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Alternate Applications of Anticancer Drugs on Cos-7 Normal Cells

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Alternate Applications of Anticancer Drugs on Cos-7 Normal Cells ALTERNATE APPLICATIONS OF ANTICANCER DRUGS ON COS-7 NORMAL CELLS by Deborah Morris A Thesis Submitted to the Faculty of The Wilkes Honors College in Partial Fulfillment of the Requirements for the Degree of Bachelor of Arts in Liberal Arts and Sciences with a Concentration in Biology Wilkes Honors College of Florida Atlantic University Jupiter, Florida April 2009 ALTERNATE APPLICATIONS OF ANTICANCER DRUGS ON COS-7 NORMAL CELLS by Deborah Morris This thesis was prepared under the direction of the candidate’s thesis advisor, Dr. Nicholas Quintyne, and has been approved by the members of her supervisory committee. It was submitted to the faculty of The Honors College and was accepted in partial fulfillment of the requirements for the degree of Bachelor of Arts in Liberal Arts and Sciences. SUPERVISORY COMMITTEE: Dr. Nicholas J. Quintyne Dr. Shree Kundalkar Dean, Wilkes Honors College Date ii Acknowledgements I would like to express my thanks to Dr. Quintyne for his guidance and advice in the completion of my thesis project. I would also like to thank Dr. Kundalkar for her comments in the editing of my thesis. I would like to acknowledge my lab group members for providing me with cells and making solutions. iii Abstract Author: Deborah Morris Title: Alternate Application of Anticancer Drugs on COS-7 Normal Cells Institution: Harriet L. Wilkes Honors College at Florida Atlantic University Thesis Advisor: Dr. Nicholas J. Quintyne Degree: Bachelor of Arts in Liberal Arts and Sciences Concentration: Biology Year: 2009 Anticancer drugs, including nocodazole and vinblastine, work by disrupting the dynamics of microtubules. Unfortunately, these drugs often produce numerous side effects, including nausea, vomiting, loss of appetite, loss of hair, increased chance of infection, and fatigue. My thesis research evaluated the efficacy of using repeated low doses of microtubule drugs instead of a single high dose, in an attempt to minimize side effects. Using nocodazole and vinblastine, I first established the minimum effective concentration that disrupts the microtubules in normal human cells grown in vitro and treated cells with those concentrations over a period of several days. I found that microtubules were increasingly depolymerized as the days progressed. Next, I tested a combination of nocodazole and vinblastine at low concentrations. iv Table of Contents Introduction ..................................................................................................................... ..1 Methods Cell Culture ..................................................................................................................... 6 Immunofluorescence ....................................................................................................... 6 Drug Treatments ............................................................................................................. 7 Fluorescence Microscopy ............................................................................................... 7 Results Nocodazole Concentration Assay ................................................................................... 9 Vinblastine Concentration Assay .................................................................................... 9 Nocodazole - 30 Minute Depolymerization and Regrowth Assay ............................... 11 Combination of Drugs................................................................................................... 12 Discussion......................................................................................................................... 14 References ........................................................................................................................ 16 v List of Illustrations Fig. 1: A 3-dimensional structure of a single microtubule ................................................. 1 Fig. 2: Nocodazole (red) binds to free tubulin dimer (green and blue) to prevent polymerization ........................................................................ 3 Fig. 3: Vinblastine bind to sites on the plus end of the microtubule .................................. 4 Fig. 4: Microtubule depolymerization at different nocodazole concentrations: (A) 33 µM, 16.5 µM, 8.25 µM, 6.6 µM, 3.3 µM, 1.65 µM (B) 825 nM (C) 666 nM (D) 333nM .................................................... 10 Fig. 5: 50nM vinblastine treatment after 1 day ................................................................. 10 Fig. 6: (A) COS-7 normal cells and 333 nM nocodazole treatment after day 1 (B), after day 4 (C) and after day 6 (D) .......................................................... 11 Fig. 7: Percentage of Cells with Various Levels of Polymerization after Exposure to Nocodazole ................................................................ .12 vi Introduction Microtubules are important cytoskeletal components of the cell that are essential for mitosis, cell signaling, motility, vesicle and organelle transport, and cell shape (Jordan & Wilson, 1998). Microtubules (Figure1) are composed of α- and β-tubulin that form heterodimers which organize head to tail to form a hollow tube of 13 parallel protofilaments (Krebs et al., 2005). An important property of microtubules is polarity, which results because of the polymerization of α- and β-tubulin heterodimers. The plus end displays only β-tubulin, and the minus end displays only α-tubulin (Howard and Hyman, 2003). There are two important microtubule dynamics called dynamic instability and treadmilling (Jordan & Wilson, 1998). Figure 1: A 3-dimensional structure of a single microtubule. Image from: Jordan and Wilson, 2004 Dynamic instability is when microtubules switch between episodes of rapid growth and shrinkage in which the plus end grows and shortens faster than the minus end. Catastrophe is the switch from growth to shrinkage, while rescue is the switch from 1 shrinkage to growth (Vasquez et al., 1997). This process is regulated by the addition and removal of the GTP cap, which is tubulin-bound GTP at the end of a microtubule. When the GTP cap is bound, the microtubule is stabilized and is able to lengthen. When the GTP cap is lost, the microtubule is unstabilized and is shortened (Jordan and Wilson, 2004). Dynamic instability is crucial for the reorganization of the cytoskeleton during mitosis and cell division. Treadmilling is net growth or gain at microtubule plus ends and net shortening or loss at minus ends. Tubulin from the plus end flows to the minus end allowing the microtubule to move. This means that there will be no net increase in microtubule length. (Jordan and Wilson, 1998). All stages of mitosis, including prometaphase, metaphase, and anaphase, require very dynamic microtubules for the mitotic spindle. Microtubules at the spindle poles must grow and shorten in order to attach to the kinetochores of chromosomes during prometaphase. If a chromosome does not have a bipolar attachment to the spindle, the cell does not proceed to anaphase in order to avoid missegregation and aneuploidy. The cell is blocked at the transition point from metaphase and anaphase (Jordan and Wilson, 2004). Anticancer drugs target and suppress microtubule dynamics which slows or stops mitosis at the metaphase-anaphase checkpoint. There is an accumulation of mitotic cells, and they eventually die by apoptosis. Cancer cells are particularly sensitive to anticancer drugs that target microtubules, because cancer cells are more proliferative and go through the stages of mitosis more often than normal cells (Jordan and Wilson, 2004). Two such chemotherapeutic drugs, nocodazole and vinblastine, target these dynamics and inhibit cell proliferation (Jordan & Wilson, 1998). 2 Nocodazole is a benzimidazole derivative that at low concentrations, equal to or greater than concentrations of tubulin, inhibits polymerization by binding to tubulin dimers (Figure 2). At higher concentrations, nocodazole depolymerizes microtubules and stops the cell cycle at mitosis (Vasquez et al., 1997). Figure 2: Nocodazole (red) binds to free tubulin dimer (green and blue) to prevent polymerization. Image from: w3.impa.br/%7Ejair/microtubule_structure.htm Vinblastine is a naturally occurring vinca alkaloid that is used to treat Hodgkin’s disease and testicular germ-cell cancer. At low concentrations, vinblastine blocks mitosis by inhibiting microtubule dynamics, while at high concentrations, vinblastine depolymerizes microtubules, breaks down mitotic spindles, and blocks the cell at mitosis. Dynamic instability and treadmilling can be reduced by 50% with the addition of only one or two molecules of vinblastine to microtubule ends. Vinblistine can bind to the β- tubulin of dimers or can bind directly onto the microtubules of the plus ends (Fig.2) (Jordan and Wilson, 2004). Vinblastine does not bind very well to tubulin on the sides of microtubules (Jordan, 2002) 3 Figure 3: Vinblastine bind to sites on the plus end of the microtubule. Image from: Jordan and Wilson, 2004 Anticancer drugs cause numerous side effects to patients, and side effects are dependent upon the type of treatment and drugs used. Common side effects of anticancer drugs include nausea, hair loss, fatigue, increased chance of bruising and bleeding, anemia, and infection (www.cancer.org). The specific common side effects of vinblastine are low blood counts, injection site reactions, fatigue, and weakness (Chemocare). It is important to minimize side effects by using lower doses of anticancer drugs because using more than what
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