Different Concentrations Of Cisplatin Research Articles

Cell cycle arrest and apoptosis in TP53 subtypes of bladder carcinoma cell lines treated with cisplatin and gemcitabine

Currently, the combination of cisplatin and gemcitabine is considered a standard chemotherapeutic protocol for bladder cancer. However, the mechanism by which these drugs act on tumor cells is not completely understood. The aim of the present study was to investigate the effects of these two antineoplastic drugs on the apoptotic index and cell cycle kinetics of urinary bladder transitional carcinoma cell lines with wild-type or mutant TP53 (RT4: wild type for TP53; 5637 and T24: mutated TP53). Cytotoxicity, cell survival assays, clonogenic survival assays and flow cytometric analyses for cell cycle kinetics and apoptosis detection were performed with three cell lines treated with different concentrations of cisplatin and gemcitabine. G(1) cell cycle arrest was observed in the three cell lines after treatment with gemcitabine and gemcitabine plus cisplatin. A significant increase in cell death was also detected in all cell lines treated with cisplatin or gemcitabine. Lower survival rates occurred with the combined drug protocol independent of TP53 status. TP53-wild type cells (RT4) were more sensitive to apoptosis than were mutated TP53 cells when treated with cisplatin or gemcitabine. Concurrent treatment with cisplatin and gemcitabine was more effective on transitional carcinoma cell lines than either drug alone; the drug combination led to a decreased cell survival that was independent of TP53 status. Therefore, the synergy between low concentrations of cisplatin and gemcitabine may have clinical relevance, as high concentrations of each individual drug are toxic to whole organisms.

Evaluation of the cytotoxicity and genotoxicity of curcumin in PC12 cells

Neurotoxicity induced by reactive oxygen species can appear as an adverse effect of chemotherapy treatment with platinum compounds, such as cisplatin. The use of this drug in clinical practice is limited due to its adverse effects, including nephrotoxicity, ototoxicity, neurotoxicity and genotoxicity. Functional foods or nutraceuticals have demonstrated potential neuroprotective activity in several experiments and models. This study aimed to investigate the possible cytotoxicity and genotoxicity/antigenotoxic effects of curcumin in PC12 cells exposed to cisplatin. Cell viability and genotoxicity/antigenotoxicity were evaluated by the MTT assay and micronucleus test, respectively. PC12 cells were treated with different concentrations of cisplatin and curcumin (0.5–128 μg/mL). Analysis of the results showed that high concentrations of curcumin were cytotoxic and increased micronuclei frequency compared to the control group. In the associated treatments, at all three concentrations evaluated, curcumin significantly reduced the total frequency of micronuclei induced by cisplatin. Determining the cytotoxic and genotoxic/antigenotoxic effects of this frequently used antioxidant in a neuronal model is important to assess possible hazards when combined with other chemical agents, including chemotherapy drugs used in cancer therapy.

Determination of volatile biomarkers for apoptosis and necrosis by solid-phase microextraction–gas chromatography/mass spectrometry: A pharmacometabolomic approach to cisplatin's cytotoxicity to human lung cancer cell lines

In order to find potential new biomarkers of cisplatin-induced apoptosis and necrosis, volatile organic compounds (VOCs) from cisplatin-treated human lung cancer cell lines were investigated. The biological system employed was human non-small cell lung carcinoma A549 cell lines. The cell lines were treated with two different concentrations of cisplatin, 100 μM and 400 μM, and apoptosis and necrosis were determined by flow cytometric analysis. For each drug concentration, the VOCs from the treated cell lines were extracted by solid-phase microextraction (SPME), and subsequently analyzed by gas chromatography/mass spectrometry (GC/MS). The compounds that change during cisplatin-induced apoptosis and necrosis of lung cancer cell lines can serve as new biomarkers. The pharmacometabolomic approach presented in this study, significantly, implicates a non-destructive, sample-thrifty and time-saving tool for finding new biomarkers for the assessment of drug-induced cell death pathways.

Cisplatin-induced hair cell loss in zebrafish ( Danio rerio) lateral line

We have used time-lapse imaging to study cisplatin-induced hair cell death in lateral line neuromasts of zebrafish larvae in vivo. We found that cisplatin-induced hair cell death occurred much more slowly than had been shown to occur in aminoglycoside-induced hair cell death. By prelabeling hair cells with FM1-43FX, and assessing hair cell damage, it was established that cisplatin causes hair cell loss in the lateral line in a dose-dependent fashion. The kinetics of hair cell loss during exposure to different concentrations of cisplatin was also assessed and it was found that the onset of hair cell loss correlated with the accumulated dose of cisplatin. These data demonstrate the feasibility and repeatability of cisplatin damage protocols in the zebrafish lateral line and set the stage for future evaluations of modulation of cisplatin-induced hair cell death.

Chemosensitizing tumor cells by targeting the Fanconi anemia pathway with an adenovirus overexpressing dominant-negative FANCA.

Fanconi anemia (FA) is a rare genetic disorder characterized by bone-marrow failure and cellular hypersensitivity to crosslinking agents, including cisplatin. Here, we studied the use of the FA pathway as a possible target for cancer gene therapy with the aim to sensitize tumor cells for cisplatin by interfering with the FA pathway. As proof-of-principle, FA and non-FA lymphoblast-derived tumors were grown subcutaneously in scid mice and treated with two different concentrations of cisplatin. As predicted, the antitumor response was considerably improved in FA tumors. An adenoviral vector encoding a dominant-negative form of FANCA, FANCA600DN, was generated that interfered with endogenous FANCA-FANCG interaction resulting in the disruption of the FA pathway as illustrated by disturbed FANCD2 monoubiquitination. A panel of cell lines, including non-small-cell lung cancer cells, could be sensitized approximately two- to three-fold for cisplatin after Ad.CMV.FANCA600DN infection that may increase upon enhanced infection efficiency. In conclusion, targeting the FA pathway may provide a novel strategy for the sensitization of solid tumors for cisplatin and, in addition, provides a tool for examining the role of the FA pathway in determining chemoresistance in different tumor types.

PTEN and p27 kip1 expression in prostate cancer — Analysis in radical prostatectomy specimens and needle biopsies

We investigated cisplatin-induced apoptosis and the effects on cell cycle-related proteins and cell cycle changes. Two human hepatoma cell lines, HepG2 (with wild-type p53) and Hep3B (with deleted p53), were treated with different concentrations of cisplatin. Cisplatin induced apoptosis in both cell lines as assessed by cell morphology, DNA fragmentation analysis,TdT-mediated dUTP nick end labeling assay and flow cytometry. HepG2 cells were more sensitive to cisplatin than Hep3B. Low-dose cisplatin induced a transient G1 arrest, S phase block and upregulation of p53 and p21WAF1/CIP1 expression in HepG2, but not in Hep3B cells. With cisplatin at a high dose, both cell lines underwent apoptosis that was accompanied by downregulation of p27KIP1 and Bcl-xL. In HepG2, upregulation of p53 and p21WAF1/CIP1 was observed before apoptosis occurred, suggesting that cisplatin-induced apoptosis in HepG2 might be p53-dependent. Expression of Fas was also increased following cisplatin treatment in HepG2. However, there was no induction of p53, p21WAF1/CIP1 and Fas observed in Hep3B cells. In conclusion, cisplatin induced apoptosis in hepatoma cells via both p53-dependent and -independent pathways.

Induction of apoptosis by cisplatin and its effect on cell cycle-related proteins and cell cycle changes in hepatoma cells

We investigated cisplatin-induced apoptosis and the effects on cell cycle-related proteins and cell cycle changes. Two human hepatoma cell lines, HepG2 (with wild-type p53) and Hep3B (with deleted p53), were treated with different concentrations of cisplatin. Cisplatin induced apoptosis in both cell lines as assessed by cell morphology, DNA fragmentation analysis,TdT-mediated dUTP nick end labeling assay and flow cytometry. HepG2 cells were more sensitive to cisplatin than Hep3B. Low-dose cisplatin induced a transient G 1 arrest, S phase block and upregulation of p53 and p21 WAF1/CIP1 expression in HepG2, but not in Hep3B cells. With cisplatin at a high dose, both cell lines underwent apoptosis that was accompanied by downregulation of p27 KIP1 and Bcl-x L. In HepG2, upregulation of p53 and p21 WAF1/CIP1 was observed before apoptosis occurred, suggesting that cisplatin-induced apoptosis in HepG2 might be p53-dependent. Expression of Fas was also increased following cisplatin treatment in HepG2. However, there was no induction of p53, p21 WAF1/CIP1 and Fas observed in Hep3B cells. In conclusion, cisplatin induced apoptosis in hepatoma cells via both p53-dependent and -independent pathways.

Apoptosis in the OC-k3 immortalized cell line treated with different agents.

The aim of this study is to outline the mechanisms leading cochlear cells to die. We utilized an immortalized cell line (OC-k3 cells) derived from the organ of Corti of transgenic mice in order to perform in-depth biochemical studies with no limitations on sample size and number. We probed these cells with cisplatin and gentamicin, two drugs which display in vivo undesired ototoxic side-effects. We investigated cell viability, reactive oxygen species (ROS) production and glutathione (GSH) levels and tested the effects of different concentrations of cisplatin and gentamicin from 0 to 48 h. Results show that cells undergo a dose- and treatment-time-dependent apoptosis characterized by nuclear fragmentation, integrity of the cell membrane and mitochondria, and absence of DNA endonuclease activity. During the early part of treatment, ROS production increases and intracellular GSH decreases, probably due to the activation of protein kinase C alpha. Use of antioxidants such as N-acetylcysteine, GSH and vitamin C rescues cells from apoptosis almost completely. Overall, these data indicate that ROS generation might play a central role in inducing inner ear cell apoptosis and may have an additive role in the ageing process.

Formation of nuclear Bax/p53 complexes is associated with chemotherapy induced apoptosis.

Mechanisms by which chemotherapeutic agents induce apoptosis are not completely understood. Current knowledge of the actual pharmacologic effects of chemotherapy and their biochemical mechanisms are better understood than the downstream events, which initiate the apoptotic cascade. The chemotherapeutic agent cisplatin causes DNA damage and can induce apoptosis in several types of human cancers. We found the formation of previously unreported nuclear complexes between the tumor suppressor protein p53 and the pro-apoptotic protein Bax, in human melanoma cell lines induced into apoptosis following cisplatin exposure. These detergent resistant complexes were detected: after wild type (wt) p53 and Bax increased in the nucleus; at the same time when active cytoplasmic apoptosis related protease, caspase 3/CPP32 appeared; and prior to the detection of apoptotic DNA fragmentation. Three channel fluorescence laser scanning confocal image microscopy revealed that the nuclear Bax/p53 complexes remained in the nucleus and localized proximal to DNA fragmentation sites as assayed by TUNEL after cisplatin exposure. Two human melanoma cell lines, expressing wt p53, were induced into apoptosis after cisplatin exposure, however they differed in the timing of this induction. In both cell lines the formation of nuclear Bax/p53 co-immunoprecipitable complexes correlated with the timing of the induction of apoptosis. The degree of apoptosis induced by different concentrations of cisplatin correlated with the amount of nuclear Bax/p53 complexes. The co-immunoprecipitation of Bax and p53 was found regardless of the antibodies tested and was specific since Bcl-xL/p53 complexes were not detected. Additionally, the human prostate cancer cell line, LNCaP, also formed nuclear Bax/p53 complexes only after apoptosis was induced by paclitaxel.

Carrier-mediated uptake of cisplatin by the OK renal epithelial cell line

The purpose of this study was to investigate whether transport of cis-diamminedichloroplatinum II (cisplatin) across renal epithelial cell line OK cells is mediated by the organic cation transport system. OK cell monolayers cultured on permeable membranes were incubated with 100 μM cisplatin on the apical or basolateral side, and the cellular accumulation and the transport of cisplatin across the monolayer were measured. The accumulation from the basolateral medium and the basolateral-to-apical transport of cisplatin were higher than the accumulation from the apical medium and the apical-to-basolateral transport, respectively. The cell monolayers were incubated with different concentrations of cisplatin (0.02∼3 mM) in the basolateral medium. The relationship between the cisplatin concentrations in the medium and in the cells revealed that cisplatin accumulation tended to be saturable. The basolateral-to-apical transport of cisplatin was increased when the pH of the apical medium was decreased, with a concomitant decrease in the accumulation of cisplatin. Coincubation of cisplatin with tetraethylammonium (TEA), a typical substrate for the organic cation transporter, significantly decreased the accumulation and transport of cisplatin from the basolateral medium. These results suggest that the uptake and basolateral-to-apical transport of cisplatin are mediated by not only simple diffusion but also by the organic cation transport system.

Induction of micronuclei and granular chromatin condensation in human skin fibroblasts influenced by cisplatin (cis-DDP) in vitro

Chromosomal damage was evaluated by quantification of micronuclei (MN) in two cell lines of human skin fibroblasts treated with different concentrations of cisplatin (ranging from 2 to 80 μmol/1) over the following time intervals: 2, 24 and 48 h. The formation of micronuclei was dependent upon the concentration of cis-DDP as well as on the duration of exposure. The dose-response curve for micronuclei in treated cells revealed a bell shape with the maximum at 12.5 μmol/1 cis-DDP. The results were compared with another toxicological phenomenon, granular condensation of nuclear chromatin. This change, which was closely related to apoptosis in the cells exposed to cis-DDP, occurred at 5 μmol/1. The degree of condensation of nuclear chromatin depended on the concentration of cis-DDP. In contrast to the observations for nuclei, the dose-effect relationship was linear up to the highest tested concentration (80 μmol/1). This change was primarily due to the duration of cis-DDP treatment. Our results showed the comparison between micronucleus formation and granular condensation of nuclear chromatin, as two morphological manifestations of cisplatin-induced DNA damage.

Effects of cisplatin on human thyrocytes in monolayer or follicle culture.

Cis-diamminedichloroplatinum (II) (cisplatin) is a widely used anticancer drug which induces many side-effects, but its action on the thyroid gland is still unknown. We have investigated the effects of this drug on human thyrocytes cultured in monolayers or in follicles and stimulated with 200 microU TSH/ml. After 72 h in culture, different concentrations of cisplatin (15, 30 and 75 microM) caused partial or total inhibition of cyclic AMP (cAMP), thyroglobulin (Tg) and tri-iodothyronine (T3) production, whereas thyroxine levels increased in the medium of thyrocytes cultured as follicles. Small doses of the drug did not affect thyrocyte production. Decreases in neutral-red uptake by thyroid cells and in intracellular lactate dehydrogenase, alpha-hydroxybutyryldehydrogenase and creatine phosphokinase activities were induced by 30 and 75 microM cisplatin. These data show that high concentrations of cisplatin had a cytotoxic effect on thyrocytes. Cisplatin also induced inhibition of the production of cAMP, Tg and T3.

Thermochemotherapy with cisplatin or carboplatin in the BT 4 rat glioma in vitro and in vivo

The effect of thermochemotherapy with cisplatin or carboplatin was compared in the BT 4-cell line. In vitro. BT 4C-cells were treated with different concentrations of cisplatin or carboplatin, with or without simultaneous hyperthermia. In vivo: Inbred BD IX rats with transplanted glioma-like BT 4A or glioblastoma-like BT 4An tumors on the hind leg were treated with cisplatin (4 mg/kg) or carboplatin (50 mg/kg), with or without local hyperthermia. In vitro the benefit of adding hyperthermia to chemotherapy was similar for cisplatin and carboplatin. For both cisplatin and carboplatin, the difference of treatment effect between thermochemotherapy and chemotherapy alone increased with higher drug concentrations. In vivo hyperthermia clearly enhanced the effect of carboplatin on BT 4A tumors. When treating BT 4An tumors, thermochemotherapy with cisplatin or carboplatin was equally effective. Both combinations were superior to treatment with hyperthermia alone. Local toxicity and weight loss following thermochemotherapy were comparable when substituting cisplatin with carboplatin.

Cytotoxicity of Cisplatin and Carboplatin Used Alone and in Combination with the Other Anticancer Drugs in the Mouse Embryo C3H10T½ Cell Line

Cytotoxicity of cisplatin and carboplatin was compared using each drug alone and in combination with either 5-fluorouracil (5-FU) or methotrexate (MTX) and with 5-FU, vincristine (VCR) and bleomycin (BLM) together in the mouse embryo fibroblast line C3H10T1/2. The survival curves, drawn on the basis of the colony-forming ability of cells, indicated that carboplatin was about 25 times less effective in cell killing compared to cisplatin. Dose-response curves obtained with different concentrations of cisplatin combined with 5-FU or MTX showed a dose modification factor (DMF) of 1.8. A DMF of 5.7 was obtained when cisplatin was combined with 5-FU + VCR + BLM. A combination of carboplatin with 5-FU and MTX resulted in DMFs of 3.4 and 2.4, respectively, while a DMF of 4.8 was obtained by combining the platinum analogue with 5-FU + VCR + BLM. Initial shoulders in the cell survival curves resulting from treatments with cisplatin, carboplatin and 5-FU used as single agents disappeared after combined drug treatments. While the data generally indicated a higher cytotoxic effectiveness of combined drug treatments, there was no great difference between the DMFs for cisplatin and carboplatin when used in different combination protocols. These results may reflect the functional similarity of the two platinum compounds and suggest that the replacement of one with the other may not significantly alter the cytotoxic effectiveness in combined treatments.