Evaluation of anti-cancer and immunomodulatory effects of carnosol in a Balb/c WEHI-164 fibrosarcoma model

Figures & data

Figure 1. Tumor volumes in treated mice. Values shown are from measures made on Days 0, 4 and 7 of the treatment regimens indicated. Values shown are mean ± SE (n = 6/group). Values significantly different from vehicle control mice at *p < 0.05; **p < 0.01 and ***p < 0.001.

Figure 2. Splenocyte (MNC) proliferation assays. Values shown are from measures made using splenocytes harvested 24 h after the final treatment (harvested on Day 7) from hosts in the indicated regimens. Values shown are mean ± SE (n = 6/group). Values significantly different from vehicle control at **p < 0.01 and ***p < 0.001.

Figure 3. Cytokine formation by isolated splenocytes (MNC). Levels of IFNγ, IL-4, and IL-10 produced after culture of splenocytes harvested 24 h after the final treatment (harvested on Day 7) from hosts in the indicated regimens. Values shown are mean ± SE (n = 6/group). Values significantly different from vehicle control at *p< 0.05 and ***p< 0.001. ^###Value significantly different from positive control (p < 0.001).

Table 1. Spontaneous cytokine release by splenocytes from mice in treatment groups.

Groups	IFNγ	IL-4	IL-10	IFNγ/IL-4
Vehicle control	2.51 ± 0.08	4.31 ± 0.07	9.73 ± 0.06	0.58 ± 0.02
Cyclophosphamide	2.18 ± 0.81	3.51 ± 0.06***	4.60 ± 0.23***	0.62 ± 0.21
Carnosol (5 mg/kg)	2.36 ± 0.09	3.38 ± 0.10***	5.04 ± 0.15***	0.70 ± 0.02*
Carnosol (10 mg/kg)	2.46 ± 0.13	3.35 ± 0.08***	4.42 ± 0.15***	0.74 ± 0.04*

Value significantly different from vehicle control at *p < 0.05 and ***p < 0.001.

Figure 4. Percentage of intra-tumor and splenic CD4⁺CD25⁺FoxP3⁺ T-cells. Levels of indicated cells among splenocytes/tumor tissues harvested 24 h after the final treatment (harvested on Day 7) from hosts in indicated regimens. All values shown are based on CD4⁺ cell levels in the sample set as a 100% value. Values shown are mean ± SE (n = 6/group). Values significantly different from vehicle control at *p < 0.05 and ***p < 0.001.

Table 2. Absolute numbers of splenic lymphocyte sub-populations/gram spleen.

Parameter	Vehicle	Cyclophosphamide	Carnosol (5 mg/kg)	Carnosol (10 mg/kg)
CD3^+ (×10^8)	4.20 ± 0.13	2.80 ± 0.25*	3.69 ± 0.19	3.61 ± 0.15
CD4^+ (×10^8)	2.72 ± 0.06	1.70 ± 0.02*	2.31 ± 0.07	2.50 ± 0.02
CD8^+ (×10^8)	1.39 ± 0.05	0.93 ± 0.06*	1.35 ± 0.07	1.11 ± 0.04
CD4^+CD25^+Foxp3^+ (×10^8)	0.38 ± 0.02	0.02 ± 0.01***	0.31 ± 0.03*	0.29 ± 0.01*

Value significantly different from vehicle control at *p < 0.05 and ***p < 0.001.

Table 3. Absolute numbers of intra-tumor lymphocyte sub-populations/gram tumor tissue.

Parameter	Vehicle	Cyclophosphamide	Carnosol (5 mg/kg)	Carnosol (10 mg/kg)
CD3^+ (×10^3)	560.5 ± 2.8	368.2 ± 7.0*	496.5 ± 25	557.3 ± 32
CD4^+ (×10^3)	279.1 ± 18.1	177.3 ± 4.1*	284.0 ± 28.3	287.9 ± 18.9
CD8^+ (×10^3)	268.8 ± 29.1	148.0 ± 9.5*	181.0 ± 16.5	252.9 ± 26.1
CD4^+CD25^+Foxp3^+ (×10^3)	17.4 ± 1.7	0.8 ± 0.5***	13.4 ± 4.00*	2.1 ± 0.2***

Value significantly different from vehicle control at *p < 0.05 and ***p < 0.001.

Figure 5. Percentage of intra-tumor and splenic CD3⁺ T-cells. Levels of indicated cells among splenocytes/tumor tissues harvested 24 h after the final treatment (harvested on Day 7) from hosts in the indicated regimens. All values shown are based on CD4⁺ cell levels in the sample set as a 100% value. Values shown are mean ± SE (n = 6/group). Values significantly different from vehicle control mice at *p < 0.05 and ***p < 0.001.

Figure 6. Percentages of CD4⁺ and CD8⁺ T-cells and CD4⁺/CD8⁺ ratios in spleens. Levels of indicated cells (and calculated ratios) among splenocytes harvested 24 h after the final treatment (harvested on Day 7) from hosts in the treatment regimens indicated. All values shown are based on CD3⁺ cell levels in the sample set as 100% value. Values shown are mean ± SE (n = 6/group). Values significantly different from vehicle control mice at *p < 0.05.

Figure 7. Percentages of CD4⁺ and CD8⁺ T-cells and CD4⁺/CD8⁺ ratios in tumor samples. Levels of indicated cells (and calculated ratios) among tumor samples harvested 24 h after the final treatment (harvested on Day 7) from hosts in indicated regimens. All values shown are based on CD3⁺ cell levels in the sample set as 100% value. Values shown are mean ± SE (n = 6/group).