Bleomycin induced sensitivity to TRAIL/Apo-2L-mediated apoptosis in human seminomatous testicular cancer cells is correlated with upregulation of death receptors.

The most common solid tumor is testicular cancer among young men. Bleomycin is an antitumor antibiotic used for the therapy of testicular cancer. TRAIL is a proapoptotic cytokine that qualified as an apoptosis inducer in cancer cells. Killing cancer cells selectively via apoptosis induction is an encouraging therapeutic strategy in clinical settings. Combination of TRAIL with chemotherapeutics has been reported to enhance TRAIL-mediated apoptosis of different kinds of cancer cell lines. The molecular ground for sensitization of tumour cells to TRAIL by chemotherapeutics might involve upregulation of TRAIL-R1 (TR/1, DR4) and/or TRAIL-R2 (TR/2, DR5) receptors or activation of proapoptotic proteins including caspases. The curative potential of TRAIL to eradicate cancer cells selectively in testicular cancer has not been studied before. In this study, we investigated apoptotic effects of bleomycin, TRAIL, and their combined application in NTera-2 and NCCIT testicular cancer cell lines. We measured caspase 3 levels as an apoptosis indicator, and TRAIL receptor expressions using flow cytometry. Both NTera-2 and NCCIT cells were fairly resistant to TRAIL's apoptotic effect. Incubation of bleomycin alone caused a significant increase in caspase 3 activity in NCCIT. Combined incubation with bleomycin and TRAIL lead to elevated caspase 3 activity in Ntera-2. Exposure to 72 h of bleomycin increased TR/1, TR/2, and TR/3 cell-surface expressions in NTera-2. Elevation in TR/1 cell-surface expression was evident only at 24 h of bleomycin application in NCCIT. It can be concluded that TRAIL death receptor expressions in particular are increased in testicular cancer cells via bleomycin treatment, and TRAIL-induced apoptosis is initiated.

Effects of curcumin on bleomycin-induced apoptosis in human malignant testicular germ cells

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. Bleomycin causes an increase in oxidative stress which has been shown to induce apoptosis in cancer cells. Curcumin (diferuloylmethane), an active component of the spice turmeric, has attracted interest because of its anti-inflammatory and chemopreventive activities. However, no study has been carried out so far to elucidate its interaction with bleomycin in testicular cancer cells. In this study, we investigated the effects of curcumin and bleomycin on apoptosis signalling pathways and compared the effects of bleomycin with H2O2 which directly produces reactive oxygen species. We measured apoptosis markers such as caspase-3, caspase-8, and caspase-9 activities and Bcl-2, Bax, and Cyt-c levels in NCCIT cells incubated with curcumin (5 ìM), bleomycin (120 ìg/ml), bleomycin + curcumin, H2O2 (35 ìM), and H2O2 + curcumin for 72 h. Curcumin, bleomycin, and H2O2 caused apoptosis indicated as increases in caspase-3, caspase-8, and caspase-9 activities and Bax and cytoplasmic Cyt-c levels and a decrease in Bcl-2 level. Concurrent use of curcumin with bleomycin decreased caspase activities and Bax and Cyt-c levels compared to their separate effects in NCCIT cells. Our findings suggest that concurrent use of curcumin during chemotherapy in testis cancer should be avoided due to the inhibitory effect of curcumin on bleomycin-induced apoptosis (AU)

N-acetyl-L-cysteine inhibits bleomycin induced apoptosis in malignant testicular germ cell tumors.

Antioxidants may prevent apoptosis of cancer cells via inhibiting reactive oxygen species (ROS). However, to date no study has been carried out to elucidate the effects of strong antioxidant N-acetylcysteine (NAC) on Bleomycin induced apoptosis in human testicular cancer (NTERA-2, NT2) cells. For this reason, we studied the effects of Bleomycin and NAC alone and in combination on apoptotic signaling pathways in NT2 cell line. We determined the cytotoxic effect of bleomycin on NT2 cells and measured apoptosis markers such as Caspase-3, -8, -9 activities and Bcl-2, Bax, Cyt-c, Annexin V-FTIC and PI levels in NT2 cells incubated with different agents for 24 h. Early apoptosis was determined using FACS assay. We found half of the lethal dose (LD50) of Bleomycin on NT2 cell viability as 400, 100, and 20 µg/ml after incubations for 24, 48, and 72 h, respectively. Incubation with bleomycin (LD50 ) and H2O2 for 24 h increased Caspase-3, -8, -9 activities, Cyt-c and Bax levels and decreased Bcl-2 levels. The concurrent incubation of NT2 cells with bleomycin/H2O2 and NAC (5 mM) for 24 h abolished bleomycin/H2O2-dependent increases in Caspase-3, -8, -9 activities, Bax and Cyt-c levels and bleomycin/H2O2-dependent decrease in Bcl-2 level. Our results indicate that bleomycin/H2O2 induce apoptosis in NT2 cells by activating mitochondrial pathway of apoptosis, while NAC diminishes bleomycin/H2O2 induced apoptosis. We conclude that NAC has antagonistic effects on Bleomycin-induced apoptosis in NT2 cells and causes resistance to apoptosis which is not a desired effect in eliminating cancer cells.

Effects of curcumin on bleomycin-induced apoptosis in human malignant testicular germ cells.

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. Bleomycin causes an increase in oxidative stress which has been shown to induce apoptosis in cancer cells. Curcumin (diferuloylmethane), an active component of the spice turmeric, has attracted interest because of its anti-inflammatory and chemopreventive activities. However, no study has been carried out so far to elucidate its interaction with bleomycin in testicular cancer cells. In this study, we investigated the effects of curcumin and bleomycin on apoptosis signalling pathways and compared the effects of bleomycin with H2O2 which directly produces reactive oxygen species. We measured apoptosis markers such as caspase-3, caspase-8, and caspase-9 activities and Bcl-2, Bax, and Cyt-c levels in NCCIT cells incubated with curcumin (5 µM), bleomycin (120 µg/ml), bleomycin + curcumin, H2O2 (35 µM), and H2O2 + curcumin for 72 h. Curcumin, bleomycin, and H2O2 caused apoptosis indicated as increases in caspase-3, caspase-8, and caspase-9 activities and Bax and cytoplasmic Cyt-c levels and a decrease in Bcl-2 level. Concurrent use of curcumin with bleomycin decreased caspase activities and Bax and Cyt-c levels compared to their separate effects in NCCIT cells. Our findings suggest that concurrent use of curcumin during chemotherapy in testis cancer should be avoided due to the inhibitory effect of curcumin on bleomycin-induced apoptosis.

Effects of N-acetylcystein on bleomycin-induced apoptosis in malignant testicular germ cell tumors

Oxidative stress has been shown to induce apoptosis in cancer cells. Therefore, one might suspect that antioxidants may inhibit reactive oxygen species (ROS) and prevent apoptosis of cancer cells. No study has been carried out so far to elucidate the effects of N-acetylcysteine (NAC) on bleomycin-induced apoptosis in human testicular cancer (NCCIT) cells. We investigated the molecular mechanisms of apoptosis induced by bleomycin and the effect of NAC in NCCIT cells. We compared the effects of bleomycin on apoptosis with H2O2 which directly produces ROS. Strong antioxidant NAC was evaluated alone and in combination with bleomycin or H2O2 in germ cell tumor-derived NCCIT cell line (embryonal carcinoma, being the nonseminomatous stem cell component). We determined the cytotoxic effect of bleomycin and H2O2 on NCCIT cells and measured apoptosis markers such as caspase-3, caspase-8, and caspase-9 activities and Bcl-2, Bax, and cytochrome c (Cyt-c) levels in NCCIT cells incubated with bleomycin, H2O2, and/or NAC. We found half of the lethal dose (LD50) of bleomycin on NCCIT cell viability as 120 ìg/ml after incubation for 72 h. Incubation with bleomycin (LD50) induced increases in caspase-3, caspase-8, and caspase-9 activities and Cyt-c and Bax protein levels and a decrease in Bcl-2 level. Co-incubation of NCCIT cells with bleomycin and 10 mM NAC abolished bleomycin-induced increases in caspase-3 and caspase-9 activities, Bax, and Cyt-c levels and bleomycin-induced decrease in Bcl-2 level. Our results indicate that bleomycin induces apoptosis in NICCT cells and that NAC diminishes bleomycin-induced apoptosis via inhibiting the mitochondrial pathway. We conclude that NAC has negative effects on bleomycin-induced apoptosis in NICCT cells and causes resistance to apoptosis, which is not a desirable effect in the fight against cancer (AU)

Effects of N-acetyl-L-cysteine on bleomycin induced oxidative stress in malignant testicular germ cell tumors.

Testicular cancer is a very common cancer in males aged 15-44 years. Bleomycin is used in chemotherapy regimens in the treatment of patients having testicular germ-cell tumor. Bleomycin generates oxygen radicals, induces oxidative cleavage of DNA strand and induces apoptosis in cancer cells. There is no study in the literature investigating effects of N-Acetyl-L-Cysteine (NAC) on bleomycin-induced oxidative stress in testicular germ cell tumors. For this reason, we studied effects of NAC on oxidative stress produced in wild-type NTera-2 and p53-mutant NCCIT testis cancer cells incubated with bleomycin and compared the results with H(2)O(2) which directly produces oxidative stress. We determined protein carbonyl content, thiobarbituric acid reactive substances (TBARS), glutathione (GSH), 8-isoprostane, lipid hydroperoxide levels and total antioxidant capacity in both testicular cancer cells. Bleomycin and H(2)O(2) significantly increased 8-isoprostane, TBARS, protein carbonyl and lipid hydroperoxide levels in NTera-2 and NCCIT cells. Bleomycin and H(2)O(2) significantly decreased antioxidant capacity and GSH levels in both cell lines. Co-incubation with NAC significantly decreased lipid hydroperoxide, 8-isoprostane, protein carbonyl content and TBARS levels increased by bleomycin and H(2)O(2). NAC enhanced GSH levels and antioxidant capacity in the NTera-2 and NCCIT cells. It can be concluded that NAC diminishes oxidative stress in human testicular cancer cells induced by bleomycin and H(2)O(2).

Effects of curcumin on bleomycin­induced oxidative stress in malignant testicular germ cell tumors.

Bleomycin is commonly used in the treatment of testicular cancer. Bleomycin generates oxygen radicals, induces the oxidative cleavage of DNA strands and induces cancer cell apoptosis. Curcumin (diferuloylmethane) is a potent antioxidant and chief component of the spice turmeric. No study investigating the effects of curcumin on intrinsic and bleomycin-induced oxidative stress in testicular germ cell tumors has been reported in the literature. For this reason, the present study aimed to examine the effects of curcumin on oxidative stress produced in wild-type NTera-2 and p53-mutant NCCIT testicular cancer cells incubated with bleomycin and the results were compared with cells treated with H2O2 which directly produces oxidative stress. The protein carbonyl content, thiobarbituric acid reactive substances (TBARS), glutathione (GSH), 8-isoprostane, lipid hydroperoxide (LPO) levels and total antioxidant capacity in the two testicular cancer cell lines were determined. Results showed that bleomycin and H2O2 significantly increased protein carbonyl, TBARS, 8-isoprostane and LPO levels in the NTera-2 and NCCIT cell lines. Bleomycin and H2O2 significantly decreased the antioxidant capacity and GSH levels in NTera-2 cells. Curcumin significantly decreased LPO, 8-isoprostane and protein carbonyl content, and TBARS levels increased in cells treated with bleomycin and H2O2. Curcumin enhanced GSH levels and the antioxidant capacity of NTera-2 cells. In conclusion, curcumin inhibits bleomycin and H2O2-induced oxidative stress in human testicular cancer cells.

Effects of N-acetylcystein on bleomycin-induced apoptosis in malignant testicular germ cell tumors.

Oxidative stress has been shown to induce apoptosis in cancer cells. Therefore, one might suspect that antioxidants may inhibit reactive oxygen species (ROS) and prevent apoptosis of cancer cells. No study has been carried out so far to elucidate the effects of N-acetylcysteine (NAC) on bleomycin-induced apoptosis in human testicular cancer (NCCIT) cells. We investigated the molecular mechanisms of apoptosis induced by bleomycin and the effect of NAC in NCCIT cells. We compared the effects of bleomycin on apoptosis with H(2)O(2) which directly produces ROS. Strong antioxidant NAC was evaluated alone and in combination with bleomycin or H(2)O(2) in germ cell tumor-derived NCCIT cell line (embryonal carcinoma, being the nonseminomatous stem cell component). We determined the cytotoxic effect of bleomycin and H(2)O(2) on NCCIT cells and measured apoptosis markers such as caspase-3, caspase-8, and caspase-9 activities and Bcl-2, Bax, and cytochrome c (Cyt-c) levels in NCCIT cells incubated with bleomycin, H(2)O(2), and/or NAC. We found half of the lethal dose (LD(50)) of bleomycin on NCCIT cell viability as 120 µg/ml after incubation for 72 h. Incubation with bleomycin (LD(50)) induced increases in caspase-3, caspase-8, and caspase-9 activities and Cyt-c and Bax protein levels and a decrease in Bcl-2 level. Co-incubation of NCCIT cells with bleomycin and 10 mM NAC abolished bleomycin-induced increases in caspase-3 and caspase-9 activities, Bax, and Cyt-c levels and bleomycin-induced decrease in Bcl-2 level. Our results indicate that bleomycin induces apoptosis in NICCT cells and that NAC diminishes bleomycin-induced apoptosis via inhibiting the mitochondrial pathway. We conclude that NAC has negative effects on bleomycin-induced apoptosis in NICCT cells and causes resistance to apoptosis, which is not a desirable effect in the fight against cancer.

Synergistic anticancer activity of curcumin and bleomycin: an in vitro study using human malignant testicular germ cells.

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 used for testicular cancer; however, side-effects are common. Bleomycin causes an increase in 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 a number of malignancies. However, to date no study has been carried out to elucidate its anticancer activity and interaction with bleomycin in testicular cancer cells. In this study, we investigated and compared the effects of curcumin, bleomycin and hydrogen peroxide (H2O2) on apoptotic signaling pathways. Curcumin (20 µM), bleomycin (400 µg/ml) and H2O2 (400 µM) incubation for 24 h decreased the viability of NTera-2 cells, and increased caspase-3, -8 and -9 activities, Bax and cytoplasmic cytochrome c levels and decreased Bcl-2 levels. The concurrent use of curcumin with bleomycin induced caspase-3, -8 and -9 activities to a greater extent in NTera-2 cells than the use of each drug alone. Our observations suggest that the effects of curcumin and bleomycin on apoptotic signaling pathways are synergistic. Therefore, we propose to use curcumin together with bleomycin to decrease its therapeutic dose and, therefore, its side-effects.

Concurrent use of antioxidants in cancer therapy: an update.

Some chemotherapeutic agents and all radiation therapy generates reactive oxygen species (ROS), which induce apoptosis in cancer cells. As ROS play a role in drug-induced apoptosis, one might suspect that antioxidants may inhibit ROS and prevent apoptosis of cancer cells. In order to find an answer to whether or not there is any interference between the concurrent use of antioxidants and chemotherapeutic agents, we have reviewed all of the recent available literature. Except for three specific interferences, considerable data exists demonstrating an increased effectiveness, as well as decreased side effects of chemotherapeutic agents when administered with antioxidants.

The effect of Topotecan on oxidative stress in MCF-7 human breast cancer cell line.

PURPOSE: Topotecan, a semisynthetic water-soluble derivative of camptothecin exerts its cytotoxic effect by inhibiting topoisomerase I and causes double-strand DNA breaks which inhibit DNA function and ultimately lead to cell death. In previous studies it was shown that camptothecin causes ROS formation. The aim of this study was to investigate if Topotecan like camptotecin causes oxidative stress in MCF-7 human breast cancer cell line. Determining the oxidant effect of Topotecan may elucidate a possible alternative mechanism for its cytotoxicity. EXPERIMENTAL DESIGN: MCF-7 cells were cultured and exposed to Topotecan for 24 h at 37 degrees C. The viability of the cells (% of control) was measured using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Lipid peroxidation (TBARS), protein oxidation (carbonyl content), sulfhydryl, glutathione (GSH) levels, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities were determined in MCF-7 cells with and without Topotecan incubation. RESULTS: We found the IC(50) concentration of Topotecan as 0.218 microM in MCF-7 cells. This concentration of Topotecan was used in the incubations of the cells. Our data indicated increased oxidative status, as revealed by increased lipid peroxidation and protein oxidation, and decreased GSH and sulfhydryl levels in MCF-7 cells exposed to Topotecan compared to control cells. In contrast, there was a slight increase in SOD and a significant increase in GPx and catalase activity in MCF-7 cells incubated with Topotecan compared to the control. CONCLUSIONS: These results support our hypothesis that Topotecan increases oxidative stress in MCF-7 cells.

The effect of quercetin on topotecan cytotoxicity in MCF-7 and MDA-MB 231 human breast cancer cells.

BACKGROUND: Topotecan, which is a Camptothecin derivative, shows a large spectrum in anti-tumor activity. Topotecan exerts its cytotoxic effect on tumor cells mainly by inhibition of topoisomerase I activity resulting in double-strand DNA breaks. In our study, we investigated the combined cytotoxic action of Topotecan and Quercetin in MCF-7 and MDA-MB 231 human breast cancer cells. To examine the possible relation between the cytotoxic activity of Topotecan and oxidative stress, we measured ROS and nitrite levels in both human breast cell lines. MATERIALS AND METHODS: MCF-7 and MDA-MB 231 cells were exposed to Topotecan, Quercetin, or a combination of both agents for 24 h at 37 degrees C. The viability of the cells was measured using the colorimetric MTT (3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. We determined reactive oxygen species and nitrite levels as indicators of oxidative stress in both cell lines with and without Topotecan and/or Quercetin incubations using fluorometric dichlorofluorescin diacetate (DCFH-DA) and diaminonaphtalene (DAN) assay. RESULTS: The IC(50) concentration of Topotecan was 100 ng/ml in MCF-7 cell line and 160 ng/ml in MDA-MB231 cell line. Treatment with Quercetin enhanced cytotoxicity of Topotecan as 1.4-fold in MCF-7 and 1.3-fold in MDA-MB-231 cell line. A significant increment on ROS and nitrite levels was found in MCF-7 and MDA-MB-231 cells following Topotecan incubation. CONCLUSIONS: Our results suggest that Topotecan has cytotoxic activity against both of the breast cancer cell lines in vitro. A combination with Quercetin increases efficacy of Topotecan in the treatment of breast cancers. Our results indicate that increased oxidative stress plays a role in the cytotoxic action of Topotecan.