Effect of Artesunate on HT-29-AK Cancer Cells

Effect of Artesunate on HT-29-AK Cancer Cells, and its Therapeutic Implications

Cancer Biology Coursework II

  1. The possible cytotoxic effect of Artesunate on the survival factors and the concentration of HT-29-AK cells over different incubation periods and its therapeutic implications.
      1. The possible cytotoxic effect of Artesunate on the survival factors and the concentration of HT-29-AK cells over different incubation periods and its therapeutic implications.
      1. Background: HT-29-AK are cancer cells, Artesunate is an antimalarial compound which could possibly be used as an anti-tumour agent. The present study attempts to confirm the incubation period most effective in decreasing the concentration of HT-29-AK cancer cells.

Aim/Hypothesis: This experiment tests the effect of Artesunate on the E-Cadherin mRNA expression, VEGF-alpha and beta mRNA expression, Survivin and on caspase-3 expression.

Methods: 96 well plates were used and HT-29-AK cells were incubated at different concentrations over different time periods to examine the effective concentration and incubation period. The E-Cadherin mRNA expression was measured using immunocytochemistry and the Survivin and VEGF-alpha and beta mRNA levels were also measured using methods such as qPCR and ELISA.

Results: We could show that at lower concentrations and a 72 hour incubation period Artesunate killed HT-29-AK cells, and decreased E-Cadherin and VEGF-alpha and beta levels. Levels.

Conclusion: The results allude to the cytotoxic effect of Artesunate and lower concentrations for 72 hour incubation periods and its effect on HT-29-AK cells with potential clinical applications.

Figure 1 shows the concentration of ART on the X-axis and the percentage of control growth on the Y axis, this graph is aimed to show the effect of ART on HT-29-AK cells over varying periods of time.

As the incubation time period increases the drug is becoming more cytotoxic, if the cells are incubated with the drug for longer, a lower concentration is required. The pharmacological index is 72hr>48hr>24hr, the IC50 is the concentration at which the cells need to be incubated to kill half the number of cells:

24hrs: incubating the cells over of 24 hours leads to an IC50 of 165µM, this alludes to the requirement for higher concentration over shorter incubation periods. The Concentration required is 100.39µM more than if the cells were incubated for 48hours and 150.56 µM more if the cells were incubated for 72 hours.

48hrs: incubating the cells over of 48 hours leads to an IC50 of 64.61µM, the concentration required to kill half the number of HT-29-AK cells is 100.39µM less if the cells were incubated for 48 hours instead of 24 hours, however the concentration required to kill half the number of cells is 50.17µM more than if the cells were incubated for 72 hours instead of 48 hours.

72hrs: incubating the cells over of 72 hours leads to an IC50 of 14.44µM, the concentration required to kill half the number of cells over 72 hours is 150.56µM less than incubation for 24hours, and 50.17µM less than incubation for 48 hours.

Figure A and B shows the relative E-cadherin mRNA levels at different ART concentration incubated at 24 hours(Left) and 72 hours(right).

Relative E-Cad mRNA levels at 24 hours

Control: The control showed a relative E-Cad mRNA level of 1, at a concentration of 82.53µM

The concentration at 82.53µM showed a relative E-cad mRNA level of approximately 1.1, this relative expression is 0.1 more than the controls relative expression, the relative expression of E-cad mRNA levels at 82.53µM was 0.9 less than the relative expression of E-Cad mRNA at 165.06µM and 1.8 less than the relative expression of E-cad mRNA at 330.12µM. The concentration at 165.06µM, showed a relative E-cad mRNA level of approximately 1.9, which was 0.8 more than the expression at 82.53µM and 1.0 less than the expression at 330.12 µM. the concentration at 330.12µM, showed a relative E-cad mRNA level of approximately 2.9, an increase of 1.8 is observed compared to the ART concentration of 82.53µM and an increase of 1.0 is observed compared to the ART concentration of 165.06µM.

Relative E-Cad mRNA levels at 72 hours

Control: The control showed a relative E-Cad mRNA level of 1, at a concentration of 82.53µM

The concentration at 7.22µM showed a relative E-cad mRNA level of approximately 0.4, this relative expression is 0.6 less than the controls relative expression, the relative expression of E-cad mRNA levels at 14.44µM equal to the relative expression of E-Cad mRNA at 7.22µM and 0.01 less than the relative expression of E-cad mRNA at 28.88µM. The concentration at 28.88µM, showed a relative E-cad mRNA level of approximately 0.41, which was 0.01 more than the expression at 7.22 µM and 14.44µM

Figure C shows the level of staining of adhesion molecules

At 24 hours the control showed the least amount of staining compared to the ART concentrations at 82.53µM, 165.06µM and 330.12µM. At a concentration of 82.53µM there is an increase in staining compared to the control but there is less staining compared to 165.06 and 330.12µM concentrations. At an ART concentration of 165.06 µM more staining is observed compared to the control and at 82.53 µM however less staining is observed compared to 330.12µM. At the final ART concentration 330.12 µM an increase in staining is observed compared to the control, 82.53µM, and 165.06µM.

At 72 hours the control showed the most amount of staining compared to the ART concentrations at 7.22µM, 14.44µM and 28.88µM. At a concentration of 7.22µM there is a decrease in staining compared to the control but there is more staining compared to 14.44µM and 28.88µM concentration. At an ART concentration of 14.44µM less staining is observed compared to the control and at 82.53µM however more staining is observed compared to 28.88µM. At the final ART concentration 28.88µM a decrease in staining is observed compared to the control, 7.22µM and 14.44µM.

Figure A and B show the relative VEGF-alpha and beta mRNA levels at different ART concentrations.

Control: The control concentration showed the same relative mRNA levels for both VEGF- alpha and beta which was a level of 1.

7.22µM: the relative VEGF- alpha concentration was approximately 0.62, and the VEGF- beta concentration was 0.39, this means that at a concentration of 7.22µM, 0.23 µM more of VEGF-alpha mRNA levels is expressed compared to VEGF-beta mRNA levels.

14.44µM: the relative VEGF- alpha concentration was approximately 0.64, and the VEGF- beta concentration was 0.35, this means that at a concentration of 14.44µM, 0.19 µM more of VEGF-alpha mRNA levels is expressed compared to VEGF-beta mRNA levels.

28.88µM: the relative VEGF- alpha concentration was approximately 0.61, and the VEGF- beta concentration was 0.05, this means that at a concentration of 28.88µM, 0.56 µM more of VEGF-alpha mRNA levels is expressed compared to VEGF-beta mRNA levels.

The control showed a relative survivin mRNA level of 2, an ART concentration of 7.22µM showed a mRNA survivin expression of approximately 4 which is 2 more than the control. At an ART concentration of 14.44 µM a relative mRNA expression of 13 is observed, an mRNA expression of 9 more than at 7.22 µM and 13 less than 28.88µM.

At an ART concentration of 28.88µM a relative mRNA Survivin expression of 26 is observed, this level of expression is 13 more than at 14.44µM and 22 more than at 7.22µM. these results show that Survivin which is an inhibitor of apoptosis is inhibited over a 72 hour incubation period and a concentration of 7.22µM.

The control showed a % cleaved caspase 3 level of 100, an ART concentration of 7.22µM relative to the control showed 300% cleaved caspase-3 level which is 200% more than the control. At an ART concentration of 14.44 µM a relative to the control 320% cleaved caspase-3 levels was observed, which is 20% more than at 7.22 µM and 5% less than 28.88µM. At an ART concentration of 28.88µM the percentage of cleaved caspase-3 relative to the control was 325%, this level of expression is 5% more than at 14.44µM and 25% more than at 7.22µM.

4. Discussion

Jiang W et al experimented with Artesunate on osteosarcoma cells, Artesunate was combined with another compound called allicin, which was derived mainly from garlic. The aim of this experiment was to investigate the synergistic effects of the combined therapy. The results of this experiment showed a decrease in concentration of osteosarcoma cells, a decrease in invasion, motility, and the colony formation of these cells, this occurred due to an increase in Caspase3/9 expression when combined. In Prof Olivera’s experiment the methods only included Artesunate and the cleaved caspase activity were all similar at different concentrations, the difference in methodology is apparent because two compounds was used in Jiang W et al’s study while only one was used in Prof Oliviera’s study.

Liu Y et al also used a combination therapy to investigate the cytotoxic effect, triptolide and Artesunate was used to inhibit the pancreatic cell line growth by inducing apoptosis, the experiment also showed a production of heat shock proteins which produce synergic effects. Similar to Prof Oliviera’s experiment Artesunate has a cytotoxic effect, however the similarity between these two experiments is that the combination therapy used in Liu Y et al and the single therapy used in Prof Oliviera’s study both were more effective in lower concentrations of Artesunate these allude to potential clinical applications.

Dong HY et al experimented the effects of Artesunate on breast cancer using tumour transplanted nude mice, cyclophosphamide or normal saline was used in combination with Artesunate and the results showed ART inhibiting the growth of the MCF-7 cancer cells by arresting the cell cycle.

In conclusion the findings of Prof Oliviera’s study is as follows:

      • Over a longer incubation period a lower concentration of Artesunate is required to kill half of the cancer cells.
      • Over 72 hours less Artesunate is reuired to reduce the relative mRNA levels. And less staining of adhesion molecules is observed over 72 hours as the concentration increases.
      • ART over 72 hours has a greater effect on decreasing the expression of VEGF-beta compared to VEGF-alpha.
      • Over 72 hours the higher the concentration of ART the increase in relative survivin mRNA levels.
      • The percentage of cleaved caspase-3 levels relative to the control increases as the concentration of ART increases.

References

      1. Dong HY et al, ‘Antitumour effects of Artesunate on human breast carcinoma MCF-7 cells and IGF-IR expression in nude mice xenografts’, 2014
      2. Liu Y et al, ‘Synergism of cytotoxicity effects of triptolide and Artesunate combination treatment in pancreatic cancer cell lines’, 2013
      3. Jiang W et al, ‘The synergistic anticancer effect of Artesunate combined with allicin in osteosarcoma cell line in vitro and in vivo’, 2013

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