Protection by Salvia extracts against oxidative and alkylation damage to DNA in human HCT15 and CO115 cells.

DNA damage induced by oxidative and alkylating agents contributes to carcinogenesis, leading to possible mutations if replication proceeds without proper repair. However, some alkylating agents are used in cancer therapy due to their ability to induce DNA damage and subsequently apoptosis of tumor cells. In this study, the genotoxic effects of oxidative hydrogen peroxide (H2O2) and alkylating agents N-methyl-N-nitrosourea (MNU) and 1,3-bis-(2-chloroethyl)-1-nitosourea (BCNU) agents were examined in two colon cell lines (HCT15 and CO115). DNA damage was assessed by the comet assay with and without lesion-specific repair enzymes. Genotoxic agents were used for induction of DNA damage in both cell lines. Protective effects of extracts of three Salvia species, Salvia officinalis (SO), Salvia fruticosa (SF), and Salvia lavandulifolia (SL), against DNA damage induced by oxidative and alkylating agents were also determined. SO and SF protected against oxidative DNA damage in HCT15 cells. SO and SL decreased DNA damage induced by MNU in CO115 cells. In addition to chemopreventive effects of sage plant extracts, it was also important to know whether these plant extracts may interfere with alkylating agents such as BCNU used in cancer therapy, decreasing their efficacy. Our results showed that sage extracts tested and rosmarinic acid (RA), the main constituent, protected CO115 cells from DNA damage induced by BCNU. In HCT15 cells, only SF induced a reduction in BCNU-induced DNA damage. Sage water extracts and RA did not markedly change DNA repair protein expression in either cell line. Data showed that sage tea protected colon cells against oxidative and alkylating DNA damage and may also interfere with efficacy of alkylating agents used in cancer therapy.

The chemopreventive effect of dimethylthiourea against carmustine-induced myelotoxicity in rats.

The possible chemopreventive role of dimethylthiourea (DMTU) against carmustine (1,3-bis(2-chloroethyl)-1-nitrosourea, BCNU)-induced myelotoxicity was assessed through evaluation of apoptosis, lipid peroxidation, glutathione (GSH) content and some antioxidant enzymes activities in bone marrow cells of rats. Thirty-six rats were randomly classified into four groups. The first group was injected i.p. with ethanol and served as a control. The second group was treated with BCNU. The third group was given DMTU, while the fourth group was co-administered with DMTU prior to BCNU administration. BCNU treatment in a single dose of 30 mg/kg significantly decreased the normal counts of RBCs, WBCs and platelets as well as hemoglobin level. In addition, BCNU exhibited marked apoptotic effect associated with significant alterations in the oxidative cascade parameters. Treatment of animals with DMTU in a single dose of 500 mg/kg 1h before BCNU injection, followed by 125 mg/kg twice daily for 5 consecutive days significantly mitigated the induced changes in the hematological parameters. The induced alterations in the oxidant and antioxidant parameters as well as apoptosis were also improved. Conclusively, DMTU treatment exhibited marked chemopreventive effect against BCNU-induced myelotoxicity; an effect which may be partially attributed to its inherently antioxidant potential.

The epidermal growth factor receptor pathway mediates resistance to sequential administration of radiation and chemotherapy in primary human glioblastoma cells in a RAS-dependent manner.

Resistance to conventional adjuvant therapies (i.e., chemotherapy and radiation) has been well documented in malignant gliomas. Unlike many other tumor types, combined modality therapy involving radiation and chemotherapy has failed to appreciably enhance outcome for glioblastoma patients compared with radiation alone. In vitro, we have observed an actual antagonistic effect between sequential administration of radiation and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) chemotherapy in three primary human glioblastoma cell lines (referred as the GBME3-5 cell lines), which also happen to demonstrate strong expression of the epidermal growth factor receptor (EGFR). Upon inhibition of EGFR with the EGFR tyrosine kinase inhibitor, AG1478, it was found that this cross-resistance between sequential administration of radiation and BCNU was abrogated. To dissect which of these pathways may be responsible for the observed antagonism, known EGFR-regulated downstream signaling pathways including RAS, phosphatidylinositol 3-kinase (PI3-K), mitogen-activated protein kinase (p44/p42), and protein kinase C were inactivated with both pharmacological inhibitors and transient transfection experiments with dominant-negative and constitutively active constructs in the presence of exogenous EGF stimulation. It was found that BCNU inhibited radiation-induced apoptosis through EGFR-mediated activation of PI3-K/AKT via RAS. On the other hand, radiation was found to inhibit BCNU-induced apoptosis through EGFR-mediated activation of both PI3-K and mitogen-activated protein kinase (p44/p42) pathways, also via RAS. Inhibition of either EGFR or RAS activity appears to not only abrogate the observed antagonism between sequentially administered radiation and chemotherapy but actually results in a greater enhancement of apoptosis in the setting of combined modality therapy than when administered with either radiation or chemotherapy as single agents. Therefore, these findings suggest that strategies to inactivate EGFR or RAS signaling may be critical to improving not only the efficacy of single-agent therapy but also of combined modality therapy in gliomas.

The antitumor drug, 1,3-bis(2-chloroethyl)-1-nitroso-urea, inactivates human nicotinamide mononucleotide adenylyltransferase.

Nicotinamide mononucleotide (NMN) adenylyltransferase (EC 2.7.7.1) from human placenta is rapidly inactivated by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). A similar inactivation is observed with other C- and N-nitroso compounds. The inactivation by BCNU is dependent on incubation time, temperature and BCNU concentration. Protective reagents for -SH groups, dithiothreitol and beta-mercaptoethanol, and the substrate NMN are very effective in protecting NMN adenylyltransferase from BCNU inactivation and in preserving its catalytic properties, while ATP is less efficient. Incubation of BCNU-inactivated and dialysed NMN adenylyltransferase with dithiothreitol results in a partial recovery of the enzymatic activity.

Reovirus type 3 chemoimmunotherapy of murine lymphoma is abrogated by cyclosporine.

We have previously shown that the combined modalities of reovirus type 3 and the chemotherapeutic agent 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) synergize to cause the rejection of various murine tumors. Resistance of surviving animals to challenge with homologous but not heterologous tumor suggested that tumor was eliminated through an immune-mediated mechanism. In this study, we showed that mice undergoing therapy-mediated rejection of tumor were able to reject subsequent weekly challenges with EL-4 but not L1210 tumor cells. The mechanism underlying this therapy was investigated using cyclosporine (CS) to suppress immune responsiveness. A dose-related inhibition of therapy was observed with total inhibition occurring at 30 mg/kg/day. Delayed administration of CS at day 14 or later after tumor administration resulted in little or no inhibitory effect. The resistance of cured mice to EL-4 tumor challenge was not affected by CS, which is consistent with the reduced ability of CS to affect secondary immune responses. In addition, CS did not alter natural killer cell activity in mice receiving the BCNU/reovirus therapy. These results suggest that there is an obligatory immune response produced by the BCNU/reovirus therapy which arises early after the administration of the therapy.

Increased survival and multilineage hematopoietic protection from delayed and severe myelosuppressive effects of a nitrosourea with recombinant interleukin-11.

The chloroethylnitrosoureas, such as 1,3-bis(2-chloroethyl)-1-nitrosourea, are alkylating agents which are thought to exert antitumor activity by initiating lethal DNA interstrand cross-links. Although nitrosoureas are among the most active agents against childhood and adult gliomas, the utility of this class of agents has been limited by severe and cumulative myelosuppression, which can be fatal. Nitrosourea-induced myelosuppression in humans is delayed and may continue after withdrawal of the agent. We have developed a murine model which mimics the delayed and cumulative myelosuppression seen in humans receiving nitrosoureas. In this model, we demonstrate that interleukin-11, a stromal-derived hematopoietic growth factor with pleiotropic effects in a number of preclinical ablation models, markedly diminishes nitrosourea-induced pancytopenia and leads to a significant reduction in chemotherapy-related mortality. These data suggest that interleukin-11 could allow significant dose intensification in the treatment of tumors which are nitrosourea sensitive.

Absence of cross-resistance between two alkylating agents: BCNU vs bifunctional galactitol.

Dianhydrogalactitol (DAG) increased the life span of both BCNU-sensitive and -resistant L1210 tumor-bearing mice. However, the BCNU-resistant strain showed slightly lower sensitivity against DAG, which could be overcome by an increase in drug dose of ca. 20%. The somewhat lower sensitivity was proportional to a slightly reduced DNA cross-linking formation induced by DAG in BCNU-resistant cells. The amount of DNA cross-links was determined by measurement of the 1,6-di(guaninyl)-galactitol content of DNA. The slight reduction in cross-links is not attributable to DNA repair but rather to other factors that seem to prevent the formation of DNA-drug adducts. The absence of cross-resistance is explained by different kinds of DNA damage caused by the two alkylating agents and the presumably different defense mechanisms developed by cells against these lesions.

Vitamin E protection against chemical-induced cell injury. II. Evidence for a threshold effect of cellular alpha-tocopherol in prevention of adriamycin toxicity.

The cardiomyopathy produced by the widely used anticancer drug adriamycin (ADR) is believed to be related to the production of reaction oxygen species and consumption of reduced glutathione (GSH) during redox cycling of the drug. Protection by vitamin E against the toxicity of ADR was studied in a model of compromised isolated hepatocytes, generated by physiological alterations in the concentration of cell calcium. A decrease in cell calcium concentration leads to a greater loss of endogenous alpha-tocopherol and enhances the intracellular hydrolysis of exogenous alpha-tocopheryl esters. With this model, vitamin E (alpha-tocopheryl succinate) at 25 microM protected the calcium-depleted hepatocytes against the toxicity of ADR, in association with greater cellular alpha-tocopherol content as compared to calcium-adequate cells. The incubation of calcium-adequate hepatocytes with increasing concentrations of alpha-tocopheryl succinate up to 200 microM demonstrated that maximal protection by vitamin E was directly dependent on the alpha-tocopherol content of the cells, regardless of the concentration of cell calcium. The viability of the cells was closely associated with the alpha-tocopherol-mediated maintenance of cellular protein thiols. Viability and protein thiol content of the cells were maximal at cellular alpha-tocopherol levels in the range 0.6-1.0 nmol/10(6) cells in both calcium-depleted and -adequate cells. It is suggested that the potential use of vitamin E as a protective agent against ADR toxicity in vivo be reevaluated with an emphasis placed on the threshold level of intracellular alpha-tocopherol in the critical target tissue.