1.2 Tumour Formation Assay

Dr. Ju

11 1.2 Tumour Formation Assay

Brief Overview

While researchers made the conclusion that vitexin reduced A549 cell viability by inducing apoptosis in the previous experiments, it’s important to note that all the experiments were in vitro. The last part of the experiment consisted of an in vivo study. In vivo experiments are a great way of seeing if the produced results in vitro are reproducible in living organisms. Tumour formation assay is an excellent way of measuring and determining the self-renewal and multipotent nature of the cancer cells. In order to determine this, healthy mice are injected with cancerous cells and treated with the drug of choice at different doses while one group remains untreated as a control group. At the end of the study, there should be a clear dosage winner which would be the most effective drug to slowing down cell proliferation.

Experiment Protocol

For the last part of the experiment, fifteen male nude mice aged 5–6 weeks were purchased from Shanghai Laboratory Animals Center. They injected the mice with 2×10⁶ A549 cells into a single side of the posterior flank of the mice. Every three days, they measured the size of the tumour using a calliper. When the tumour size reached approximately 100 mm^3, the mice were randomized into three groups. The mice in low dose were treated with 1 mg/kg vitexin, and the high dose group was treated with 2 mg/kg vitexin while the mice in the control group received 0.1% DMSO daily for four weeks. At the end of the study (Day 19), the tumours were excised and weighed.

Results

The treatment of vitexin was shown to reduce the rate of growth of the A549 cell-derived tumours. In figure 2, vitexin at higher doses was able to show an evident decrease in the rate of growth when compared to tumours not treated. When measuring the weight of the tumours after the experiment ended, there are similar results. The end weight of the tumours is significantly lower with treatment of vitexin, as seen in figure 3.

Figure 2. Treatment with vitexin on A549 tumours reduces their growth. A high dose of vitexin is 2mg/kg and a low dose is 1mg/kg.

Figure 3. Treatment of vitexin for A549 cell derived tumours in mice reduced their weight after 19 days. Error bars represent one standard deviation (SD) from the mean. An asterisk (*) indicates a statistical significance compared to untreated tumours (P<0.05).

Western blot analysis done of the tumour cells show similar outcomes as the cells treated in vitro. Vitexin treatment significantly increased the expression of pro-apoptotic genes, seen as a decrease in Bcl-2/Bax ratio and an increase in cleaved caspase-3.

Figure 4. Western blot analysis shows that vitexin treatment on A549 cell derived tumours in mice have greater expression of pro-apoptotic proteins. Error bars represent one standard deviation (SD) from the mean. An asterisk (*) indicates a statistical significance compared to untreated tumours (P<0.05).

Statistical Analysis

All the statistical analyses for this experiment were performed using GraphPad Prism 6.0. All experimental data are shown as the mean standard deviation and analyzed using a one-way analysis of variance, which is a technique that can be used to compare means of two or more samples. Also, Dunnett’s post hoc test was used, and a P value less than 0.05 was considered to indicate a statistically significant difference.

Summary

The researchers conducted the following in vivo experiments to determine the effects of vitexin on tumours:

A549 cells were injected in mice and were able to grow to form tumours.
Tumours were treated with two different doses of vitexin.
Western blot analysis showed a decrease in Bcl-2/Bax ratio and an increase in cleaved caspase-3.