Use of clonogenic assays to investigate the cytotoxic effects of drugs used in cancer therapy

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Use of clonogenic assays to investigate the cytotoxic effects of drugs used in cancer therapy

Introduction

The clonogenic or colony-forming assay was first developed and described in a technical paper written by Puck and Marcus (1956). Puck and Marcus (1956) generated the first radiation dose response curve for x-ray irradiated mammalian cells using the now infamous HeLa cell line. Whilst the aims of this initial landmark study differ somewhat from the aims of this experiment, the method used remains relatively unchanged. The clonogenic assay enables the researcher to assess the differences in the reproductive vitality of cells that have been subjected to various forms of treatment, which can include radiation or cytotoxic agents, and compare them to untreated controls. Rafehi et al (2011) state that the clonogenic assay has become the most widely accepted and used technique for evaluating the radiation sensitivity of different cell lines and for determining the effects of cytotoxic agents or other anti-cancer therapeutics on colony forming cell lines.

According to Rafehi et al (2011), a typical clonogenic assay experiment, which uses adherent cell lines, relies upon three separate components. These are the treatment of the cell monolayer within the cell culture flasks, the development of single cell suspensions and their plating into petri dishes and finally, the fixing and staining of the colonies formed on these petri dishes after a set incubation period (usually between 1 and 3 weeks). In this experiment, we aimed to test the effects of different anti-cancer drugs on the clonogenic propensity of three human cell lines.

Methods

Single cell suspensions of HCT116 and SW620 cell lines were prepared by trypsinization. The growth medium of each of the cell lines was added to 5ml of chlorine based bleach before 1ml of trypsin (0.5% w/v) / EDTA (0.22 % w/v) was added and allowed to incubate for 5 - 10 mins. 4ml of serum-containing growth medium was added to neutralise the trypsin and the cells were detached by triturating the mixture up and down before transferring the cell suspension to a plastic tube. Cells were then counted using a haemocytometer and dilutions carried out accordingly to achieve 20ml of cells at 100 cells/ml. Dilutions of the three drugs (Taxol, Cisplatin and Etoposide) were carried out to achieve final concentrations of 0.01, 0.1 and 1uM.

2x6-well dishes were then labelled with the drug used and the triplicate wells were labelled with control, 0.01, 0.1 and 1uM. 3ml of growth medium containing 20% foetal calf serum was added to each of the wells and 1ml of each of the drug dilutions was added to the correspondingly labelled well. After resuspending the cells in the cell suspension, 1 ml of this suspension was then added evenly across each of the wells. These were then incubated at 37oC for 10-14 days or until colonies could be seen on one or more of the plates.

After establishing the formation of colonies, the medium from each dish was discarded into bleach. Approximately 2ml of undiluted methanol was then added and the plates were left at room temperature for 10 minutes. After discarding the methanol, crystal violet solution (0.4% w/v in PBS) was added to cover the cells and left to stain for 30 minutes at room temperature. Plates were then washed and left inverted to drain and dry overnight. Colonies were then counted by hand and the results recorded.

Results

Figure 1. Box & Whisker plot showing the mean, standard deviation and range of Taxol treated HCT116 cells. This plot shows that colony numbers gradually increased at low drug concentrations, before falling sharply at 1uM,

Figure 2.Box & Whisker plot showing the mean, standard deviation and range of Cisplatin treated HCT116 cells.This plot shows a gradual decline in colonies with the rise in drug concentration.

Figure 3.Box & Whisker plot showing the mean, standard deviation and range of Etoposide treated HCT116 cells.This plot shows a gradual decline in colonies with the rise in drug concentration.

Figure 4.Box & Whisker plot showing the mean, standard deviation and range of Taxol treated SW620 cells.This plot shows that colony numbers gradually increased at low drug concentrations, before falling sharply at 1uM,

Figure 5.Box & Whisker plot showing the mean, standard deviation and range of Cisplatin treated SW620 cells.This plot shows a gradual decline in colonies with the rise in drug concentration.

Figure 6.Box & Whisker plot showing the mean, standard deviation and range of Etoposide treated SW620 cells.This plot shows a gradual decline in colonies with the rise in drug concentration.

By carrying out a Kruskal-Wallis statistical test, comparisons between the average number of colonies formed after treatment with each drug were made.

Kruskal-Wallis: all pairwise comparisons (Dwass-Steel-Chritchlow-Fligner) for HCT116 treated cells

Variables: Taxol, Cisplatin, Etoposide

Groups = 3

df = 2

Total observations = 312

T = 4.88149

P = 0.0871

Adjusted for ties:

T = 4.90188

P = 0.0862

Critical q (range) = 3.314493

Taxol vs. Cisplatinnot significant

(|-2.361118| > 3.314493)P = 0.217

Taxol vs. Etoposidenot significant

(|0.057359| > 3.314493)P = 0.9991

Cisplatin vs. Etoposidenot significant

(|-2.944388| > 3.314493) P = 0.0936

Kruskal-Wallis: all pairwise comparisons (Dwass-Steel-Chritchlow-Fligner) for SW620 treated cells

Variables: Taxol, Cisplatin, Etoposide

Groups = 3

df = 2

Total observations = 360

T = 21.037559

P < 0.0001

Adjusted for ties:

T = 21.143118

P < 0.0001

Probability value suggests that at least one of the sample populations tends to yield larger observations than at least one other sample population.

Critical q (range) = 3.314493

Taxol vs. Cisplatinsignificant

(|5.553061| > 3.314493)P = 0.0003

Taxol vs. Etoposidesignificant

(|-5.833213| > 3.314493)P = 0.0001

Cisplatin vs. Etoposidenot significant

(|-0.452553| > 3.314493) P = 0.9451

Discussion

The box and whisker plots show that cisplatin and etoposide both behave in a similar manner with the number of colonies reducing as the concentration of the drug increases. However, the number of colonies forming on the Taxol treated plates showed a slight increase between the 0.01uM and 0.1uM concentrations, before falling sharply with the 1uM concentration. This mimics the findings presented by Liebmann et al (1993); however, Liebmann et al (1993) found that the effective range of Taxol was much lower than our study suggests. Liebmann et al (1993) found that the fraction of surviving cells fell sharply after 24 hours exposure to Taxol at concentrations between 0.0025uM and 0.0075uM.

The similarity in action between cisplatin and etoposide is also shown by the Kruskal-Wallis test. Whilst the first of these tests, which compares the action of the three drugs on HCT116 cells, shows no significant difference between them, the second test, which compares the action of the three drugs on SW620 cells, shows that the number of colonies forming on the Taxol treated plates were significantly less than both Etoposide and Cisplatin. These latter two drugs were not significantly different. These results are supported by findings published by Kelland and Abel (1992), who found that Taxol was more than 1000 times more cytotoxic than either cisplatin or etoposide. The mode of action of Taxol was considered to be due to its ability to induce mitotic arrest, however, recent studies have identified that intratumoural concentrations are too low to cause mitotic arrest but do induce multipolar divisions (Weaver, 2014). In contrast, cisplatin causes DNA damage and lesions, which induces the apoptosis response (Siddik, 2003). However, recent studies have shown that certain cells can become resistant to this mode of action (Galluzzi et al, 2011).

Overall, this experiment has shown that all three drugs display a cytotoxic effect, which limits the colony forming ability of cancer cells. However, whilst all three worked in a similar fashion on HCT116 cells, Taxol showed improved results on the SW620 cell line when compared to cisplatin and etoposide.

Word count: 1278(exclude reference)

References

Galluzzi, L., Senovilla, L., Vitale, I., Michels, J., Martins, I., Kepp, O., &Kroemer, G. (2011).Molecular mechanisms of cisplatin resistance.Oncogene,31(15), 1869-1883.

Kelland, L. R., & Abel, G. (1992). Comparative in vitro cytotoxicity of taxol and Taxotere against cisplatin-sensitive and-resistant human ovarian carcinoma cell lines. Cancer Chemotherapy and Pharmacology, 30(6), 444-450.

Liebmann, J. E., Cook, J. A., Lipschultz, C., Teague, D., Fisher, J., & Mitchell, J. B. (1993). Cytotoxic studies of paclitaxel (Taxol) in human tumour cell lines. British Journal of Cancer, 68(6), 1104.

Puck, T. T., & Marcus, P. I. (1956). Action of X-rays on mammalian cells. The Journal of Experimental Medicine, 103(5), 653-666.

Rafehi, H., Orlowski, C., Georgiadis, G. T., Ververis, K., El-Osta, A., &Karagiannis, T. C. (2011). Clonogenic assay: adherent cells. Journal of Visualized Experiments, (49), 2573.

Siddik, Z. H. (2003). Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene, 22(47), 7265-7279.

Weaver, B. A. (2014). How Taxol/paclitaxel kills cancer cells. Molecular Biology of the Cell, 25(18), 2677-2681.

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