Mujgan Timur

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.

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 bleomycin‑induced 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.

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. J. Cell. Biochem. 114: 1685–1694, 2013.

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 TRAILs 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.