In these experiments, the effects of two drugs, IP6 and PG490 will be combined in various concentrations to various cancer cell lines, and their effects on the cancer cells will be recorded. IP6 is a drug which prevents the cell proliferation in the cells, thus slowing the growth of the cancer cells. PG490 will induce apoptosis on the cancer cells, thus effectively killing them. By combining these two drugs, the investigator will look for synergy between them, with the two drugs working together to kill the cancer cells more effectively.

The cells will be prepared on a microtiter plate, with a specific number of cells per well, depending on the proliferation rate of each cell line, so as to control the number of cells in the wells at the end. IP6 and PG490 will be applied to the various cancer cell lines in varying concentrations and synergism will be determined. This will be done by applying an MTS Assay on the treated cancer cells after five days, and then passing them through a plate reader. The plate reader records the amount of living cells in each well of microtiter plates by shining a light through them, and the living cells will absorb the MTS Assay, which in turn absorbs this light. By measuring how much light is absorbed in each well, the information from the plate reader can be interpreted to how many cells are living in each well. This data will then be plotted and analyzed, and synergy between the drugs will be investigated.

For this experiment, different cell lines will be used in each experiment to determine if the drugs have the same effect on them. The amount of cells for each cell line set up in each well of the microtiter plate will be controlled, and cells will be kept in medium and in an incubator at 37°C and 5% CO2. Cell lines that will be tested include: A549 (Lung Cancer Cell Line), MCF7 (Breast Cancer Cell Line), 983M (Melanoma Cell Line), LNCaP (Prostate Cancer Cell Line), Colo (Colon Cancer Cell Line), HEK (Epithelium Cell Line), T47D (Breast Cancer Cell Line), HT1080 (Fibrosarcoma Cell Line), DU145 (Prostate Cancer Cell Line), and DLD2 (Colon Carcinoma Cell Line).

Experiments 3 – 6 will focus on a much smaller range of concentrations for the drugs, since a specific range in experiments 1 – 2 will be expanded and investigated. The concentration range of IP6 in experiments 1 and 2 range from 0.001 to 10, increasing 10-fold, and the concentration of PG490 in experiments 1 and 2 range from 0.001 to 100, increasing 10-fold as well. The concentration range of IP6 in experiments 3 to 6 range from 0.037 to 3, increasing by 3-fold, and the concentration of PG490 in experiments 3 to 6 range from 0.016 to 5, increasing by 5-fold.

Materials

PG490, IP6, PBS and trypsin(10x dilute) solutions for splitting cells, DMEM with 10% fetal bovine serum (FBS), L-glutamine, and antibiotics, MTS Cell titer 96 Aqueous non-radioactive cell proliferation assay, humidified 37°C incubator with 5% CO2, pipettes, including multichannel pipette, hemocytometer (with Trypan Blue Assay), Crystal Violet Stain and cell lines (A549, HEK, MCF7, T47D, 983M, HT1080, LNCaP, Colo, DLD2)

General Procedure

1. Trypsinize cells. Resuspend cells in 5mL DMEM complete and count on hemocytometer and calculate the amount cell suspension needed to distribute the desired amount of cells/
well. Add medium to the cell suspension to a total volume of 15mL.

2. Set up 96-well microtiter plate, by adding 50uL of the following cell dilution to each well of B through H (50 uL medium only in A).

3. Set up PG490 drug dilution microtiter plate:
a .Experiments 1 and 2: Prepare a PG490 stock solution of 400ng/mL, and then reduce this to 100ng/mL. Attain the following concentrations by diluting it by 10-fold: 0.001, 0.01, 0.1, 1, 10, 100ng/mL
b.Experiments 3 to 6: Prepare a PG490 stock solution of 50 ng/mL. Attain the following concentrations by diluting it by 5-fold: 0.016, 0.08, 0.4, 2, 10, 50 ng/mL

4. Set up IP6 drug dilution plate:
a.Experiments 1 and 2: Prepare an IP6 stock solution of 40mM, and then reduce this to 10 mM. Attain the following concentrations by diluting the IP6 by ten-fold: 0, 0.001, 0.01, 0.1, 1, 10 mM
b.Experiments 3 to 6: Prepare an IP6 stock solution of 3mM. Attain the following concentrations by diluting the IP6 by three-fold: 0, 0.037, 0.111, 0.33, 1, 3 mM.

5. Add 25 uL from each well of the PG490 and IP6 dilution plate to the corresponding wells on the cell plates.

6. Incubate plates for 5 days. Observe cells on day 5. Add MTS assay: Add 20 uL of reagent (20 uL PMS + 2 mL MTS + 0.2 mL medium) to each well and incubate for 2 hours. Use plate reader at OD 492 to attain results.

7. Conduct Crystal Violet Stain on cell lines. (Empty medium from microtiter plates and rinse. Then apply Crystal Violet Stain solution.)

Sub-culturing Procedure

Medium Ingredients
Dulbecco’s Modified Eagle’s Medium (DMEM)
- medium needed for splitting/growing cell
Antibiotic – Antimycotic
- decontaminate cell samples
L-Glutamine – 200mM
- amino acid
- for volatile mediums (to maintain)
Fetal Bovine Serum Qualified
PBS à buffer

Procedure
1. Take cells out of incubation and aspirate the medium with a long Pasteur
2. Rinse with 10mL PBS twice (aspirating each time)
3. Add 1.5mL trypsin and incubate at 37 C and 5% CO2 until cells become detached
4. Add 10mL DMEM and pipette out into a labeled tube.

Buffering Procedure

To fix the cells onto microtiter plate and stain with Crystal Violet stain.
1. Dump cell medium in to bin
2. Wash twice with 10x PBS to protect the cells
3. Pipette 50µL of Crystal Violet Stain in to each well of the cell and leave for 1H.
4. Rinse with water and allow to dry.

MTS Assay Procedure

To apply the MTS Assay onto the cells and read with a plate reader.
1. Prepare the reagent by mixing 20 µl PMS, 2 ml MTS and 0.2 ml medium.
2. Add 20 µl of the reagent to each well in the cell plates. (They should begin to turn yellow/orange)
3. Incubate the cells for 2 hours at 37ºC and 5.0% CO2.
4. Read the plate with the plate reader set at OD 492. Plate reader is found in room 1-19.

Hemocytometer Procedure

To use the hemocytometer and the Trypan Blue Assay to count the cells in the cell suspension.
1. Apply Trypan Blue Assay to 5mL of cell suspension.
2. Take 25 µL of this and apply it to a hemocytometer.
3. Count the number of cells in 4 to 5 of the 4x4 grids and take the average number of cells in a 4x4 grid
4. The number of cells in one quadrant is equal to that number x 104 cells/mL. (The cell suspension dilutions to obtain the desired number of cells per well can then be calculated)

Specific Procedures for Each Experiment

Experiment 1 - A549 and MCF7 Cells
Experiment 2 - LNCaP and 983M Cells
Experiment 3 - HEK and Colo Cells
Experiment 4 - LNCaP and MCF7 Cells
Experiment 5 - 983M and T47D Cells
Experiment 6 - HT1080, DLD2, and DU145 Cells