Low Concentrations Of Cisplatin Research Articles

Using Single‐Cell Amperometry To Reveal How Cisplatin Treatment Modulates the Release of Catecholamine Transmitters during Exocytosis

AbstractThe pretreatment of cultured pheochromocytoma (PC12) cells with cis‐diamminedichloroplatinum (cisplatin), an anti‐cancer drug, influences the exocytotic ability of the cells in a dose‐dependent manner. Low concentrations of cisplatin stimulate catecholamine release whereas high concentrations inhibit it. Single‐cell amperometry reflects that 2 μm cisplatin treatment increases the frequency of exocytotic events and reduces their duration, whereas 100 μm cisplatin treatment decreases the frequency of exocytotic events and increases their duration. Furthermore, the stability of the initial fusion pore that is formed in the lipid membrane during exocytosis is also regulated differentially by different cisplatin concentrations. This study thus suggests that cisplatin influences exocytosis by multiple mechanisms.

Using Single‐Cell Amperometry To Reveal How Cisplatin Treatment Modulates the Release of Catecholamine Transmitters during Exocytosis

The pretreatment of cultured pheochromocytoma (PC12) cells with cis-diamminedichloroplatinum (cisplatin), an anti-cancer drug, influences the exocytotic ability of the cells in a dose-dependent manner. Low concentrations of cisplatin stimulate catecholamine release whereas high concentrations inhibit it. Single-cell amperometry reflects that 2 μm cisplatin treatment increases the frequency of exocytotic events and reduces their duration, whereas 100 μm cisplatin treatment decreases the frequency of exocytotic events and increases their duration. Furthermore, the stability of the initial fusion pore that is formed in the lipid membrane during exocytosis is also regulated differentially by different cisplatin concentrations. This study thus suggests that cisplatin influences exocytosis by multiple mechanisms.

Abstract AS30: BRCA1-IRIS inactivation sensitizes ovarian tumors to cisplatin

Abstract Ovarian cancer is the first in mortalities among gynecological cancers in the USA, often due to late diagnosis and/or acquired platinum-resistant recurrences. This study investigates whether BRCA1-IRIS is a novel treatment target for ovarian cancers and their platinum-resistant recurrences. Here we show that more than half of ovarian cancer samples analyzed showed BRCA1-IRIS and survivin overexpression and lacked nuclear FOXO3a expression. Normal ovarian epithelial cells overexpressing BRCA1-IRIS formed metastasis in mice when injected in the peritoneal cavity, whereas aggressive ovarian cancer cell lines failed to form tumors or metastases in mice when BRCA1-IRIS was silenced in them. We show that BRCA1-IRIS activates two autocrine signaling loops, BDNF/TrkB and NRG1/ErbB2. These loops are involved in anoikis resistance and metastasis promotion. These loops operate in several ovarian cancer cell lines and BRCA1-IRIS silencing or inactivation using a novel inhibitory peptide renders both non-functional and promoted cell death. In a mouse xenograft model BRCA1-IRIS inactivation using this novel inhibitory peptide resulted in significant reduction in ovarian tumor growth. More importantly, this treatment sensitized ovarian tumors to low Cisplatin concentrations. Taken together, these data strongly suggest that BRCA1-IRIS and/or BDNF/TrkB and NRG1/ErbB2 could serve as rational therapeutic targets for advanced ovarian cancers. Citation Format: Bibbin T. Paul, Zannel Blanchard, Mildred Ridgway, Wael M. ElShamy. BRCA1-IRIS inactivation sensitizes ovarian tumors to cisplatin [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr AS30.

The role of mTOR during cisplatin treatment in an in vitro and ex vivo model of cervical cancer.

Cisplatin is used as a cytotoxic agent for the management of cervical cancer. However, the severity of the side-effects limits the use of this drug, particularly at high doses. Resistance to cisplatin is often attributed to a disruption in the normal apoptotic response via aberrant activation of pathways such as the mTOR pathway. Here we assess the role of mTOR and its effect on cell death sensitization and autophagy in response to a low concentration of cisplatin in cervical cancer cells. Additionally we measured the expression profile of mTOR in normal, low- and high-grade squamous intraepithelial (LSIL and HSIL) lesions and cancerous tissue. An in vitro model of cervical cancer was established using HeLa and CaSki cells. mTOR protein expression as well as autophagy-related proteins were evaluated through Western blotting. Inhibition of mTOR was achieved with the use of rapamycin and RNA silencing. A low concentration of cisplatin administered as a single agent induces autophagy, but not apoptosis. Cisplatin cytotoxicity was greatly enhanced in cancer cells when mTOR had been inhibited prior to cisplatin treatment which was likely due to autophagy being increased above cisplatin-induced levels, thereby inducing apoptosis. Cervical tissue samples revealed an increase in mTOR protein expression in LSIL and carcinoma tissue which suggests a change in autophagy control. Our data suggest that utilising a lower dose of cisplatin combined with mTOR inhibition is a viable treatment option and addresses the challenge of cisplatin dose-dependent toxicity, however future studies are required to confirm this in a clinical setting.

Lysine-specific demethylase 1 inhibitors protect cochlear spiral ganglion neurons against cisplatin-induced damage

Cisplatin is a widely used chemotherapeutic drug, but one of its side effects is ototoxicity. Epigenetic-related drugs, such as lysine-specific demethylase 1 (LSD1) inhibitors, have been reported to protect against cisplatin-induced hair cell loss by preventing demethylation of histone H3K4 (H3K4me2). However, the protective effect of LSD1 inhibitors in spiral ganglion neurons (SGNs) remains unclear. To investigate whether LSD1 inhibitors exert similar protective effects on SGNs, we treated mouse cochlear explant cultures with LSD1 inhibitors (2PCPA, S2101, or CBB1007) together with cisplatin. Low concentrations of cisplatin damaged SGNs much more than high concentrations, and blocking the demethylation of H3K4me2 with LSD1 inhibitors prevented the SGNs from injury. Reactive oxygen species are also involved in the injury process, and LSD1 inhibitors protected SGNs by increasing the expression level of the antioxidant gene Slc7a11 and decreasing the level of the pro-oxidant gene lactoperoxidase (Lpo). Our findings show that LSD1 inhibitors prevent cisplatin-induced SGN loss by regulating the demethylation of H3K4 and preventing increases of reactive oxygen species levels, which might provide a potential therapeutic strategy for cisplatin-induced hearing loss.

BRCA1-IRIS inactivation sensitizes ovarian tumors to cisplatin.

Ovarian cancer is the first in mortalities among gynecologic cancers in the United States, often due to late diagnosis and/or acquired platinum-resistant recurrences. This study investigates whether BRCA1-IRIS is a novel treatment target for ovarian cancers and in platinum-resistant recurrences. Here we show that more than half of the ovarian cancer samples analyzed showed BRCA1-IRIS and survivin overexpression and lacked nuclear FOXO3a expression. Normal ovarian epithelial cells overexpressing BRCA1-IRIS formed metastasis in mice when injected in the peritoneal cavity, whereas aggressive ovarian cancer cell lines failed to form tumors or metastases in mice when BRCA1-IRIS was silenced in them. We show that BRCA1-IRIS activates two autocrine signaling loops, brain-derived neurotrophic factor/tyrosine kinase B receptor (BDNF/TrkB) and neuregulin 1 (NRG1)/ErbB2. These loops are involved in anoikis resistance and metastasis promotion. These loops operate in several ovarian cancer cell lines, and BRCA1-IRIS silencing or inactivation using a novel inhibitory peptide renders both non-functional and promoted cell death. In a mouse xenograft model, BRCA1-IRIS inactivation using this novel inhibitory peptide resulted in significant reduction in ovarian tumor growth. More importantly, this treatment sensitized ovarian tumors to low cisplatin concentrations. Taken together, these data strongly suggest that BRCA1-IRIS and/or BDNF/TrkB and NRG1/ErbB2 could serve as rational therapeutic targets for advanced ovarian cancers.

Low-doses of cisplatin injure hippocampal synapses: A mechanism for ‘chemo’ brain?

Chemotherapy-related cognitive deficits are a major neurological problem, but the underlying mechanisms are unclear. The death of neural stem/precursor cell (NSC) by cisplatin has been reported as a potential cause, but this requires high doses of chemotherapeutic agents. Cisplatin is frequently used in modern oncology, and it achieves high concentrations in the patient's brain. Here we report that exposure to low concentrations of cisplatin (0.1μM) causes the loss of dendritic spines and synapses within 30min. Longer exposures injured dendritic branches and reduced dendritic complexity. At this low concentration, cisplatin did not affect NSC viability nor provoke apoptosis. However, higher cisplatin levels (1μM) led to the rapid loss of synapses and dendritic disintegration, and neuronal—but not NSC—apoptosis. In-vivo treatment with cisplatin at clinically relevant doses also caused a reduction of dendritic branches and decreased spine density in CA1 and CA3 hippocampal neurons. An acute increase in cell death was measured in the CA1 and CA3 neurons, as well as in the NSC population located in the subgranular zone of the dentate gyrus in the cisplatin treated animals. The density of dendritic spines is related to the degree of neuronal connectivity and function, and pathological changes in spine number or structure have significant consequences for brain function. Therefore, this synapse and dendritic damage might contribute to the cognitive impairment observed after cisplatin treatment.

Abstract P3-01-07: Metformin has an additive effect to cisplatin in triple negative breast cancer cells with high lactate dehydrogenase B (LDHB) expression

Abstract Background; Glycolysis is one of the major metabolic pathways in cancer cells. The conversion of glucose to lactate in the presence of oxygen is a critical aerobic pathway that allows cancer cells to proliferate rapidly and the amount of lactate produced is correlated with tumor aggressiveness. Moreover, the increase in glycolysis is partially mediated by hypoxia-inducible transcription factor (HIF-1) and a metabolic enzyme, lactate dehydrogenase B (LDHB). However, the mechanism behind this metabolic shift is not well understood. Metformin reduces the overall risk of cancer incidence by 31%, improves cancer-related mortality and enhances response to chemotherapy in diabetic patients pre-operatively. This study aimed to evaluate metabolism in triple negative breast cancer. Methods; Breast cancer cell lines (estrogen receptor positive MCF7 and triple negative MDA-MB 231 and MDA-MB468) were analysed for protein expression by western blotting and mRNA expression by qRT-PCR with or without cisplatin and metformin. To study the biological mechanism, we evaluated cell growth using proliferation assay in the presence of metformin and cisplatin. The contribution of LDHB to glycolysis was evaluated using MDA-MB-231 and MDA-MB468 cell lines. Results; We first examined the basal expression levels of LDHB and HIF1a in these cancer cell lines. LDHB expression was high in MDA-MB 231, and HIF1α was high in MDA-MB468. Both metformin and cisplatin treatment resulted in the down-regulation of LDHB and HIF1α in MDA-MB231, metformin led to downregulation of LDHB in MDA-MB 468, while MCF7 demonstrated no significant changes. Inhibition of cell proliferation was dose-dependent and significant at low concentrations of cisplatin and metformin in only MDA-MB468 but not in MCF7 and MDA-MB231. The combination of cisplatin in with metformin had an added inhibitory effect on cell proliferation in these MD-MB231 cells. Conclusion; These studies suggest that combination treatment of metformin and cisplatin have potent anticancer activity in LDHB highly expressed triple negative breast cancer cells. Identification of a new targeted therapeutic approach could contribute to the new therapy to the breast cancer patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-01-07.

Anvirzel™ in combination with cisplatin in breast, colon, lung, prostate, melanoma and pancreatic cancer cell lines

BackgroundPlatinum derivatives are used widely for the treatment of many cancers. However, the toxicity that is observed makes imperative the need for new drugs, or new combinations. Anvirzel™ is an extract which has been demonstrated with experimental data that displays anticancer activity. The aim of the present study is to determine whether the combination of Cisplatin and Anvirzel™ has a synergistic effect against different types of cancer.Materials and methodsTo measure the efficacy of treatment with Cisplatin and Anvirzel™, methyl-tetrazolium dye (MTT) chemosensitivity assays were used incorporating established human cancer cell lines. Measurements were performed in triplicates, three times, using different incubation times and different concentrations of the two formulations in combination or on their own. t-test was used for statistical analysis.ResultsIn the majority of the cell lines tested, lower concentrations of Anvirzel™ induced a synergistic effect when combined with low concentrations of Cisplatin after an incubation period of 48 to 72 h. The combination of Anvirzel™/Cisplatin showed anti-proliferative effects against a wide range of tumours.ConclusionThe results showed that the combination of Anvirzel™ and Cisplatin is more effective than monotherapy, even when administered at low concentrations; thus, undesirable toxic effects can be avoided.

Relationship of STAT3 activity with chemosensitivity to cisplatin in prostate cancer cell lines

Objective To investigate the relationship between STAT3 activity and sensitivity to cisplatin in prostate cancer cell lines.Methods STAT3 activity was examined by immunocytochemistry and Western blotting analysis in three prostate cancer cell lines: LNCaP,PC3 and DU145.Androgen-independent cell lines PC3 and DU145 were selected to examine the inhibitory effect of various concentrations of cisplatin(2 ng/ml,20 ng/ml,200 ng/ml,2 μg/ml and 20 μg/ml).STAT3 activity of DU145 cell line was re-examined by Western blotting analysis after treatment with low concentration of cisplatin.Results The STAT3 activity of androgen-independent cell lines PC3 and DU145 was higher than that of androgen-dependent cell line LNCaP.The sensitivity of DU145 cells to cisplatin was lower than that of PC3 cells with lower STAT3 activity.Treatment with low concentration of cisplatin for a long period caused STAT3 activation in DU145.Conclusion Our results suggest that STAT3 may play a role in regulating the sensitivity of prostate cancer cells to cisplatin.

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.

The interactions of cisplatin and DNA studied by atomic force microscopy

The interactions of the several linear DNA fragments and cisplatin at different concentrations and incubation time have been studied by atomic force microscopy. When equal volume of 2 mM cisplatin and 5 ng/μl 1134 bp DNA fragment were incubated for 30 min, 1 h, 2 h and 3 h, the observed DNA were gradually changed from the extended chains to the bending structures, the worm-like structures and flat particles. The formation of these structures can be attributed to the coordination and electrostatic interactions by binding of cisplatin on DNA. When longer DNA fragments such as pBR322/pstI DNA (4361 bp) and λ DNA (48502 bp) were used for interacting with cisplatin in the same condition, the observed DNA molecules tended to form many loop structures. The differences of the interactions between long and short DNA can be ascribed to the strong intramolecular interactions within the long DNA fragment. When the relative low concentration of cisplatin (100 μmol/l) was used in the experiments, the observed DNA tended to bend along its chains. Furthermore, the measured contour length and the calculated persistent length of cisplatin–DNA were gradually decreased with the incubated time. All of these AFM results revealed that the cisplatin–DNA interactions in vitro can be influenced by the concentration and the initial state of cisplatin, the length of DNA fragment and the reaction time.

Bcl‐XL and MCL‐1 constitute pertinent targets in ovarian carcinoma and their concomitant inhibition is sufficient to induce apoptosis

In ovarian carcinomas, recurrence and acquired chemoresistance are the first leading causes of therapeutic failure and are responsible for the poor overall survival rate. Cisplatin exposure of sensitive cells has been previously associated with a down-regulation of Bcl-X(L) expression and apoptosis, whereas recurrence was systematically observed when Bcl-X(L) expression was maintained. Bcl-X(L) down-regulation could thus constitute an interesting chemosensitizing strategy. We showed that a Bcl-X(L)targeted RNA interference strategy efficiently sensitized chemoresistant ovarian carcinoma cells to cisplatin, but some of them were still able to re-proliferate. Considering the possible cooperation between Bcl-X(L)and MCL-1, we investigated the possibility to avoid recurrence in vitro using a multi-targeted RNAi strategy directed against these two anti-apoptotic proteins. We showed that their concomitant inhibition lead to massive apoptosis in absence of cisplatin, this multi-targeted RNAi approach being much more efficient than conventional chemotherapy. We thus demonstrated that Bcl-X(L) and MCL-1 cooperate to constitute together a strong molecular "bolt", which elimination could be sufficient to allow chemoresistant ovarian carcinoma cells apoptosis. Moreover, we demonstrated that in presence of a low concentration of cisplatin, the concomitant down-regulation of Bcl-X(L) and MCL-1 allowed a complete annihilation of tumour cells population thus avoiding subsequent recurrence in vitro in cell lines highly refractory to any type of conventional chemotherapy. Therefore, Bcl-X(L) and MCL-1 targeted strategies could constitute an efficient therapeutic tool for the treatment of chemoresistant ovarian carcinoma, in association with conventional chemotherapy.

Specific Apoptosis Induction in Human Papillomavirus-Positive Cervical Carcinoma Cells by Photodynamic Antisense Regulation

Human papillomavirus type 18 (HPV18) is frequently detected in cervical cancer cells. The viral proteins E6 and E7 are expressed consistently and have oncogenic activities. The E7 protein binds to a tumor suppressor, the retinoblastoma gene product (pRB), however, leading to the stabilization of tumor suppressor, p53 protein. On the other hand, another viral product, E6, forms complexes with p53 and abrogates its function, resulting in tumor progression. These facts imply that the E6 oncogene is one of the ideal targets for directed gene therapy in HPV-positive cervical cancer. In this study, we tried photodynamic antisense regulation of the antiapoptotic E6 expression using a photocross-linking reagent, 4,5',8-trimethylpsoralen, conjugated oligo(nucleoside phosphorothioate) (Ps-S-Oligo). This photodynamic antisense strategy effectively elicited the apoptotic death of HPV18-positive cervical cancer cells through the selective repression of E6 mRNA and consequent stabilization of p53 protein. E7-mediated signals potentially activated the p53 function and mobilized the p53 pathway to deliver pro-apoptotic signals to the cancer cells, leading to the suppression of in vivo tumorigenesis. An extremely low concentration of cisplatin in addition to Ps-S-Oligos further up-regulated p53 activity, provoking massive apoptotic induction. These results suggest that the photodynamic antisense strategy has the great therapeutic potential in HPV-positive cervical cancers.

Acute apoptosis by cisplatin requires induction of reactive oxygen species but is not associated with damage to nuclear DNA

Cisplatin is a broad-spectrum anticancer drug that is also widely used in experimental studies on DNA damage-induced apoptosis. Induction of apoptosis within 24-48 hr requires cisplatin concentrations that are at least one order of magnitude higher than the IC(50). Here, we show that such high, apoptosis-inducing cisplatin concentrations induce cellular superoxide formation and that apoptosis is inhibited by superoxide scavengers. The same concentration limit and the requirement for superoxide are also true for induction of caspase activation in enucleated cells (cytoplasts), showing that cisplatin-induced apoptosis occurs independently of nuclear DNA damage. In contrast, lower cisplatin concentrations, which do not induce acute apoptosis, are sufficient for induction of DNA damage signaling. We propose that the antiproliferative effects of cisplatin at IC(50) doses involve premature senescence and secondary, nonstress-induced apoptosis. The higher doses currently used in in vitro studies lead to acute, stress-induced apoptosis that involves induction of superoxide but is largely DNA damage-independent.

Protective effect of cyclic AMP against cisplatin-induced nephrotoxicity

We reported earlier that reactive oxygen species are implicated in necrotic injury induced by a transient exposure of cultured renal tubular cells to a high concentration of cisplatin but not in apoptosis occurring after continuous exposure to a low concentration of cisplatin. We report here the protective effect of cyclic AMP against cisplatin-induced necrosis in cultured renal tubular cells as well as cisplatin-induced acute renal failure in rats. Several pharmacological agents that stimulate cyclic AMP signaling, including the nonhydrolyzable cyclic AMP analogue dibutyryl cyclic AMP, forskolin, 3-isobutyl-1-methylxanthine, and a prostacyclin analogue, beraprost, prevented cisplatin-induced cell injury in a protein kinase A-dependent manner. Cisplatin enhanced lipid peroxidation, decreased CuZn superoxide dismutase (SOD) while enhancing MnSOD activity, and increased cellular tumor necrosis factor-α (TNF-α) content. The elevation of TNF-α content and cell injury induced by cisplatin were attenuated by p38 mitogen-activated protein kinase (MAPK) inhibitors including SB203580 and PD169316. Indeed, cisplatin increased the number of phosphorylated p38 MAPK-like immunoreactive cells. These intracellular events were all reversed by antioxidants such as N-acetylcysteine (NAC) and glutathione or cyclic AMP analogues. The in vivo acute renal injury after cisplatin injection was associated with the elevation of renal TNF-α content. The cisplatin-induced renal injury and the increase in TNF-α content were reversed by NAC or beraprost. These findings suggest that cyclic AMP protects renal tubular cells against cisplatin-induced oxidative injury by obliterating reactive oxygen species and subsequent inhibition of TNF-α synthesis through blockade of p38 MAPK activation.

Cisplatin-Induced Apoptosis Is Enhanced by Hypoxia and by Inhibition of Mitochondria in Renal Collecting Duct Cells

Cisplatin is a widely used chemotherapeutic agent. Here we show that cisplatin induces apoptosis in renal collecting duct-derived cells (MDCK-C7 cells, resembling principal cells) in a dose-dependent manner. Additionally, we studied the role of mitochondria in this process by inhibition of the mitochondrial respiratory chain, the F1F(o)-ATP synthase or by uncoupling. The role of intra- and extracellular pH in apoptosis induction was investigated. Activation of caspase-3 and DNA ladder formation were used to monitor the apoptotic response. When cells were incubated with inhibitors of the mitochondrial respiratory chain or an inhibitor of the ATP-synthase, cisplatin-induced apoptosis was markedly enhanced. Mitochondrial blockade led to enhanced production of lactic acid. Also, anoxia potentiated the cisplatin-induced caspase-3 activation. Neither intra- nor extracellular pH had an influence on caspase-3 activation at low cisplatin concentrations. Acidic conditions (pH 6.8) potentiated the caspase-3 activation when high (100 microM) cisplatin concentrations were used. We demonstrate that intact mitochondria are important to prevent cisplatin-induced apoptosis in MDCK-C7 cells and that acidic conditions can aggravate the toxic effects of cisplatin.

Cisplatin triggers apoptotic or nonapoptotic cell death in Fanconi anemia lymphoblasts in a concentration-dependent manner

Cells derived from Fanconi anemia (FA) patients are hypersensitive for cross-linking agents, such as cisplatin, that are potent inducers of programmed cell death (PCD). Here, we studied cisplatin hypersensitivity in FA in relation to the mechanism of PCD in lymphoblastoid cells representing FA groups A and C. In FA cells, a low concentration of cisplatin caused chromatin condensation, phosphatidylserine (PS) externalization, and the expression of an 18-kDa variant of Bax, all indicators of apoptotic cell death, and the latter suggesting the involvement of a mitochondrial route. However, procaspases-3, -8, and -9, and PARP were not cleaved, although small increases in caspase activity could be detected. At a high concentration of cisplatin, both FA and corrected cells showed a robust cleavage of procaspases and PARP. DNA fragmentation was clearly visible under high cisplatin conditions and to some extent at a low concentration in FA-A cells, but not in the FA-C cell line regardless of the presence of functional FANCC, suggesting an unknown deficiency in these cells. We conclude that hypersensitivity in FA cells is associated with a mixture of necrotic and apoptotic features that is best described as apoptotic-like cell death, and that a defective FA pathway does not interfere with the proper activation of caspase-mediated cell death.

The influence of very low doses of Cisplatin on tumor cell proliferation in vitro and on some hematological and enzymatic parameters of healthy rats.

Healthy rats had been treated for 2 or 6 weeks with 1.0 mL of 10(-8) and 10(-16) mg/mL of cisplatin. After 2 weeks of treatment, a significant increase in leukocyte and erythrocyte count and also in hematocrit was observed. Among leukocytes the number of neutrophils and eosinophils significantly increased. Biochemical analyses indicated a decrease in the glycogen content in the liver and kidneys after 2 weeks of treatment with low doses of cisplatin but at the end of the experiment (8th week of experiment) the stores of glycogen increased significantly. Biochemical analyses concerning the activity of some enzymes in the liver revealed a significant increase of peroxidase and acid phosphatase as well as catalase activities after 2 weeks of treatment. However, catalase was induced by a very low concentration of cisplatin, 10(-16) mg/mL. After the cessation of cisplatin treatment the activity of enzymes returned to normal values.Human lung carcinoma cell line A(549) (ECACC No 86012804) was also studied after treatment with the same doses of cisplatin and inhibition of its growth was observed. The results of these experiments strongly indicated that low doses of cisplatin could be stimulating for healthy cells but cytostatic for tumor cells.Possible mechanisms involved in the biological activity of very low cisplatin concentrations are discussed.

Possible Involvement of Oxidative Stress in Cisplatin-Induced Apoptosis in LLC-PK1 Cells

Use of cisplatin, a chemotherapeutic agent, is associated with toxicity as a significant number of patients develop a decline in renal function. The mechanisms by which cisplatin produces renal injury are not well understood. It has been suggested that free radical-catalyzed lipid peroxidation can induce apoptosis or necrosis leading to renal injury. This study examined whether low concentrations of cisplatin induce apoptosis in LLC-PK1 cells and whether caspases 1, 2, 3, 8, and 9 are activated during this event. Our results show a dose- and time-dependent induction of apoptosis by micromolar concentrations of cisplatin. Expression of oncogenes c-myc and p53 was induced, and except for caspase 1, all the other caspases tested were activated. Z-VAD, the broad-spectrum inhibitor of caspases, prevented caspase activation and apoptosis, but not c-myc and p53 induction. On the other hand, N-acetylcysteine prevented cisplatin-induced apoptosis as well as c-myc induction but not p53 induction. The antioxidant trolox also prevented cisplatin-induced apoptosis. The results suggest that antioxidants and caspase inhibitors may alleviate cisplatin-associated nephrotoxicity.