane. In conclusion, our data indicate that quercetin may be useful in mitigating daunorubicin-induced cardiotoxicity.

Effects of static magnetic field on human leukemic cell line HL-60.

A number of structures with magnetic moments exists in living organisms that may be oriented by magnetic field. While most experimental efforts belong to the area of effects induced by weak and extremely low-frequency electromagnetic fields, we attempt to give an attention to the biological effects of strong static magnetic fields. The influence of static magnetic field (SMF) on metabolic activity of cells was examined. The metabolic activity retardation is observed in human leukemic cell line HL-60 exposed to 1-T SMF for 72 h. The retardation effect was observed as well as in the presence of the mixture of the antineoplastic drugs 5 fluorouracil, cisplatin, doxorubicin and vincristine.

Modulation of the phototoxic effect of hypericin in human leukemia CEM cell line by N-ethylmaleimide, amiloride and omeprazole.

Hypericin is a photosensitizing plant pigment from Hypericum perforatum with multiple modes of light-induced biological activities due to production of singlet oxygen and/or excited-state proton transfer with consequent pH drop in the hypericin environment. In the present work, we studied the effects of three inhibitors of crucial mechanisms responsible for intracellular pH (pHi) regulation on hypericin phototoxicity: N-ethylmaleimide (NEM), an inhibitor of H+-ATPase, 5'-(N,N-dimethyl)-amiloride (DMA), an inhibitor of Na+/H+ exchanger, and omeprazole (OME), an inhibitor of H+K+-ATPase. Our experiments show that the effect of hypericin at 1 x 10(-5) and 1 x 10(-6) mol x l(-1) was significantly potentiated by NEM (1 x 10(-7)-1 x 10(--9) mol x l(-1)) and DMA (1 x 10(-6) and 1 x 10(-7) mol x l(-1)) in leukemic CEM cell line. On the other hand, OME had no significant effect on hypericin cytotoxicity. Our results support the hypothesis that the excited-state proton transfer and the consequent acidification of hypericin environment could play a role in the biological activity of hypericin.

Protective effect of quercetin on ischemia/reperfusion-induced gastric mucosal injury in rats.

This study was designed to determine the gastroprotective properties of quercetin in ischemia/reperfusion-induced gastric mucosal injury and the involvement of endogenous prostaglandins in this process. Oral pretreatment of rats with quercetin (100 mg x kg(-1)) 30 min before surgery significantly decreased the length of gastric mucosal lesions. However, lower doses of quercetin (25 and 50 mg x kg(-1)) only slightly decreased the gastric mucosal injury. Intraperitoneal application of indomethacin (5 mg x kg(-1)) had no effect in control (sham-operated) animals, but significantly worsened gastric injury in non-treated animals after ischemia/reperfusion. Furthermore, indomethacin only slightly reversed protective effect of quercetin. Non-treated animals showed a marked decrease in adherent mucus after ischemia/reperfusion. On the other hand, application of quercetin prevented this significant decrease even in animals pretreated with indomethacin. It can be concluded that antioxidant properties of quercetin and its mucus protective effect might be the main factors responsible for its protective effect against ischemia/reperfusion-induced gastric mucosal injury.

The effect of 5'-(N,N-dimethyl)-amiloride on cytotoxic activity of doxorubicin and vincristine in CEM cell lines.

Intracellular pH (pHi) plays an important role in anticancer drug accumulation in cancer cells. Resistant cells often express membrane P-glycoprotein responsible for active drug extrusion and participating in increased pHi. In the present paper, we report on the influence of Na+/H+-exchanger inhibitor, 5'-(N,N-dimethyl)-amiloride (AMI), on the cytotoxic effects of doxorubicin (DOXO) and vincristine (VCR) in the parental CEM, and resistant CEM/DNR and CEM/VCR cell lines. The obtained results revealed a potentiating effect of AMI to both anticancer drugs in parental CEM line. However, AMI did not significantly potentiate the effect of DOXO or VCR in resistant CEM cell lines. We conclude, that inhibition of Na+/H+-exchanger by AMI is not sufficient for reversal of drug resistance in the tested CEM/DNR and CEM/VCR cell lines and the possible change in pHi does not affect the mechanisms of cell resistance.

Diazepam enhances etoposide-induced cytotoxicity in U-87 MG human glioma cell line.

Various approaches might be employed in an effort to increase efficacy of the chemotherapeutic treatment of cancer. Recently, various modulators of anticancer therapy effectiveness have been studied. Antiproliferative effects of peripheral benzodiazepine receptor (PBR) ligands might be exploited to enhance cytotoxic effect of a chemotherapeutic drug towards cancer cells. In this work, we sought to enhance cytotoxic effect of etoposide (VP-16) by a PBR ligand, diazepam (DZ) in U-87 MG human glioma cells. Cytotoxicity of VP-16, DZ and their combinations was assessed by using the microculture MTT assay. Cell survival, effective concentrations (EC) and the onset of cytotoxic effect were determined. After 72 h of cultivation, survival of U-87 MG cells was reduced to 57 +/- 7% in the presence of VP-16 at 12.5 microg/mL alone, whereas DZ at 10-4 mol/L alone caused 28 +/- 6% reduction in cell survival. Coincubation of VP-16 at 12.5 microg/mL with DZ at 10-4 mol/L led to a further decrease in cell survival to 45 +/- 6%. Furthermore, DZ at 10-4 mol/L significantly decreased effective concentrations, EC10, EC30 and EC50, of VP-16 and the dose-response curves were shifted to the left. Addition of DZ at 10-4 mol/L to VP-16 also facilitated the onset of its cytotoxic effect. The same decrease in survival was thus achieved approximately 30 h earlier in comparison with VP-16 alone. However, DZ at 10-9 mol/L failed both to exert any effect on glioma cells survival and enhance cytotoxic effect of VP-16. DZ at 10-4 mol/L was capable of both reducing U-87 MG glioma cells survival when applied alone and also enhancing the cytotoxic effect of VP-16. No such observation was made for the lower concentrations of DZ. Potential implementation of diazepam in the antiglioma/anticancer armamentarium awaits further experimentation but phase I and phase II clinical trials could be suggested.

The effect of quercetin on light-induced cytotoxicity of hypericin.

Protective effect of quercetin, a natural antioxidant compound, on hypericin-induced cytotoxicity was studied in human promyelocytic leukemia cells (HL-60). Hypericin (10(-5) mol x l(-1)) alone significantly decreased cell survival to 21% that found in the controls, whereas in combination with quercetin (10(-5) mol x l(-1)) this decrease was diminished to 46%. Lower concentrations of quercetin had no protective effect. These findings indicate that oxygen radicals can play an important role in hypericin-induced phototoxic effects.

Role of mucus in ischemia/reperfusion-induced gastric mucosal injury in rats.

Gastric mucus plays an important role in gastric mucosal protection. Apart from its "barrier" function, it has been demonstrated that mucus protects gastric epithelial cells against toxic oxygen metabolites derived from the xanthine/ xanthine oxidase system. In this study, we investigated the effect of malotilate and sucralfate (mucus production stimulators) and N-acetylcysteine (mucolytic agent) on ischemia/reperfusion-induced gastric mucosal injury. Gastric ischemia was induced by 30 min clamping of the coeliac artery followed by 30 min of reperfusion. The mucus content was determined by the Alcian blue method. Sucralfate (100 mg/kg), malotilate (100 mg/kg), and N-acetylcysteine (100 mg/kg) were given orally 30 min before surgery. Both sucralfate and malotilate increased the mucus production in control rats. On the other hand, N-acetyloysteine significantly decreased mucus content in control (sham) group. A significant decrease of mucus content was found in the control and the N-acetylcysteine pretreated group during the period of ischemia. On the other hand, sucralfate and malotilate prevented the decrease the content of mucus during ischemia. A similar result can be seen after ischemia/reperfusion. In the control group and N-acetylcysteine pretreated group a significant decrease of adherent mucus content was found. However, sucralfate and malotilate increased mucus production (sucralfate significantly). Sucralfate and malotilate also significantly protected the gastric mucosa against ischemia/reperfusion-induced injury. However, N-acetylcysteine significantly increased gastric mucosal injury after ischemia/reperfusion. These results suggest that gastric mucus may be involved in the protection of gastric mucosa after ischemia/reperfusion.