Cytotoxic Effect Of Bleomycin Research Articles

Electroporation-based treatments in minimally invasive percutaneous, laparoscopy and endoscopy procedures for treatment of deep-seated tumors.

Electroporation (EP) techniques, used alone (Irreversible Electroporation, IRE) or in combination with anti-cancer drugs (Electrochemotherapy, ECT), have been shown to be effective in the treatment of several types of cancers. The efficacy of ECT and IRE is well demonstrated for the treatment of non-superficial tumor metastases, and it depends on the applied electrical parameters. Particularly, ECT is an effective local therapy that uses electroporation to enhance the cytotoxic effect of bleomycin or cisplatin injected intravenously or intratumorally. Pre-clinical investigations to test alternative anti-cancer drugs, explore new combinations of treatment modalities, and evaluate different sets of pulse protocols for effective tissue electroporation, are ongoing. Further ECT developments include the treatment of deep-seated tumors with percutaneous, laparoscopy, and endoscopy approaches, with the aim of establishing a less invasive approach. ECT is highly effective in the treatment of tumors of any histology, in minimizing the damage of critical normal tissue or organs, and in reducing pain and muscular contractions. This work describes the new technological advances in the field of ECT treatment for deep-seated tumors.

A Novel Method for Controlled Gene Expression via Combined Bleomycin and Plasmid DNA Electrotransfer.

Electrochemotherapy is an efficient method for the local treatment of cutaneous and subcutaneous metastases, but its efficacy as a systemic treatment remains low. The application of gene electrotransfer (GET) to transfer DNA coding for immune system modulating molecules could allow for a systemic effect, but its applications are limited because of possible side effects, e.g., immune system overactivation and autoimmune response. In this paper, we present the simultaneous electrotransfer of bleomycin and plasmid DNA as a method to increase the systemic effect of bleomycin-based electrochemotherapy. With appropriately selected concentrations of bleomycin and plasmid DNA, it is possible to achieve efficient cell transfection while killing cells via the cytotoxic effect of bleomycin at later time points. We also show the dynamics of both cell electrotransfection and cell death after the simultaneous electrotransfer of bleomycin and plasmid DNA. Therefore, this method could have applications in achieving the transient, cell death-controlled expression of immune system activating genes while retaining efficient bleomycin mediated cell killing.

Abstract 4910: Apoptotic effects of curcumin and bleomycin alone or in combination on malignant testicular germ cells

Abstract Aims: Testicular cancer is the most common cancer among young men of reproductive age. Bleomycin is a frequently used drug for the treatment of several malignancies and is part of the chemotherapy protocols in testicular cancer, but side effects are common. Bleomycin increases oxidative stress which has been shown to induce apoptosis in cancer cells. Curcumin (diferuloylmethane), an active component of the spice turmeric, has been demonstrated to induce apoptosis in several types of malignancies. However, no study has been carried out so far to elucidate its possible anticancer activity and interaction with Bleomycin in testicular cancer cells. Methods: In this study, we investigated the effects of Curcumin and Bleomycin alone or in combination on apoptosis-signalling-pathways. We determined the cytotoxic effect of Bleomycin and Curcumin on NTera-2 (NT2) cells using 3-(4,5) dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and measured apoptosis signalling pathway markers such as Caspase-3, Caspase-8, Caspase-9 activities and Bcl-2, Bax and Cyt-c levels in NT2 cells incubated with Bleomycin in the presence or absence of Curcumin. Results: Median lethal dose (LD50) of Bleomycin on NT2 cell viability as 400 μg/ml after an incubation of 24 h and LD50 of Curcumin as 20 μM. Treatment with Bleomycin (400 μg/ml) and Curcumin (20 μM) alone for 24 h caused 3.2-fold and 3-fold increase in caspase-3 activity, 3.4-fold and 2-fold increase in caspase-8 activity, 2.9-fold and 2.8 fold increase in caspase-9 activities respectively. These treatments alone also caused 2-fold increase in Bax and 2-fold increase in cytoplasmic Cyt-c levels while leading 2-fold decrease in Bcl-2 levels. Combination of Curcumin with Bleomycin induced Caspase-3,-8,-9 activities more than their separate effects in NT2 cells. Specifically combination of Curcumin (20 μM) with Bleomycin (400 μg/ml) induced 4.2-fold increase in Caspase-3, 4.3-fold increase in caspase-8 activity, 4-fold increase in caspase-9 activity, 3-fold increase in cytoplasmic Cyt-c levels and 3-fold decrease in Bcl-2 levels.Conclusion: Bleomycin has proven to be an essential component of the cisplatin-based chemotherapy regimens, used in the treatment of testicular cancer. However, the use of Bleomycin is limited because of Bleomycin-induced pneumonitis, a complication occurring in approximately 10% of patients treated with Bleomycin and fatal in approximately 10% of the cases. Our observations suggest that the effects of Bleomycin on apoptosis-signalling-pathways were improved by Curcumin. Co-administration of Bleomycin with Curcumin could be a new strategy to use less dosage of Bleomycin and prevent patients from Bleomycin-induced side effects. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4910. doi:1538-7445.AM2012-4910

Bleomycin and the skin

Bleomycin is frequently used as a chemotherapeutic agent to treat various kinds of malignancy. However, the cytotoxic effects of bleomycin cause a number of adverse responses, in particular in the lung and the skin. Bleomycin is used by dermatologists as a treatment for various skin cancers, recalcitrant warts, keloid and hypertrophic scars. This article discusses the use of bleomycin for various skin disorders, as well as the risk factors and cutaneous side-effects resulting from its use.

703. Combination Therapy with Bleomycin and IL-12 Gene Induces Regression of Breast Cancer

Electrochemotherapy has been used extensively in clinical studies for treating melanoma and squamous cell carcinoma, demonstrating that a low dose of drug such as bleomycin (BLM) has anti-tumor activity when it is delivered by electroporation. However, many tumors re-grow from the escaped tumor cells causing metastasis. Electrochemo-gene immunotherapy combines the local cytotoxic effect of BLM with the induction of cell-mediated immune response by cytokines. In this study, we used the IL-12 gene and BLM combination therapy delivered intratumorally by electroporation for treating breast cancer in Balb/c mice.

Histological Evaluation of the Effects of Electropermeabilization after Administration of Bleomycin on Bladder Cancer in the Rat

Objective: The aim of this study was to examine the usefulness of an electropermeabilization method in the administration of a chemotherapeutic agent in bladder cancer in the rat. Materials and Methods: N-Butyl-N-(4-hydroxybutyl)-nitrosamine was used to induce bladder tumors in Wistar rats. Rats in group 1 were given a sham treatment. Rats in group 2 received an injection of bleomycin (BLM). Rats in group 3 received a series of eight 100-µs square-wave pulses at an electric field intensity of 1,000 V/cm (electropermeabilization) applied to the bladder. Rats in group 4 were treated with square-wave pulses after the injection of BLM. Nine days after the various treatments, all the bladders were removed and examined histopathologically. Results: Histopathological examination revealed severe damage to tumors in group 4 exclusively. Conclusion: The cytotoxic effect of BLM on bladder cancer tissue was enhanced by electropermeabilization.

Type I procollagen production and cell proliferation is mediated by transforming growth factor-beta in a model of hepatic fibrosis.

Fibrosis is a significant component of advanced chronic inflammatory liver diseases and is caused by the accumulation of extracellular matrix, including type I procollagen. The mechanism by which fibrosis develops in liver tissue remains unknown. We tested the effects of transforming growth factor beta 1 (TGF-beta), a cytokine that alters cell differentiation and proliferation, and bleomycin, a cytotoxic glycopeptide antibiotic, on cultured isolated rat hepatocytes. TGF-beta (1 ng/ml) inhibited radiolabeled thymidine incorporation 39% at 24 h and 69% at 48 h. Inhibition of hepatocyte proliferation was dose dependent. Bleomycin (1 microgram/ml) significantly inhibited radiolabeled thymidine incorporation at 48 h (44%). Neutralizing antibody to thymidine incorporation at 48 h (44%). Neutralizing antibody to TGF-beta (TGF-beta-Ab) attenuated the inhibition of proliferation by TGF-beta and bleomycin in a concentration-dependent manner. The addition of either TGF-beta or bleomycin increased immunostaining of type I procollagen in hepatocytes. The addition of TGF-beta-Ab alone increased cell proliferation, suggesting that neutralization of endogenous TGF-beta may attenuate the inhibition of hepatocyte proliferation. These data suggest that the hepatocyte contains type I procollagen and, under some conditions, produces TGF-beta. We propose that procollagen production in rat hepatocytes is induced by TGF-beta and may be related to endogenous production of this cytokine in response to cell injury. The cytotoxic effect of bleomycin is mediated by TGF-beta and inhibition of TGF-beta and bleomycin with TGF-beta-Ab attenuates the additive effects of those compounds on isolated rat hepatocytes. These data provide a model of collagen expression in isolated rat hepatocytes.

Effect of hypoxia on the frequency of bleomycin-induced micronuclei.

Normal G(0) pig lymphocytes were maintained in a hypoxic or aerobic environment for one hour and then concurrently exposed to bleomycin at varying concentrations for a second hour. The cytochalasin-block micronucleus assay was used to determine cytotoxicity. Exposure to transient hypoxia significantly decreased the frequency of bleomycin-induced micronuclei per cell as compared to that observed in oxygenated cells. Furthermore, the frequency of micronuclei per hypoxic cell did not correspond to any increase in bleomycin dose as observed in oxygenated cells. These data demonstrate the significance of transient hypoxia in the resistance of G(0) cells to the cytotoxic effects of bleomycin and may explain the observed selectivity of tumor tissue response to bleomycin.

Fluorodeoxyglucose cell incorporation as an index of cell proliferation: evaluation of accuracy in cell culture.

The use of fluorodeoxyglucose (FDG) and positron emission tomography (PET) is recognized as an accurate tool for the specific diagnosis and staging of cancer. It has also been proposed for the monitoring of anticancer therapy. FDG cell incorporation reflects glycolytic activity whereas inhibition of cell proliferation corresponds to an efficient cancer treatment. The relationship between FDG incorporation and cell proliferation has yet to be demonstrated. Therefore, we aimed to correlate the effects of the toxic agents bleomycin and unlabelled meta-iodobenzylguanidine (mIBG) on cellular metabolism and proliferation. We determined the in vitro metabolic and cytotoxic effects of bleomycin and mIBG by measuring the incorporation of fluorine-18 FDG (%UFDG) and hydrogen-3 thymidine (%UTHY) in cells of the human premonocytic line U937 in the presence of increasing concentrations of these agents. Proliferation rate of these cells was studied by means of limiting dilution analysis. %UTHY appeared more sensitive to bleomycin or mIBG-mediated cell injury than %UFDG. After 1 h of exposure to 0.5 microM bleomycin, %UTHY was significantly reduced to 62.0% +/- 10.4% of control value whereas %UFDG remained unchanged (91.6% +/- 5.3%). Similar results were obtained after 1 h of exposure to increasing concentrations of mIBG (1 microM to 1 mM). After 20 h of exposure to bleomycin, %UTHY and %UFDG were significantly reduced as a function of concentration. After 20 h of exposure to mIBG, a transient increase in %UFDG up to 149.3% +/- 11.2% with 50 microM mIBG was further followed by a reduction to 20.1% +/- 6.7% with 0.5 mM (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction between the kinetics of thermotolerance and effect of cis-diamminedichloroplatinum(II) or bleomycin given at 37 or 43 degrees C.

The interaction between the cytotoxic effect of bleomycin (BLM) or cis-diamminedichloroplatinum(II) (cis-DDP) and the kinetics of thermotolerance was studied in cultured Chinese hamster ovary (CHO) cells. Pre-heated cells were treated with cis-DDP or BLM at 37 or 43 degrees C for various times after heating. Pre-heating enhanced cis-DDP cytotoxicity given immediately after heating, but this enhancement decreased within 24 h to an additive level. Cell survival following the initial heating and the second treatment of 'cis-DDP at 43 degrees C was minimal when cis-DDP at 43 degrees C was given immediately after the initial heating, but became higher with increasing treatment interval and reached 'less than additive' level when the treatment interval was extended to more than 24 h. This alteration in cell survival appeared to follow the kinetics of thermotolerance. The interaction between BLM treatment and the kinetics of thermotolerance was similar to that of cis-DDP. However, pre-heating enhanced BLM cytotoxicity much less extensively than cis-DDP cytotoxicity. These results indicate that: (a) pre-heating of cells enhanced drug-toxicity when the drug was given shortly after heating, but the magnitude of this enhancement depended on the drug; (b) pre-heating did not influence the cytotoxicity of drugs given at 37 degrees C; and (c) pre-heating decreased the magnitude of thermal sensitization of drug cytotoxicity. The magnitude of the decrease in thermal sensitization appeared to be parallel to the kinetics of thermotolerance. In this study it was also demonstrated that pre-treatment of CHO cells by cis-DDP or BLM did not alter sensitivity to subsequent drug treatment, hyperthermia or thermochemotherapy.

Comparative flow cytometric analysis of bleomycin toxicity on normal and tumour sheep cell kinetics in vitro

The cytotoxic effects of bleomycin on cell proliferation, morphology and cycle progression were compared in a sheep tumour cell line and in normal sheep sinus cells. They were concentration and time dependent, irreversible, and were accompanied by increased cell diameter and cytosolic synthetic activity. Cell accumulation in the G2M phase, without much change in the cell cycle progression through the S phase, was observed using flow cytometry. Although the tumour cells showed a greater sensitivity to low bleomycin concentrations compared to the normal cells, both cell types exhibited equal sensitivity to higher drug concentrations. Provided that tumour cells are also preferentially affected by bleomycin in vivo, these findings raise the possibility that bleomycin cytotoxicity can be reduced or avoided in normal tissue.

Comparison of bleomycin and peplomycin toxicity on clonogenic tumor cells from various human tumors

The cytotoxic effect of bleomycin and peplomycin was compared using a methylcellulose monolayer assay for the cultivation of human tumor cells. In 3 out of 4 samples from human malignant melanomas peplomycin proved to be more cytotoxic than bleomycin. Peplomycin was more cytotoxic than bleomycin in 1 of 5 myosarcoma samples, whereas 2 samples from squamous cell carcinomas of the lung showed identical dose response curves. In 1 carcinoma of the gall bladder peplomycin was more toxic than bleomycin.

The cytotoxicity of Bleomycin A 2 on adult rat liver epithelial cell lines at different stages of spontaneous cell transformation

Cytotoxic effects of Bleomycin A 2 on adult rat liver epithelial cell lines were evaluated by three methods: the incorporation rate of ( 3H)thymidine for DNA biosynthesis, the incorporation rate of L-( 3H)leucine for protein biosynthesis and Giemsa dye staining of surviving cells. Chromosome investigations at successive passages of cell lines have shown that spontaneous chromosome abnormalities in distribution and structure after 15–20 passages, i.e. 50 to 60 cell generations, were the earliest morphological sign of spontaneous transformation. In this study a highly spontaneously transformed cell line was very sensitive to the drug. Another cell line at the beginning of spontaneous transformation appeared to be insensitive although on further passage it became more sensitive. The use of microtitration plates made it easier for us to undertake a comparative study of the different parameters. Following Bleomycin A 2 exposure, Giemsa staining gave the best evaluation of cell killing whereas thymidine incorporation allowed the estimation of cell recovery. The antineoplastic effect of Bleomycin A 2 can probably be used to evaluate the malignant potential of different rat liver epithelial cell lines.

Alteration of bleomycin cytotoxicity by glutathione depletion or elevation

In part, some of the cytotoxicity of bleomycin may be lessened or enhanced by modulation of glutathione (GSH) concentrations. Enhancement of bleomycin cytotoxicity was observed when GSH levels were low and protection was observed when GSH levels were elevated. Since H202 is one of the reactive species produced by bleomycin catalyzed oxygen activation, we studied the effects of H202 exposure after GSH depletion. H202, like bleomycin, shows enhanced cytotoxicity in GSH depleted cells. It has been proposed that bleomycin cytotoxicity requires reducing equivalents from non-protein bound thiols (such as GSH) to activate the bleomycin-metal complex, which in turn reacts with oxygen to generate free radicals and peroxides. Our data suggest that either GSH is not required to cycle reducing equivalents to the oxidized bleomycin-metal complex, or the low levels of depleted GSH attained (less than 5% of control) were still sufficient to effect reduction. Further, our data shows that GSH in fact provides a means of protection and detoxification from the cytotoxic effects of bleomycin. Our data suggest that caution should be exercised clinically when one uses drugs that modulate GSH because there may be either enhancement of normal tissue toxicity or decreases in tumor targeted cytotoxicity resulting from bleomycin treatment.

Enhancement of antitumor activity of bleomycin by benzamide in vitro and in vivo.

The cytotoxic effects of bleomycin on HeLa cells in culture were enhanced by incubation of the cells with benzamide, a potent inhibitor of poly(ADP-ribose) polymerase, at concentrations at which benzamide alone did not show any cytotoxicity. Benzamide plus bleomycin display enhanced therapeutic effects against Ehrlich ascites tumor cells in vivo. On daily treatment with various doses of bleomycin plus benzamide for 10 days, mice with Ehrlich ascites tumors survived longer than mice on treatment with bleomycin alone.

Differential survival of cell subpopulations in spheroids after treatment with bleomycin

The cytotoxic effect of bleomycin (BLM) on Chinese hamster V79-cells grown as spheroids is compared to that on monolayer cultures in exponential vs. plateau phase of growth. Cells treated as intact spheroids with BLM (50 micrograms/ml for 1 h) are more sensitive than monolayer cultures. Cell subpopulations derived from different zones of the spheroids survive BLM-treatment to different degrees: When treated as single cells in suspension, the formerly "outer" cells are less affected than the "inner" cells. Within intact spheroids, however, progressive cellular resistance with increasing distance from the outer rim is found.