Effects triggered by platinum nanoparticles on primary keratinocytes.

The platinum (Pt)-group elements (PGEs) represent a new kind of environmental pollutant and a new hazard for human health. Since their introduction as vehicle-exhaust catalysts, their emissions into the environment have grown considerably compared with their low natural concentration in the earth crust. PGE emissions from vehicle catalysts can be also in the form of nanometer-sized particles (Pt nanoparticles [PtNPs]). These elements, both in their metallic form or as ions solubilized in biological media, are now recognized as potent allergens and sensitizers. Human skin is always exposed to toxic particles; therefore, in the present study we addressed the question of whether polyvinylpyrrolidone-coated PtNPs may have any negative effects on skin cells, including predominantly epidermal keratinocytes. In this study, PtNPs of two sizes were used: 5.8 nm and 57 nm, in concentrations of 6.25, 12.5, and 25 µg/mL. Both types of NPs were protected with polyvinylpyrrolidone. Primary keratinocytes were treated for 24 and 48 hours, then cytotoxicity, genotoxicity, morphology, metabolic activity, and changes in the activation of signaling pathways were investigated in PtNP-treated cells. We found that PtNPs trigger toxic effects on primary keratinocytes, decreasing cell metabolism, but these changes have no effects on cell viability or migration. Moreover, smaller NPs exhibited more deleterious effect on DNA stability than the big ones. Analyzing activation of caspases, we found changes in activity of caspase 9 and caspase 3/7 triggered mainly by smaller NPs. Changes were not so significant in the case of larger nanoparticles. Importantly, we found that PtNPs have antibacterial properties, as is the case with silver NPs (AgNPs). In comparison to our previous study regarding the effects of AgNPs on cell biology, we found that PtNPs do not exhibit such deleterious effects on primary keratinocytes as AgNPs and that they also can be used as potential antibacterial agents, especially in the treatment of Escherichia coli, representing a group of Gram-negative species.

Effect of silver nanoparticles on human primary keratinocytes.

Silver nanoparticles (AgNPs) have many biological applications in biomedicine, biotechnology and other life sciences. Depending on the size, shape and the type of carrier, AgNPs demonstrate different physical and chemical properties. AgNPs have strong antimicrobial, antiviral and antifungal activity, thus they are used extensively in a range of medical settings, particularly in wound dressings but also in cosmetics. This study was undertaken to examine the potential toxic effects of 15 nm polyvinylpyrrolidone-coated AgNPs on primary normal human epidermal keratinocytes (NHEK). Cells were treated with different concentrations of AgNPs and then cell viability, metabolic activity and other biological and biochemical aspects of keratinocytes functioning were studied. We observed that AgNPs decrease keratinocyte viability, metabolism and also proliferatory and migratory potential of these cells. Moreover, longer exposure resulted in activation of caspase 3/7 and DNA damage. Our studies show for the first time, that AgNPs may present possible danger for primary keratinocytes, concerning activation of genotoxic and cytotoxic processes depending on the concentration.

The effect of tyrphostins AG494 and AG1478 on the autocrine growth regulation of A549 and DU145 cells.

We employed two selective EGFR tyrosine kinase inhibitors: AG494 (reversible) and AG1478 (irreversible) for growth regulation of human lung (A549) and prostate (DU145) cancer cell lines, cultured in chemically defined DMEM/F12 medium. Both tested tyrphostins significantly inhibited autocrine growth of the investigated cell lines. The action of AG494 was dose dependent, and at highest concentrations led to complete inhibition of growth. AG1478 seemed to be more effective at lower concentrations, but was unable to completely inhibit growth of A549 cells. Inhibition of EGFR kinase activity by AG494 in contrast to AG1478 had no effect on the activity of ERK in both cell lines. Both EGFR's inhibitors induced apoptosis of the investigated lung and prostate cancer cell lines, but the proapoptotic effect of the investigated tyrphostins was greater in A549 than in DU145 cells. The tyrphostins arrested cell growth of DU145 and A549 cells in the G1 phase, similarly to other known inhibitors of EGFR. The influence of AG494 and AG1478 on the activity of two signaling proteins (AKT and ERK) was dependent upon the kind of investigated cells. In the case of DU145 cells, there was an evident decline in enzymatic activity of both kinases (stronger for AG1478), while in A549, only AG1478 effectively inhibited the phosphorylation of Akt. Tyrphostins AG494 and AG1478 are ATP-competitors and are supposed to have a similar mechanism of action, but our results suggest that this is not quite true.

Cytostatic and cytotoxic effects of tyrphostin AG1296 on RMS cells.

AIM OF THE STUDY: The aim of the work was to determine the influence of tyrphostin AG1296, an inhibitor of platelet-derived growth factor receptor (PDGFR) tyrosine kinase, on autocrine growth of rhabdomyosarcoma (RMS) cells. MATERIALS AND METHODS: RMS cells were cultured in serum-free DMEM/F12 medium. Modified crystal violet (CV) and MTT methods were used to determine the RMS cells' proliferation and viability. Influence of the investigated inhibitor on cell apoptosis or necrosis was determined by differential staining with Hoechst No. 33258 and propidium iodide. RESULTS: The AG1296 inhibitor affects RMS cell proliferation in a dose-dependent way at the concentration range 1-100 µM. At concentrations above 25 µM there was 100% inhibition of growth of these cells and a cytotoxic effect was noticed. 50% inhibition of RMS cells proliferation (IC50) was observed at concentration 6.65 ±0.44 µM (determined by CV method) and 7.30 ±0.26 µM (determined by MTT method). The differential staining method shows that this inhibitor causes a cytotoxic effect. CONCLUSION: The results of these experiments indicate that autocrine growth of RMS cells is regulated by at least one autocrine loop, involving platelet-derived growth factor (PDGF) and its receptor (PDGFR). The fact that tyrphostin AG1296 is able to complete inhibition of RMS cell growth in vitro gives a chance for providing a new group of antitumor drugs, which may be more effective than the medicines used so far.

Iron inhibits respiratory burst of peritoneal phagocytes in vitro.

Objective. This study examines the effects of iron ions Fe(3+) on the respiratory burst of phagocytes isolated from peritoneal effluents of continuous ambulatory peritoneal dialysis (CAPD) patients, as an in vitro model of iron overload in end-stage renal disease (ESRD). Material and Methods. Respiratory burst of peritoneal phagocytes was measured by chemiluminescence method. Results. At the highest used concentration of iron ions Fe(3+) (100 µM), free radicals production by peritoneal phagocytes was reduced by 90% compared to control. Conclusions. Iron overload may increase the risk of infectious complications in ESRD patients.

Growth factor/growth factor receptor loops in autocrine growth regulation of human prostate cancer DU145 cells.

Autocrine growth factors produced by epithelial cells mediate the development and proliferation of neoplastic human prostate tissue. Various approaches have been used to down-regulate neoplastic growth of prostate cancer using natural flavonoids, soluble receptors, pseudo-ligands, monoclonal antibodies and tyrosine kinase inhibitors (tyrphostins). Selected growth factor/growth factor receptor loops (mainly TGFα/EGFR and IGFs/IGFIR) have been proposed as regulators of prostate cancer cell growth. We have previously determined that blockade of IGFIR or VEGF2R signaling pathways by tyrphostin AG1024 and SU1498 inhibits autocrine growth and viability of DU145 cells in vitro. Recently, we compared the activity of AG1024 and SU1498 with the inhibiting effect of tyrphostin A23 (a selective inhibitor of EGFR). The results described in this paper confirm that DU145 cells do not produce IGFI or EGF. In contrast, DU145 cells produce a great amount of VEGF, much more than TGFα (about 60-fold), and VEGF may be the real autocrine growth factor of the investigated cells. The results indicate that the growth of DU145 may be regulated by at least three autocrine loops: TGFα/EGFR, IGFII/IGFIR and VEGF/VEGFR2. Neither AG1024 nor SU1498 affected the production of TGFα substantially, which excludes the possibility that IGFRs or VEGFR2 inhibitors arrest the growth of these cells by inhibition of synthesis and/or secretion of TGFα. The obtained data indicate that all tree investigated tyrphostins (AG1024, SU1498 and A23) inhibit signal transmission by Akt (PKB), ERK(1/2), Src and STAT in a similar manner. A comparison of the effects of the investigated tyrphostins indicates that TGFα, IGFII and VEGF stimulate cell growth by affecting the same signaling pathway. The hypothesis was confirmed by the effect of the investigated tyrphostins on activation of EGFR. All these inhibitors decreased phosphorylation of EGFR to the same extent, and after the same time of incubation with cell culture. These results strongly suggest that stimulation of EGFR kinase is the main step in the initiation of mitogen signaling in DU145 cells, regardless of the type of ligand (TGFα, IGFs or VEGF) and their specific receptors.

Sodium orthovanadate affects growth of some human epithelial cancer cells (A549, HTB44, DU145).

Within the concentration range of 1-20 microM, orthovanadate (Na3VO4) demonstrated a time and dose-dependent inhibition of autocrine growth of the human carcinoma cell lines A549 (lung), HTB44 (kidney) and DU145 (prostate), as compared to appropriate controls (without Na3VO4). The investigation was conducted by two methods: staining with N-hexa-methylpararosaniline (crystal violet=CV) or bromide3-(4,5-dimethyltio-azo-2)-2,5-diphenyl-tetrazole (MTT). In 5, 10 and 20 microM of Na3VO4 in serum-free medium, the mean values of these two tests for A549 were approximately 40%, 45% or 65% as compared to the appropriate controls. HTB44 had the greatest opportunity (statistically insignificant) at lower vanadium concentrations (up to 10 microM), whereas at 20 microM growth inhibition of these cells was approximately 50% of the controls. DU145 showed approximately 33%, 65% and 98% growth inhibition for 5, 10 and 20 microM of Na3VO4, respectively Additionally, hypothetical curves obtained by a MANOVA test based on the CV results after 72 h incubation with Na3VO4 in serum-free medium, and an example of a time-dependent effect of Na3VO4 on A549 cells, were also presented. Sodium orthovanadate was also examined for its cytotoxic capabilities, especially its ability to induce tumor cell apoptosis; the results were compared with the effect of paclitaxel. The target cells were dyed by differential staining (HOECHST33258 and propidium iodide) after 3 h and 24 h (DU145) or 3 h and 72 h (A549) of incubation with the vanadium compound. Contrary to the two cancer cell lines (viable, apoptotic or necrotic in experimental conditions), the renal HTB44 cells were insensitive up to 15 microM Na3VO4 concentrations. After 3 h incubation with Na3VO4, both lung (A549) and prostate (DU145) cancer cells showed a slight but significant reduction in the percentage of viable cells, and an increased amount of apoptotic cells. In contrast to the lung cells, DU145 prostate cells after 24 h were more sensitive to paclitaxel than to sodium orthovanadate. In the case of lung cells, the time of incubation was prolonged (to 72 h) to allow for a study of the effect of orthovanadate in greater detail. After 72 h of incubation with Na3VO4 or paclitaxel, A549 showed a similar level of viable cells (25-32% of total cultured cells); however, the percentage of apoptotic cells was higher in the case of A549 cells--ca 36% for both drugs, but the concentration of Na3VO4 was significantly greater than paclitaxel levels.

The effect of vanadyl sulphate (VOSO4) on autocrine growth of human epithelial cancer cell lines.

Numerous studies have focused on the growth regulation effect of vanadium compounds. In our preliminary investigation we have observed growth inhibition of rat hepatoma cell line H35-19 by inorganic vanadium salts. The aim of the present study was to determine the effect of vanadyl sulphate (VOSO4) on autocrine growth and survival of tumorogenic lung (A549) and prostate (DU145) human cell lines. Additionally, non-carcinogenic human cell lines BEAS-2B (as a lung control) and PNT-2 (as a prostate control) were investigated. MTT, modified crystal violet staining, differential staining (HOECHST33258 and PI) methods and assay for anchorage-independent colony formation were used to investigate the effect of vanadyl sulphate. The results showed that VOSO4 significantly inhibited autocrine growth, decreased carcinoma cells viability and increased the ratio of apoptotic and necrotic cells compared to the controls. However, it should be noted that the examined "drug" significantly decreased viability of non-carcinogenic human cell lines (BEAS-2B, PNT-2).

The effect of tyrosine kinase inhibitors, tyrphostins: AG1024 and SU1498, on autocrine growth of prostate cancer cells (DU145).

It is well established that autocrine growth of human prostate cancer cell line DU145 is dependent on TGF (EGF)/EGFR loop. However, the participation of several other growth factors in proliferation of DU145 cells has been also proposed. We employed two selective tyrosine kinase inhibitors (tyrphostins): AG1024 (an IGFIR inhibitor) and SU1498 (a VEGFR2 inhibitor) for growth regulation of DU145 cells, cultured in chemically defined DMEM/F12 medium. Both the tested compounds inhibited autocrine growth of DU145 cells at similar concentration values (IC50 approximately 2.5 microM). The tyrphostins arrested cell growth of DU145 in G1 phase, similarly as inhibitors of EGFR. However, in contrast to selective inhibitors of EGFR, neither AG1024, nor SU1498 (at concentration < or =10 microM) decreased the viability of the investigated cells. These results strongly suggest that autocrine growth of DU145 cells is stimulated by, at least, three autocrine loops: TGFalpha(EGF)/EGFR, IGFII/IGFIr and VEGF/VEGFR2(VEGFR1). These data support the hypothesis of multi-loops growth regulation of metastatic prostate cancer cell lines.

The ability of HDL to inhibit VCAM-1 expression and oxidized LDL uptake is impaired in renal patients.

OBJECTIVES: This study examines the ability of HDL from hemodialysis (HD) and continuous ambulatory peritoneal dialysis (CAPD) patients to suppress the expression of adhesion molecules in endothelial cells (ICAM-1, VCAM-1) and in monocytes (LFA-1, VLA-4) and to inhibit the uptake of oxidized LDL by macrophages. DESIGN AND METHODS: Gene expression and the uptake of oxidized LDL were determined in 12 HD patients, 12 CAPD patients and 14 healthy volunteers. RESULTS: HDL from renal patients were less effective than control lipoproteins in reducing VCAM-1 expression. HDL from CAPD patients inhibited LFA-1 expression to the highest extent. The ability of HDL from renal patients to reduce oxidized LDL uptake was lower compared to control group. CONCLUSIONS: Decreased ability of HDL to suppress expression of VCAM-1 in endothelial cells and the uptake of oxidized LDL by macrophages can be one of the risk factors for atherosclerosis development in patients with renal failure.

Comparison of the effect of VOSO4, Na3VO4 and NaVO3 on proliferation, viability and morphology of H35-19 rat hepatoma cell line.

This study presents the investigation and comparison the influence of VOSO4 [V(IV)I, Na3VO4 and NaVO3 [V(V)] in the range of 0.5-20.0 microM on the rat hepatoma cell line H35-19. The cells were tested with crystal violet (N-hexamethylpararosaniline), and counted in a Bürker chamber to determine their rate of proliferation, while the survival level was established with neutral red and MTT [bromide 3-(4,5-dimetyltioazo-2)-2,5-diphenyl-tetrazole]. Parallel independent pathomorphological studies with electron microscopic examinations were done. We found progressive growth inhibition of rat hepatoma H35-19 cells within the range 0.5-20.0 concentrations of three vanadium salts. The most effective (and/or toxic) was NaVO3, whereas VOSO4 showed a relatively mild action. As compared with metavanadate or vanadyl sulphate and especially organic vanadium derivatives, previously studied by the same authors under similar experimental conditions, sodium orthovanadate showed an intermediate effect. Electron microscopic examinations confirmed these results. Vanadium salts in low concentration in medium (0.5 microM) were observed to normalize cell morphology. Higher doses of vanadium salts (greater than 2.5 or 5.0 microM) resulted in damaging cell organelles and the more cytotoxic the compounds seemed to be.

Does streptozotocin [STZ] exert an influence on rat hepatoma H35-19 cell line?

The authors investigated the effect of streptozotocin (STZ) in low--micromoles (up to 500 microM)--or higher --millimoles (1-10 mM)--concentrations in culture media of the H35-19 cell line. Up to 500 microM, STZ did not show any cytotoxic or cytostatic action in the investigated cells; on the contrary, it triggered an "improved growth" of these cells, as an antibiotic effect of the drug was observed. The concentration of 1-10 mmoles of STZ in the medium inhibited proliferation and viability of the studied cells. This action depended (proportionally) on drug concentration and time (up to 72 h) of experiment. Statistical analysis of the results obtained by four methods: staining with MTT, neutral red (NR) or crystal violet (CV) and Biirker chamber counting (BC), demonstrated no significant difference in STZ impact between 48 h and 72 h of incubation, according to the Benferoni post-hock test. The results obtained by MTT showed an extremely high statistical significance (p<0.001) of the effect of concentration on the results, with a non-significant interaction (p=0.2236) and general time effect (p=0.3600). An extremely significant (p<0.001) interaction of the effect of time and concentration was observed in the results obtained by neutral red method, whereas a significant effect of general time and concentration was also observed, but according to [17] it is difficult to explain. The results obtained by crystal violet staining showed a highly statistical significance (p<0.001) in time and concentration effect on the data, without a significant interaction between the above-mentioned factors (p>0.05). Cell counting in a Biirker chamber demonstrated a highly significant time and concentration effect on the results, but the interaction was mildly significant (0.01

LDL susceptibility to oxidation and HDL antioxidant capacity in patients with renal failure.

OBJECTIVES: This study examines the susceptibility to oxidation and the ability to stimulate reactive oxygen species of LDL from hemodialysis (HD) and continuous ambulatory peritoneal dialysis (CAPD) patients. It was also designed to evaluate the antioxidant activity of HDL from uremic patients. DESIGN AND METHODS: Lipoprotein properties were determined in 28 HD patients, 30 CAPD patients and 30 control subjects by spectrophotometric, chemiluminescence and electrophoresis methods. RESULTS: CAPD LDL were more resistant to oxidation than control LDL. HD and control LDL, in contrast to CAPD LDL, stimulated reactive oxygen species generation in granulocytes. The HDL ability to protect LDL against oxidation was impaired in renal patients. CONCLUSIONS: The risk of atherosclerosis development in patients with renal failure does not appear to be related to less resistance of LDL to oxidation, but rather to the decreased HDL antioxidant capacity.

Peritonitis-induced antitumor activity of peritoneal macrophages from uremic patients.

The macrophages belong to the effector cells of both nonspecific and specific immune response. These cells generally express little cytotoxicity unless activated. The present work was intended to determine if peritoneal macrophages collected from patients on Continuous Ambulatory Peritoneal Dialysis (CAPD) during episodes of peritonitis were active against human tumor cell lines without further in vitro stimulation. We also compared macrophage antitumor potential with effectiveness of drugs used in cancer therapy (taxol and suramin). Conditioned medium (CM) of macrophages collected during inflammation-free periods did not exhibit cytostatic and cytotoxic activity against both tumor (A549 and HTB44) and non-transformed (BEAS-2B and CRL2190) cells. Exposure of tumor cells to CM of macrophages harvested during peritonitis resulted in significant suppression of proliferation, impairment of viability and induction of apoptosis, in contrast to non-transformed cells, which remained unaffected. The efficacy of CM of inflammatory macrophages as an antitumor agent appeared to be comparable to cytostatic and cytotoxic potency of taxol and suramin or, in the case of HTB44 cells, even higher. The results obtained suggest that activated human macrophages might represent a useful tool for cancer immunotherapy.