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 , work by disrupting the dynamics of . 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 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 , 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 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 , , 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 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 . 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 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 is needed can be harmful to the organism (Smurova et al., 2008).

Cancer cells also form resistance to anticancer drugs. is normally administered to the patient in maximum tolerated doses (MTD) followed by a rest period between treatments. The cells repair and recover from the harmful side effects during the rest period and can form a resistance to the anticancer drug (Klement et al., 2000). This can be overcome by metronomic chemotherapy which is using low doses of an anticancer drug weekly so that there is no prolonged rest period. The repair process will not be as

4 efficient and with lower doses, patients will not experience severe side effects (Kerbel et al., 2002).

The use of metronomic chemotherapy is less toxic to the individual and especially beneficial to the elderly and children. It has been shown in previous studies that a high proportion of breast and ovarian cancer patients who were treated with a anti- cancer drug began responding to low dose chemotherapy administered weekly after they had stopped responding to a maximum tolerated dose (MTD) chemotherapy with rest periods of 3 weeks (Kerbel et al., 2002). This demonstrated that although the patients had formed a resistance to the taxane anti-cancer drug when given at high doses, they were able to overcome the resistance with lower doses of the chemotherapeutic drug. Another benefit, in addition to reducing toxicity and overcoming resistance, is cost of treatment.

Since there is decreased toxicity, there is the possibility of using outpatient therapy which would reduce personal costs and economical costs to the patients by increasing quality of life and decreasing visits to health care providers (Colleoni et al., 2002).

I wanted to evaluate the efficacy of using repeated low doses of microtubule drugs instead of a single high dose, in an attempt to minimize side effects and resistance.

I used concentration assays to determine the minimum effective concentration of nocodazole and vinblastine that caused partial depolymerization of microtubules. I then treated normal cells with the minimum effective concentration of nocodazole 30 minutes each day over a period of 8 days. I evaluated the effectiveness of nocodazole by counting the number of fully depolymerized, partially depolymerized, and normal cells on certain days. I also examined the combination effects of nocodazole and vinblastine at various concentrations.

5 Methods

Cell Culture

I grew COS-7 (American Type Cell Culture, Manassas, VA) cells in Dulbecco’s

Modified Eagle Media (DMEM) (500 mL; Sigma Chemical Company, St. Louis, MO) supplemented with fetal bovine serum (FBS) (55 mL; Fisher Scientific, Pittsburgh, PA) and penicillin-streptomycin (PS) (5.5 mL; MP Biomedicals, Solon, OH). The cells were grown in an incubator at 37°C in a 5% CO2 environment. I split the cells by first washing the cells with phosphate buffered saline) (PBS). After the addition of 2 mL of 0.05% trypsin/EDTA (MP Biomedicals) for 5-10 minutes, 8 mL of media was added to the plate. Cells were then distributed into different plates or used to seed coverslips at a ratio of 1:5.

Immunofluorescence

I fixed the cells on the coverslips in -20°C MeOH for 10 minutes. The cells were then re-hydrated with PBS for 5 minutes and blocked with PBST/1.5% BSA for 15 minutes at room temperature. The coverslips were then covered with 150 µL of primary

(Mouse anti-α-tubulin, Clone DM1A (Sigma) 1:200 dilution) and secondary (Alexa Fluor

488-conjugated goat anti-mouse (Invitrogen Molecular Probes Corporation, Carlsbad,

CA) 1:250 dilution) antibodies for 30 and 15 minutes, respectively. I used PBS to wash the cells 3X for 2 minutes after the addition of each antibody. I applied 100 µL of 4, 6- diamidino-2-phenylindole (DAPI, Sigma) to the coverslips for 1 minute and afterwards, distilled water was used to wash the cells 3X for 30-60 seconds. The coverslips were mounted on slides using 3:1 ratio of Mowiol 4-88 (Fluka, Seelze, Germany) and n-propyl gallate (MP Biomedicals) antifade mounting medium.

6 Drug Treatments

Nocodazole and Vinblastine Treatment-Concentration Assay

I prepared different concentrations of nocodazole (Sigma) by diluting the appropriate amount of 33 mM stock solution into DMEM. The coverslips were immersed in 2 mL of the nocodazole solutions for 30 minutes in the incubator. The cells were fixed and prepared for immunofluorescence. I observed the slides through fluorescence microscopy. The same procedure was used for vinblastine (Sigma) except that different concentrations were prepared by diluting the appropriate amount of 10mM stock solution into DMEM.

Nocodazole Treatement-30 Minute Depolymerization and Regrowth Assay

I applied 2 mL of 333 nM nocodazole to the coverslips for 30 minutes in the incubator. Normal media was used to wash the coverslips 3X after the drug treatment.

Coverslips were fixed each day and prepared for immunofluorescence. I took samples on days 1, 4, 6, and 8.

Combination of Drugs

I prepared low concentrations of nocodazole and vinblastine by diluting the appropriate amount of stock solution for each drug into DMEM. I made two combinations and immersed a coverslip with 2 mL of each solution for 30 minutes in the incubator. The cells were fixed and prepared for immunofluorescence.

Fluorescence Microscopy

I used an Olympus IX 81 inverted fluorescence microscope (Olympus America,

Center Valley, PA) to analyze the slides using 100X objective with a N.A. of 1.65 and the appropriate filters. Pictures were takes on an Orca 285 Cooled CCD digital camera

7 (Hamamatsu, Bridgewater, NJ). The software that was used was Slidebook 4.1 Digital

Microscopy (Intelligent Imaging Innovations, Denver, CO).

8 Results

Nocodazole Concentration Assay

A concentration assay was used in order to determine the lowest effective concentration of nocodazole that partially disrupts the microtubules. Normal COS-7 cells were exposed to nocodazole concentrations of 33 µM, 16.5 µM, 8.25 µM, 6.6 µM,

3.3 µM, 1.65 µM, 825 nM, 666 nM, and 333 nM for 30 minutes. Cells that were treated with nocodazole concentrations of 33 µM to 1.65 µM were 100% depolymerized (Figure

4A). When exposed to a concentration of 825 nM, ~87% of cells exhibited asters with a small amount of microtubule formation (Figure 4B), while ~13% had no aster formation.

When exposed to a concentration of 666 nM, ~91% of cells formed asters with increased microtubule growth (Figure 4C), while ~9% had no aster formation. When exposed to a concentration of 333 nM, ~95% of cells formed asters with microtubules covering a majority of the cell (Figure 4D), while ~5% had no aster formation. The concentration that was determined to be the lowest effective concentration was 333 nM.

Vinblastine Concentration Assay

Using the same methods as nocodaozle, various concentrations of vinblastine

(1 µM, 500 nM, 250 nM, 100 nM, 50 nM, and 10 nM) were applied to normal COS-7 cells in order to determine the lowest effective concentration. At concentrations of 1 µM and 500 nM, cells were almost fully depolymerized. When cells were exposed to 250 nM and 100 nM, cells were depolymerized with microtubules surrounding the periphery. The concentration of 50 nM was chosen, because there was partial depolymerization (Figure

5). At a concentration of 10 nM, cells were mostly normal with very slight depolymerization.

9 A B

C D

Figure 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)

333 nM. Green =microtubules, Blue = chromatin

Figure 5: 50nM vinblastine treatment after 1 day. Green =microtubules, Blue = chromatin

10 Nocodazole - 30 Minute Depolymerization and Regrowth Assay

Normal cells were treated with the minimum concentration of 333 nM nocodazole each day for 8 days in order to determine the effectiveness of repeated low doses of nocodazole over several days. In Figure 6A, the control COS-7 cells were not exposed to any anticancer drug. After day 1, there was partial microtubule depolymerization (Figure

6B). After day 4, there was progressive microtubule depolymerization (Figure 6C). After day 6, a majority of microtubules were depolymerized (Figure 6D). Cells did not survive to day 8, falling off the coverslip and presumably undergoing apoptosis.

A B

C D

Figure 6: (A) COS-7 normal cells and 333 nM nocodazole treatment after day 1 (B), after day 4 (C) and after day 6 (D). Green =microtubules, Blue = chromatin

11 After making a visual assessment of the microtubules, 150-300 cells were counted on days 1, 4, and 6 in order to determine the stages of polymerization (Figure 7). Cells on day 1 and 4 had similar percentages of normal, partially depolymerized, and fully depolymerized microtubules. On day 6, there was a sharp increase in the number of fully depolymerized cells (62%), a sharp decrease in the number of normal cells (1%), and an intermediate decrease in the number of cells that had undergone partial depolymerization

(37%).

Figure 7. Percentage of Cells with Various Levels of Polymerization after Exposure to Nocodazole

70%

60%

50%

40% Normal % Partial Depolymerization % 30% Full Depolymerization % 20%

10% % Microtubule Polymerization 0% 1 4 6 Day

Combination of Drugs

Two combinations of vinblastine and nocodazole were prepared with the concentrations: 25 nM vinblastine/167 nM nocodazole and 50 nM vinblastine/333 nM nocodazole. After the cells were treated with the 25 nM/167 nM mixture, microtubules were all normal except with a very small amount of depolymerization on the periphery of the cell. After exposing cells with the 50 nM/ 333 nM mixture, microtubules again were

12 all normal except there was greater depolymerization on the periphery of the cell. There were ~95% normal cells and ~5% cells that had partial depolymerization of microtubules.

13 Discussion

The minimum effective concentrations for nocodazole and vinblastine were visually determined to be 333 nM and 50 nM, respectively. Microtubules were classified as partially depolymerized if there was about half depolymerization and half polymerization. After treating cells for 8 days with the lowest effective concentration of nocodazole, qualitative and quantitative measurements were made in order to determine the effectiveness of the drug on a non-cancerous cell line. There was a distinct amount of depolymerization with the progression of time with cells not surviving after day 8. This demonstrates that repeated low doses of nocodazole over several days could have the same effect as a single high dose.

The results obtained from the combination of nocodazole and vinblastine were inconclusive, because it would be expected that combining the minimum concentrations of both drugs would have a greater effect on microtubules than each concentration used individually. It was demonstrated that after cells were treated with the 333 nM nocodazole only for one day, there was ~15% normal cells and ~62% partially depolymerized cells. My results showed that there were ~95% normal cells and ~5% partially depolymerized cells when exposed to 50 nM vinblastine/333 nM nocodazole which is not in agreement with the results of the nocodazole depolymerization and regrowth assay. I would predict that cells exposed to both combinations of vinblastine and nocodazole would have more microtubule depolymerization, yet this was not the case. This could be due to making the incorrect concentrations. Alternatively, it could be that the effects of these two anti-microtubule agents are not additive. Given the difference

14 in how they induce depolymerization, one agent could block the activity of the other agent without altering microtubule dynamics at such low concentrations.

There is further research that could be done in order to expand on this project. The only cell line that was used throughout my project was COS-7 cells. In order to determine the full effectiveness of nocodazole and vinblastine on microtubules, because these drugs target all cells in the body including tumor and normal tissue, cancer cells should be utilized and exposed to low concentrations of anticancer drugs in order to determine the efficacy. Other expansions of this project include alternating the drugs over several days, exposing the cells to the drugs for a longer time, or treating every other day instead of every day.

Using a daily low dose of nocodazole over several days has similar effects as a single high dose on normal noncancerous cells. This is important, because it has been shown that using repeated low doses of anticancer drugs has the therapeutic benefit of decreasing side effects, toxicity, and resistance when administered to patients. Although utilizing low dose chemotherapy on patients can reduce side effects, there is no current evidence that it can increase human survival rates (Kerbel et al., 2002). Clinical trials comparing long term low dose chemotherapy versus short term higher dose chemotherapy needs to be evaluated, because the purpose of anticancer drugs should be to achieve the greatest tumor killing effect while limiting toxicity.

15 References

Colleoni, M., Rocca, A., Sandri, M.T., Zorzino, L. Masci, G., Nole, F., et al. (2002). Low-dose oral and in metastatic breast cancer: antitumor activity and correlation with vascular endothelial growth factor levels. Annals of Oncology, 13, 73-80.

Howard, J. & Hyman, A.A. (2003). Dynamics and mechanics of the microtubule plus end. Nature, 422, 753-758.

Jordan, M.A. & Wilson, L. (1998). Microtubules and actin filaments: dynamic targets for cancer chemotherapy. Current Opinion in , 10, 123-130.

Jordan, M.A. (2002). Mechanism of action of antitumor drugs that interact with microtubules and tubulin. Curr. Med. Chem. –Anti-Cancer Agents, 2(1), 1-17.

Jordan, M.A. & Wilson L. (2004). Microtubules as a target for anticancer drugs. Nature Reviews, 4, 253-265.

Kerbel, R.S., Klement, G., Pritchard, K.I., & Kamen, B. (2002). Continuous low-dose anti-angiogenic/metronomic chemotherapy: from the research laboratory into the oncology clinic. Annals of Oncology, 13, 12-15.

Kerbel, R.S. & Kamen, B.A. (2004). The anti-angiogenic basis of metronomic chemotherapy. Nature Reviews, 4, 423-436.

Klement, G., Baruchel, S., Rak, J., Man, S., Clark, K., Hicklin, D.J., et al. (2000). Continuous low-dose therapy with vinblastine and VEGF receptor-2 antibody induces sustained tumor regression without overt toxicity. The Journal of Clinical Investigation, 105(8), 15-24.

Krebs, A, Goldie, K.N., & Hoenger, A. (2005). Structural rearrangements in tubulin following microtubule formation. European Molecular Biology Organization, 6(3), 227-232.

Smurova, K.M., Birukova, A.A., Verin, A.D., & Alieva, I.B. (2008). Dose-dependent effect of nocodazole on endothelial cell cytoskeleton. Membrane and Cell Biology, 2(2), 199-127.

Vasquez, R.J., Howell, B., Yvon, A.C., Wadsworth, P., & Cassimeris, L. (1997). Microtubule dynamic instability in vivo and in vitro. Molecular Biology of the Cell, 8, 973-985.

Vinblastine – Chemotherapy Drugs. (2005). Retrieved April 13, 2009, from http://www.chemocare.com/bio/paclitaxel.asp.

16