General Conclusion

From Experiment 2, it was found that the two drugs, IP6 and PG490, work synergistically against the 983M cells. In the cell line, the IP6 was able to increase the efficiency of triptolide by 100 times, and in turn, the triptolide was able to increase the efficiency of the IP6 by 1000 times. From this, it is deduced that the triptolide is a stronger drug. However, it is inconclusive to whether IP6 and PG490 work synergistically against other cancer cell lines. Of the numerous other cell lines tested, the drugs only proved to have synergistic effects on the 983M cells.

General Discussion

Synergism was only found in the 983M cells, but not in the other cancer cell lines. In experiment 1, the A549 and the MCF7 were inconclusive whether they were synergistic because the concentration of the IP6 did not allow for the calculation of its IC50. This was because of the precipitate formation and high pH in columns 8 – 12, which would affect results.
However, when this problem was fixed in experiment 2, the 983M cells were found to be synergistic. On the other hand, in the LNCaP cells, the drugs did not work together synergistically. In looking at the differences between the LNCaP cells and the 983M cells, a noticeable difference between them is the speed at which the cells proliferate. The 983M cells have an increased proliferation rate as they were set up to have 2000 cells in one well. The LNCaP, however, had 4000 cells per well, due to a decreased proliferation rate. From our research, the drugs specifically target rapid proliferating cells, and thus, cells that proliferate faster are more affected by the drugs.
In Experiments 3 – 5, none of the cell lines tested had drugs that worked synergistically. In Experiment 3, the drugs had little effect on the HEK cells, because this cell line is adopted from the epithelial human cell, which has a slow proliferation rate. This confirms our hypothesis in Experiment 2, about why the drugs were more effective against the 983M cells, which is because they had a high proliferation rate.
In Experiment 6, when the 983M cells were tested under the new concentrations, synergism of the drugs could not be found. The 983M cells were proved to be synergistic in Experiment 2, but were not in this experiment. The difference in these experiments was that different concentrations were applied on the 983M cells. The concentrations in Experiment 3 - 6 focused on a higher range than that of Experiments 1 – 2. Since the 983M cells were synergistic under these low concentrations, we conclude that synergism between the drugs is only possible at lower concentrations.

Experiment-Specific Conlusions/Discussions

Experiment 1 - A549 and MCF7 Cells

From both cell lines, when IP6 is graphed alone against the absorbance, no IC50 could be detected. However, if the IP6 drug concentrations were increased, to incorporate a larger range, eventually all the cells will all be inhibited, and cannot reproduce; they will die. The prediction can be seen in the graph in which there is a parabolic like curve that is leveling off. Logically, with increased drug concentrations the curve will go down and an IC50 will be achieved to prove synergism.

Experiment 2 - LNCaP and 983M Cells

From the results that were collected, there was a clear decrease in the absorbance of the samples in the MTS Assay as the concentrations of the IP6 and the PG490 were raised. There were steep drops in absorbance levels between the concentrations of 0.1mM and 1mM of IP6 and 0.1ng/mL and 1ng/mL of PG490. To better understand this decrease in absorption, it would be most beneficial to investigate this range of concentrations 0.1mM – 1mM of IP6 and 0.1ng/mL – 1ng/mL of PG490 in more depth.

After retrieving the IC50 levels for both the IP6 and the PG490, it was determined that the activity of these two drugs for the LNCaP was not synergistic. However, with the 983M cells synergy was detected. Both the drugs were said to work on any rapidly proliferating cell, one being caner cells, so they should behave either synergistically of additively for both cell lines; which they do not. However, it should be noted that the 983M has a higher rate of proliferation than the LNCaP as it was more confluent (Refer to the Procedure). It is possible that the drugs are more effective against cancer cell lines that rapidly proliferate as in this scenario both drugs are needed to fight the cancer. With cells that proliferate slower, the IP6 is not needed to control the growth and is not as effective in targeting and inhibiting proliferating cells, whereas with the 983M cells, which proliferate quicker, it is easier for the IP6 to target and inhibit cells. The drugs might only be synergistic with rapidly proliferating cells.

Experiment 3 - HEK and Colo Cells

From the results, by expanding the range of concentrations, there was no longer a drastic drop between drug concentrations of IP6 and PG490. Instead there was a smooth killing of cells.
For the HEK cell lines, no trend line can be drawn in the graphs and as a result the IC50s could not be determined. This is probably because the HEK cells are epithelium cells that do not proliferate rapidly like cancer cells. We tested the cell line to see if the drugs would affect the human epithelium cells, but they do not. The HEK cells in culture proliferate faster than in humans, so the drugs should have no affect on these cells.

Experiment 4 - LNCaP and MCF7 Cells

From the results, by expanding the range of concentrations, there was no longer a drastic drop between drug concentrations of IP6 and PG490. Instead there was a smooth killing of cells. This trend has been confirmed.

No synergy was detected in the LNCaP cells. This goes back to the hypothesis made in the conclusion of Experiment 2. Since the LNCaP cells were set in 4000/well, relative to other cell lines such as the 983M cells which were set to be 2000/well, the LNCaP cells proliferate slowly.

It is possible that the drugs are more effective against cancer cell lines that rapidly proliferate as in this scenario both drugs are needed to fight the cancer. With cells that proliferate slower, the IP6 is not needed to control the growth and is not as effective in targeting and inhibiting proliferating cells. Whereas with the 983M cells, which proliferate quicker, it is easier for the IP6 to target and inhibit cells. The drugs might only be synergistic with rapidly proliferating cells.

This hypothesis is confirmed in this experiment. As well as the LNCaP cells behaved similar in both Experiment 2 and 4, confirming the received results.

The MCF7 cells were set to be 2000/well and proliferated relatively quickly (like the 983M cell line which was proven to be synergistic), however since the range of IP6 was too small, and IC50 for IP6 could not be determined and neither could synergism.

Experiment 5 - 983M and T47D Cells

The 983M cells were not synergistic at these new concentrations. In looking at the data, there is no longer a jump from cells to no cells with increasing concentrations. Rather there is a smoother killing of cells (Refer to the stained and fixed cells on the microtiter plate).

The 983M cells in Experiment 2 were proven to have a synergistic affects with IP6 and PG490. However, in this experiment there is no synergism between the drugs. The only difference is that there are different concentrations. Perhaps, synergism can only occur at lower concentrations as with this experiment, the increased range allowed for a general increase in the concentration of drugs. All the cells tested at this new concentration did not show synergism and neither did the 983M; this is sufficient evidence.

Experiment 6 - HT1080, DLD2, and DU145 Cells

N/A (Since only observations were made)