Table 2.
Grade classification for metastasis on the liver surface and in the liver
| n | No. of each metastatic grade on the liver surface1 | No. of each metastatic grade in the liver2 | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Grade 0 | Grade 1 | Grade 2 | Grade 3 | Grade 0 | Grade 1 | Grade 2 | Grade 3 | ||
| Negative control3 | 8 | 8 | 0 | 0 | 0 | 8 | 0 | 0 | 0 |
| Control4 | 7 | 1 | 2 | 3 | 1 | 1 | 2 | 2 | 2 |
| MSE (50 mg/kg per day)5 | 8 | 5 | 1 | 2 | 0 | 6 | 1 | 0 | 1 |
| MSE (100 mg/kg per day)5 | 8 | 4 | 2 | 2 | 0 | NT | NT | NT | NT |
Assessment of metastasis: On day 21, mice were euthanized, and liver tissues were extirpated, weighed, and fixed with 10% neutral-buffered formalin. The formalin-fixed, paraffin-embedded tissues were sectioned (4 μm) and stained with H&E to evaluate the effect of MSE on intraliver micrometastases. The metastatic index was estimated by calculating the mean of metastatic grades on the liver surface in each group.
Statistics: For the in vivo assessment of metastasis index on the liver surface and in the liver, Dunnett’s multiple comparison test was used after ANOVA to analyze differences between the MSE-treated and control groups. All data analyses were performed with GraphPad Prism 5 statistical software (San Diego, CA). n = number mice per group; NT, not tested. Statistical comparison of the distribution of metastatic grades of control versus each of the MSE treatments in liver and liver surface shows that there is significant inhibition of liver metastasis (P < 0.05). However, there is only a moderate inhibition in the distribution of grades in the treatment versus control on the liver or the liver surface metastasis. Future studies will be designed with larger sample size (n) to obtain firm conclusion on the inhibition of the metastatic grades.
Grade classification: grade 0, no metastatic nodule; grade 1, one to three metastatic nodules; grade 2, four to six metastatic nodules; grade 3, seven or more metastasis nodules.
Grade classification: grade 0, no metastatic focus; grade 1, one to five metastatic foci; grade 2, six to ten metastatic foci; grade 3, eleven or more metastatic foci.
Mice with PBS-only fed with rodent chow without MSE (melinjo seed extract).
Mice with colon-26 cells fed with rodent chow without MSE.
Mice with colon-26 cells fed with rodent chow mixed with MSE.