BACKGROUND

Colon cancer is the second most frequently diagnosed cancer in the U.S. Surgery is the most common treatment; however, recurrence after surgery is a huge problem, and death usually occurs after recurrence. Successful treatment of colon cancer is limited to tumors that are localized in the bowel and present only in the primary stage. Currently, chemotherapy is not very effective for cancer that has undergone migration away from the bowel, or entered the metastatic stage.(1) Thus, a study of a different form of treatment is critical for the successful treatment of colon cancer. Platinum and palladium compounds were tested as potential anit-cancer agents.

The three malignant colon lines studied were SW-480, a primary tumor isolated from a 51-year-old Caucasian male; SW-620, a metastatic tumor isolated from the lymph node of the same individual from which the SW-480 was extracted; and HCT-15, a primary tumor from a 75-year-old male. These three cancerous colon cell lines were exposed to the following nine metal compounds synthesized by Dr. Robert Granger (Professor of Chemistry at Sweet Briar College) and Yen Nguyen (a 2001 Sweet Briar College graduate): [Pd(dione)phen Cl2]Cl2, Pd(bipy)Cl4 , Pd(phen)Cl4 , [Pt(dione)2 ](PF6 )2 II, Pd(dione)Cl2 II, Cisplatin, [Pd(dione)bipy Cl2]Cl2, [Pt(dione)phen Cl2]Cl2. It was determined which compound was the most effective against each malignant line.

The mechanism of action of these compounds is currently unknown and is under investigation. The same compounds were applied to the normal colon cell line CCD-33Co, in order to determine which compound was the safest for the normal cells.

A soft agar assay was also performed on the three malignant cell lines. The soft agar simulates human tissue. A comparative study of how many distinct colonies each line produced in the soft agar, as well as a characterization of the aggressive behavior of each line, was performed. Finally, a cell adhesion assay was performed using fluorescence microscopy to analyze the results. Data on a cell type's adhesive qualities provides a quantitative measure of the cell line's invasive properties. In this assay, cells were dyed with calcein stain and photographed. Cells that adhered were then illuminated with ultraviolet light and photographed. The photographs were used to determine which cell line was relatively most adhesive.

METHODS

Culturing Colon Lines

The SW-480 cells were grown in Liebovitz' L15 media with 10% fetal bovine serum in T-25 cm2 flasks. The cells were fed every 2-3 days and were transferred in 1:4 and 1:8 ratios to new flasks once the cells were confluent. The SW-620 cells were grown using the same protocol. The HCT-15 cells were grown in RPMI media with 10% fetal bovine serum in T-25 cm2 flasks. The cells were fed every 3-4 days and transferred in 1:5 and 1:10 ratios to new flasks once the cells were confluent. The CCD-33Co cells were supplemented with DMEM media with 10% fetal bovine serum. To transfer, or pass, the cells to another flask, they were trypsinized to remove them from the original flask. This was done by removing the old media and washing the cells with 5 mL of PBS buffer solution. One milliliter of trypsin was added to the flask, and the cells were allowed to detach from the flask. Four milliliters of media were added to halt the trypsin action. The SW-480 and SW-620 cell lines were incubated at 37 degrees Celsius in a non-CO2 incubator. The HCT-15 and CCD-33Co cell lines were incubated at 37 degrees Celsius in a 5% CO2 incubator.

Photographs of malignant and normal colon cells

Exposing Cells to Compounds and Performing
3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyl tetrazolium bromide (MTT) Assay

A colorimetric assay that measures the reduction of MTT by mitochondrial succinate dehydrogenase was performed. MTT is absorbed into the cells and into the mitochondria, where it is then reduced to a formazan product.(2) Each cell line (with the exception of CCD-33Co due to time constraints) was exposed to each of the nine compounds three times. To prepare the cells for exposure, they were trypsinized from the flask. The cells were plated in microwell plates. The final concentration of DMSO (dimethyl suloxide) was 1%, and the compound was diluted to 1.0 x 10-5M. The microplate was incubated for 48 hours. The medium was then removed using a vacuum pump, and 100 µL of DMSO was added to each well to lyse the cell membranes and dissolve the blue formazan. The plates were placed on a mixer for 5 minutes and then were inserted into a Tecan Spectra Reader. The percent survival was determined using Microsoft WinSelect and Excel.

Soft Agar Assay

To make the base layer, a 3% agar stock was made in PBS and sterilized in the autoclave. At 45 degrees Celsius, 80 mL of the medium were mixed with 20 mL of the 3% agar. Five milliliters per plate of this solution were aliquoted and allowed to solidify. To make the cell layer, the cells were counted using trypan blue dye and a hemacytometer. The cell concentration was adjusted to 1 x 106 cells/mL. The cells were resuspended in separate tubes, in which 6.5 mL of agar medium were mixed with 2.7 mL of cells. One- and-a-half milliliters of cell-agar mixture were dispensed on top of the base layer. The plates were then incubated for 2 1/2 weeks. Five plates for each cell line were used. The colonies each cell line produced were counted. (This is explained in more detail in the discussion of results below.)

Cell Adhesion Assay

First, a monolayer was made to provide a layer to which test cells might adhere. The volume needed for the monolayer was determined as follows: each chamber was measured to be approximately 3 cm2. Since the T-25 cm2 flasks were used for culturing the cells, and these flasks allow for 75 cm2 of cells at a 1:3 ratio, it was determined that one T-25 flask could seed six 4-chambered slides. The cells were trypsinized and brought to a 24-mL volume. One milliliter of cell/media solution was added to each chamber and incubated. The top layer was then prepared. The cells were trypsinized and transferred to polypropylene tubes. Five milliliters of media were added. The cells were washed twice with PBS and resuspended in serum-free RPMI media at 5 x106 cells/mL. To get this concentration, cells were counted using the hemacytometer, and the concentration was adjusted accordingly. Five microliters of calcein AM (Molecular Probes) per milliliter of cell suspension were added. The cells were then incubated at 37 degrees Celsius for 30 minutes. Next, the cells were washed with pre-warmed RPMI media and re-suspended in RPMI media at 5 x 106 cells/mL, and 100 µL of the calcein-labeled cell suspension was added to the chambers. The slides were incubated for 2 hours. The non-adherent cells were then removed by adding pre-warmed RPMI media to each chamber. The chamber was swirled gently and then inverted onto a paper towel. This process was performed four times. After the fourth washing, 200 µL of PBS were added to each chamber. Fluorescence microscopy, or exposing the cells to ultraviolet light, was used to photograph the emissions, and the amount of fluorescent cells shown for each cell line was compared. To determine the cells' viability, a portion of the non-calcein-stained cells were dyed with trypan blue and counted with a hemacytometer.

RESULTS

Exposure of Cell Lines to Compounds

Soft Agar Assay

*All cell lines produced a layer of small colonies. Only larger aggregations of cells were considered significant colonies. Colonies were counted from 5 plates for each cell line.

DISCUSSION OF RESULTS