"Enhancement of Cancer Cell Sensitivity to Radiotherapy through Increas" by Yong Zhao

Yong Zhao

Document Type

Dissertation - University Access Only

Award Date

2008

Degree Name

Doctor of Philosophy (PhD)

Department / School

Pharmaceutical Sciences

Abstract

Cancer causes about 13 % of all deaths in the world. According to the American Cancer Society 7.6 million people died from cancer worldwide in 2007. Radiotherapy, one of the major therapies for cancer treatment, is used as a cancer therapy for more than 100 years. It uses the ionizing radiation as part of cancer treatment to control malignant cells and may be used as the primary therapy for most cancers. But the major issue with radiotherapy in clinic is radiotherapy resistance. The mechanisms by which tumor cells develop radiation resistance are complex. It may be due to 1) local tissue hypoxia; 2) over expression of antioxidant enzymes such as catalase, superoxide dismutase (SOD), glutathione peroxidase (GP), glutathione reductase (GR) and so on; 3) over production of antioxidants. The major intracellular antioxidants are thiols [proein thiol (P-SH) and nonprotein thiol (NP-SH)]; 4) inherent factors such as p53 status, bcl-2 activity and levels, and so on. In recent years, several pharmacologically based approaches have been successfully combined with radiation treatment to reduce or modify radio resistance. Strategies involving radio sensitizing drugs or inhibitors of cellular repair processes and/or tumor cell proliferation are well established and have been proven effective in many instances. Specifically, these approaches include 1) modulation of the intracellular thiol pool; 2) blockage of bcl-2; 3) inhibition of topoisomerase II or activated Ras; 4) combination of radiotherapy with antiangiogenesis or vascular targeting agents. Thiols play an important role in cancer cell resistance to radiotherapy and oxidative stress. The mechanism of cancer resistance caused by thiols is attributed to the thiols' ability to terminate free radicals. Glutathione (GSH) is the most prevalent NP-SH in mammalian cells and the most abundant low molecular weight tri-peptide present in eukaryotic cells. The GSH content of cancer cells is particularly relevant in regulating mutagenic mechanisms, DNA synthesis, growth, and multidrug and radiation resistance. GSH as a determinant of radiation sensitivity could result from its role in the detoxification of radiation-induced free radicals. In view of GSH's role in cancer resistance to radiotherapy, extensive efforts have been made to identify agents that can reduce intracellular GSH and increase cancer sensitivity. Intracellular GSH levels can be affected by numerous factors but are primarily maintained by two biochemical pathways: a). the de nova biosynthesis of GSH and b). The reduction of glutathione disulfide (GSSG) catalyzed by GR. Inhibition of glutathione biosynthesis or GSSG reduction will be expected to reduce intracellular GSH. The most extensively investigated approach to reduce intracellular GSH is the use of buthionine sulphoximine (BSO), an inhibitor of GSH biosynthesis, which has been demonstrated to effectively reduce intracellular GSH and reverse GSH-mediated cancer resistance (intrinsic or acquired) to radiation. Glutathione reductase (GR, EC 1.8.1. 7) catalyzes the reduction of GSSG back to GSH to maintain a reducing intracellular environment. The enzyme GR plays a key role in the cellular defense against oxidative stress. High levels of GR activity are often associated with tumor growth and/or resistance mechanisms against chemo- and radiotherapy. Inhibitors of GR are found to be promising agents for the treatments of malaria and cancer. Two mechanisms are known by which the inhibition of GR could impair tumor growth: (a) the resulting build-up of the substrate GSSG could inhibit protein synthesis; (b) the tumor cells may become unable to cope with the cytotoxic reactive oxygen species (ROS) launched against them by activated macrophages. Therefore, inhibition of GR could increase oxidative stress. This project was aimed to investigate whether oxidatively stressed cancer would be more sensitive to radiotherapy. Specifically, oxidative stress was created through inhibition of GR by an irreversible inhibitor 2-AAP A which was developed from this laboratory with Ki and kinact values of 56 μM and 0.1 min-1 respectively. The effect of GR inhibition on cancer sensitivity to radiotherapy was investigated in four human cancer cell lines. The impact of GR inhibition on intracellular redox and related systems were investigated in CV-1 cells-a monkey kidney cell line. In addition, the effect of a combined inhibition of GR by 2-AAP A and GSH biosynthesis by BSO was also studied. Our data demonstrated that 2-AAP A inhibited 90-97% GR activities leading to a 5 to 7 fold increase in GSSG and ~30% decrease in GSH in the normal cell line CV cells. The other changes observed were an increase in the ratio of NADPH/NADP+ and NADHINAD\ and a significant increase in protein glutathionylation. It produced no significant effect on ROS formation, gene and protein expression of enzymes involved in antioxidant defense systems and in GSH biosynthesis pathway. It did not affect intracellular energy ATP content. These data indicated that 2-AAP A established a thiol oxidative stress with limited effects on other redox systems. The cancer sensitivity study revealed that 2-AAP A increased the sensitivity of all four studied cancer cell lines to X-ray radiation. X-ray dose response curves were shifted left- and downwards by 2-AAP A which indicated that 2-AAP A increased cancer cell sensitivity to X-ray. The IC50 values of X-ray alone for A431, MCF7, NCI-H226 and OVCAR-3 cells were 24.4Gy, 42.5Gy, 43.0Gy, 27.8Gy, which were reduced to 6.75Gy, 8.1 Gy, 6. 75Gy, and 12.1 Gy respectively when the cells were first treated by 2-AAP A followed by X-ray. Under the condition which produced the best sensitizing effect in OVCAR-3 cell line, 80% of GR was inhibited, and a 3 to 4 fold increase in GSSG was observed by 2-AAP A while an additional increase in GSSG was noticed by 2-AAP A plus X-ray indicating that this drug combination produced additional oxidative stress in the cells. The combination of 2-AAPA and X-ray significantly increased the content of total disulfides by 25.8% which appears to be in agreement with the effect of the drug combination on GSSG. Together, these data demonstrated that a prior establishment of thiol oxidative stress by inhibition of GR can increase cancer sensitivity to radiation. A combined inhibition of GR and GSH biosynthesis by 2-AAPA and BSO respectively further increased oxidative stress in cancer cells and, as a result, further enhanced cancer cell sensitivity to X-ray irradiation than 2-AAP A alone or BSO alone. The dose response curves of X-ray with four cell lines shifted further left- and downwards by 2-AAPA plus BSO than these two drugs used alone. The IC50 values of X-ray alone for A431, MCF7, NCI-H226 and OVCAR-3 cells were 24.4Gy, 42.5Gy, 43.0Gy, and 27.8Gy, which were reduced to less than 6.7Gy for all four cell lines by the combination of BSO and 2-AAPA. The ratio of GSSG/GSH increased in OVCAR-3 cells when BSO and/or 2-AAPA were used together with X-ray. 2-AAP A+ BSO + X-ray produced the highest GSSG/GSH ratio, which indicated that the combination treatment of the three (2-AAP A, BSO and X-ray) produced additional oxidative stress in the cancer cells. In conclusion, inhibition of GR by 2-AAP A itself increased cancer cell sensitivity to X-ray radiation. The combined inhibition of GR and GSH biosynthesis further increased oxidative stress in cancer cells and, as a result, produced a further enhancement in cancer cell sensitivity to X-ray radiation was observed.

Library of Congress Subject Headings

Cancer -- Radiotherapy

Oxidative stress

Radiation-sensitizing agents

Drug resistance in cancer cells

Format

application/pdf

Number of Pages

168

Publisher

South Dakota State University

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