Wei Chen

Document Type

Dissertation - University Access Only

Award Date

2010

Degree Name

Doctor of Philosophy (PhD)

Department / School

Pharmaceutical Sciences

Abstract

Chemotherapy is one of the most common therapeutic strategies in cancer treatment. To improve the efficacy of cancer treatment, and lower the toxicity against normal cells, as well as overcome the drug resistance problem, one of the main focuses of anticancer drug discovery has been to develop anticancer agents that exhibit selectivity and act through novel mechanisms of action. In this study, anticancer agents with novel mechanisms of action were identified. N-Acetyl-S-(p-chlorophenylcarbamoyl)cysteine (NACC) was identified as a metabolite of sulofenur which was one of the diarylsulfonylureas developed as an anticancer agent by Eli Lilly Research Laboratories for solid tumors. Sulofenur was demonstrated to have broad activities against solid tumors in preclinical studies but exhibited disappointing clinical responses due to its high protein binding related adverse effects. NACC was found to exhibit low protein binding and excellent activity against a sulofenur sensitive human colon cancer cell line (GC3/cl). Based on this finding, seventeen analogs ofNACC were rationally designed and synthesized. Their anticancer activities were investigated with the GC3/cl, UACC-62 (human melanoma), and OVCAR-3 (human ovarian cancer) cells. The results demonstrated that two of the NACC analogs (analog 1 and analog 7) showed excellent activities against UACC-62, while NACC remained to be the most potent in this series. The anticancer activities were compared to dacarbazine, a drug extensively used in the treatment of melanoma and various other cancers. NACC, analog 1 and analog 7 were found to be 490, 328 and 37- fold more active than dacarbazine respectively against UACC-62 cells. The selective activity against melanoma was further confirmed later with SK-MEL-2 cells, another human melanoma cell line. NACC, analog 1 and analog 7 were found to be 16000, 279, and 1000 more active than dacarbazine respectively against SK-MEL-2 cells. More impressively, NACC was shown to induce apoptosis without affecting the cell cycle, a unique and novel feature that distinguishes NACC from other conventional anticancer agents that can arrest cells at a specific phase, mostly at the G1/S or G2/M boundaries. Further, NACC exhibited low toxicity against monkey kidney cells (CV-1), a normal cell line. The selective anticancer activity, selective low toxicity, unique anticancer mechanism and ready obtainability through synthesis make NACC and its analogs promising anticancer agents. The second part of the project is related to oxidative stress, microtubule Sglutathionylation, and cancer growth inhibition. Oxidative stress has been reported to inhibit cancer growth by inducing apoptosis. 2-Acetylamino-3-[ 4-(2-acetylamino-2-carboxyethylsulfanylcarbonylamino)phenylcarbamoylsulfanyl]propionic acid (2-AAPA) has been found to induce intracellular thiol oxidative stress and protein Sglutathionylation. Protein S-glutathionylation is a reversible protein structural modification and is considered to be a protein posttranslational modification. In situations of oxidative stress, it serves as a mechanism to protect protein thiols from being irreversibly oxidized. 2-AAP A was also found to show activity against various human cancer cell lines with very similar IC50 values (22-75 μM). These cell lines included human ovarian, skin, prostate, lung, breast, colon, melanoma and renal cancers. Although the IC50 values of 2-AAP A are not impressive, the fact that it exhibits similar cytotoxicity against all the tested cancer cell lines is interesting. Based on the fact that 2-AAP A can cause significant protein S-glutathionylation and microtubules are rich in thiols, it is expected that microtubules are likely to be S glutathionylated in the presence of 2-AAPA. In view of the critical role microtubules play in mitosis, S-glutathionylation of microtubules is likely to lead to cell growth inhibition. This study was aimed to determine whether 2-AAPA causes microtubule S-glutathionylation and whether the Sglutathionylation contributes to cell growth inhibition of cancer cells. The significance of this investigation is that it can establish microtubule S-glutathionylation as a novel mechanism to develop antimitotic drugs. The investigation started with confirmation of protein S-glutathionylation in cancer cells. Our HPLC assay revealed that proteins were S-glutathionylated when cancer cells were treated with 0.1 mM 2-AAP A. Protein S-glutathionyaltion of 2.19 ± 0.30 nmol GSH/million cells and 2.15 ± 0.12 nmol GSH/million cells were determined in UACC-62 and OVCAR-3 cells respectively. A pull-down assay using glutathione S-transferase (GST)-agarose beads followed by Western blot analysis demonstrated that the thiol functional groups of microtubules were S-glutathionylated in the presence of 0.1 mM 2- AAPA in UACC-62 cells. The thiol functional groups of microtubules are critical to microtubule functions, such as cell shape maintenance and cell division. Consistently, the morphology of the cells was found to be altered and cellular microtubules depolymerized in UACC-62 cells through the use of microscopy and immunofluorescence microscopy respectively. These data confirmed that the functions of microtubules were affected. The microtubule depolymerization effect induced by 2-AAPA was more significant than another microtubule depolymerizing drug vinblastine. In line with these results, flow cytometry analysis revealed that UACC-62 cells were significantly arrested at the G2/M phase and underwent apoptosis after treated with 35 and 50 μM 2-AAPA. The data from this investigation demonstrated that microtubule S-glutathionylation contributed to the antimitotic effects of 2-AAPA. Our findings point to the potential of microtubule Sglutathionylation as a novel mechanism to develop new antimitotic agents. In summary, this dissertation reports two novel findings: a) a class of anticancer agents with a unique mechanism of drug action that is inhibition of cancer cell growth without affecting the cell cycle; b) microtubule S-glutathionylation as a potential novel mechanism to develop new antimitotic agents.

Library of Congress Subject Headings

Antineoplastic agents

Cancer -- Chemotherapy

Format

application/pdf

Number of Pages

136

Publisher

South Dakota State University

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