The effect of Benzamide Riboside on VX2 model of Liver Cancer in Rabbits
Purpose: Benzamide Riboside (BR) causes apoptosis in
multiple tumor cell lines by its inhibition of guanylate biosynthesis. The purpose of this study was to determine
the feasibility of using BR as a therapeutic agent for hepatic artery
infusions.
Materials &
Methods: VX2 tumor was injected into the left lobe of the liver of 14 New
Zealand White rabbits & allowed to grow for 19 + 3 days. Selective catheterization of the hepatic artery
was performed using a 3 French catheter via right femoral cut-down. The animals
were then treated with an infusion of 0.9% Saline (n=2), 1 mg/kg (n=4), 5 mg/kg
(n=4), or 10 mg/kg (n=4) of BR into the hepatic artery. One animal treated with 5mg/kg failed to develop
tumor. In this animal only 1 of 76
cells (0.65%) were apoptotic in the area of the injection scar. The animals were allowed to survive for 24
hours & were sacrificed to explant the liver. The liver was sectioned to produce a section through the tumor
and a section through normal liver. Terminal
deoxynucleotidyl Transferase Mediated dUTP Nick End Labeling (TUNEL) staining
was performed on the slides and imaged on a Zeiss LSM 510 confocal microscope
to detect apoptotic cells. Four random
fields were obtained from each slide and the percentage of apoptotic cells was
calculated by dividing the number of TUNEL positive cells by the total number
of cells. Sections of liver not
involved with tumor were section in 5 animals ; 2 that received 1 mg/kg, 1 at
5mg/kg, and 2 at 10 mg/kg.
Results: Results from a one-way ANOVA model showed
strong evidence of dose effect. In particular, the Fisher’s LSD post-hoc test
showed that means of tumor apoptosis in treated animals was significantly
greater than in animals treated with saline and the mean tumor apoptosis was
greater with 10mg/kg than with 1mg/kg. The table demonstrates the percentage of
apoptotic cells at each dose of BR administered. There were no apoptotic cells
in normal liver treated with 1mg/kg & 10 mg/kg. The animal that received 5mg/kg demonstrated 10.5% apoptotic
cells in the field examined (8/76 cells).
Conclusions: BR induces apoptosis in VX2 tumor in the
rabbit model with limited apoptosis in normal liver.
Table:% apoptosis
of Liver Cancer Cells
|
Dose of BR (mg/kg) |
% Apoptosis |
SD |
|
0 |
1.3%a |
0.1% |
|
1 |
44.8%b |
12.8% |
|
5 |
52.7%bc |
1.9% |
|
10 |
70.7%c |
21.7% |
Note: Groups identified with the same superscript letter are not significantly different (P>0.05).
Appendix: (Results from ANOVA and Fisher’s LSD by Jun)
ANOVA
Apoptosis
|
|
Sum of Squares |
df |
Mean Square |
F |
Sig. |
|
Between Groups |
.652 |
3 |
.217 |
10.232 |
.003 |
|
Within Groups |
.191 |
9 |
.021 |
|
|
|
Total |
.844 |
12 |
|
|
|
Multiple
Comparisons
Dependent Variable: Apoptosis
LSD
|
(I) GP |
(J) GP |
Mean Difference (I-J) |
Std. Error |
Sig. |
95% Confidence Interval |
|
| Lower Bound |
Upper Bound |
|||||
|
0 |
1 |
-.434650(*) |
.1262475 |
.007 |
-.720242 |
-.149058 |
| 2 |
-.514133(*) |
.1330766 |
.004 |
-.815173 |
-.213093 |
|
| 3 |
-.693550(*) |
.1262475 |
.000 |
-.979142 |
-.407958 |
|
|
1 |
0 |
.434650(*) |
.1262475 |
.007 |
.149058 |
.720242 |
| 2 |
-.079483 |
.1113399 |
.493 |
-.331352 |
.172385 |
|
| 3 |
-.258900(*) |
.1030807 |
.033 |
-.492085 |
-.025715 |
|
|
2 |
0 |
.514133(*) |
.1330766 |
.004 |
.213093 |
.815173 |
| 1 |
.079483 |
.1113399 |
.493 |
-.172385 |
.331352 |
|
| 3 |
-.179417 |
.1113399 |
.142 |
-.431285 |
.072452 |
|
|
3 |
0 |
.693550(*) |
.1262475 |
.000 |
.407958 |
.979142 |
| 1 |
.258900(*) |
.1030807 |
.033 |
.025715 |
.492085 |
|
| 2 |
.179417 |
.1113399 |
.142 |
-.072452 |
.431285 |
|
* The mean difference is significant at the .05 level.