Effect of Thuja occidentalis and its polysaccharide on cell-mediated immune responses and cytokine levels of metastatic tumor-bearing animals

Figures & data

Table 1.  Primer sequences.

Genes	Primer sequence		Product size
IL-6	Forward	5′-ATGAAGTTC CTCTCTGCAAGAGAC-3′	638bp
	Reverse	5′-CACTAGGT TTGCCGAGTAGATCTC-3′
IL-1β	Forward	5′-ATGGCAACTG TTCC TGAACTCAACT-3′	563bp
	Reverse	5′-CAGGACAGGTATAGAT TCTTTCCTTT-3′
TNF-α	Forward	5′-ACTCCCAGAAAAGCAAGCAA-3′	688bp
	Reverse	5′-T G GAAGACTCCTCCCAGGTA-3′
GM-CSF	Forward	5′-TGTGGTCTACAGCCTCTCAGCAC 3′	368bp
	Reverse	5′-CAAAGGGGATATTCAG TCAGAAAGGT 3′
GAPDH	Forward	5′-CGTCCCGTAGACAAAATGGT-3′	557bp
	Reverse	5′-CCTTCCACAATGCCAAAGTT-3′

bp, base pairs; GAPDH, reduced glyceraldehyde phosphate dehydrogenase; GM-CSF, granulocyte monocyte-colony stimulating factor; IL, interleukin; TNF, tumor necrosis factor.

Figure 1.  Effect of Thuja occidentalis and T. occidentalis L. (Cupressaceae) polysaccharide (TPS) on natural killer cell activity in metastasis tumor-bearing animals. Mice were treated with T. occidentalis or TPS and spleen cells were obtained. The spleen cells were mixed with chromium labeled K-562 cells and % cell lysis was determined by 4-h chromium release assay (n = 3/point).

Figure 2.  Effect of Thuja occidentalis and T. occidentalis L. (Cupressaceae) polysaccharide (TPS) on antibody-dependent cell-mediated cytotoxicity in metastasis tumor-bearing animals. Mice were treated with T. occidentalis or TPS and spleen cells were obtained. The spleen cells were incubated with chromium labeled sheep erythrocytes and the % cell lysis was determined by 4-h chromium release assay (n = 3/point).

Figure 3.  Effect of Thuja occidentalis and T. occidentalis L. (Cupressaceae) polysaccharide (TPS) on antibody-dependent complement-mediated cytotoxicity in metastasis tumor-bearing animals. Mice were treated with T. occidentalis or TPS and blood was obtained. Sera were incubated with fresh rabbit serum (complement) and B16F-10 cells. The cytotoxicity was assessed by Trypan blue exclusion method (n = 3/point).

Table 2.  Effect of T. occidentalis and TPS on CTL generation (in vivo).

Treatment	Survival rate of animals
	Control	T. occidentalis	TPS
EL-4 alone	26.8 ± 4.4
EL-4 + normal spleen	36.8 ± 4.2
EL-4 + normal spleen + compound		46.9 ± 3.7^a	43.1 ± 3.7^ns
EL-4 + treated spleen		43.5 ± 4.0	40.3 ± 5.1^ns
EL-4 + treated spleen + compound		54.4 ± 5.8^aΨ	50.2 ± 3.9^aΨ

Alloimmunization was carried out by injecting the spleen (2 × 10⁷) cells from C57BL/6 mice to normal, Thuja occidentalis and TPS-treated BALB/C mice. Winns neutralization test was carried out according to the method. The cells were injected intraperitoneally to treated and untreated mice. The animals were observed daily for 80 days after tumor inoculation.

^ap < 0.001, ^bp < 0.01 compared to EL-4 + normal spleen. ^Ψp < 0.01 compared to EL-4 + treated spleen + compound.

Table 3.  Effect of Thuja occidentalis and TPS on pro-inflammatory cytokine profile of metastasis-induced animal.

Cytokine	Normal	Control		T. occidentalis		TPS
	Day 0	Day 7	Day 21	Day 7	Day 21	Day 7	Day 21
IL-1β	16 ± 3.5	44.8 ± 4.9	59.4 ± 5.7	38.9 ± 2.5^b	20.3 ± 2.7^a	39.4 ± 3.1^b	23.9 ± 2.6^a
TNF-α	20 ± 1.8	241.8 ± 20.7	317.2 ± 26.7	134.4 ± 12.4^a	31.1 ± 2.3^a	149.4 ± 17.1^a	42.4 ± 3.6^a
IL-6	35.3 ± 6.5	353.4 ± 39.1	593 ± 47.3	73.9 ± 6.5^a	255.3 ± 29.7^a	92.9 ± 7.3^a	273.4 ± 29.8^a
GM-CSF	18 ± 0.94	38.38 ± 2.7	20.8 ± 1.5	30.7 ± 2.9^b	16.6 ± 0.5^a	34.1 ± 4.7^ns	19.3 ± 1.5^ns
IL-2	23 ± 2.7	22.4 ± 2.0	20.9 ± 2.4	38.0 ± 2.7^a	36.1 ± 3.0^a	29.8 ± 2.4^a	28.5 ± 2.8^b
TIMP	652 ± 46.8	514 ± 69.8	468 ± 13.6	656 ± 41.3^b	701 ± 93.4^a	593.1 ± 21.7^b	623.4 ± 82.1^b

All the values are mean ± SD. Blood was collected from the metastasis-induced animals by injecting B16F-10 melanoma cell after seventh and twenty-first day. Serum was separated by centrifugation and cytokine levels were estimated by ELISA method. ^ap < 0.001, ^bp < 0.01 compared to respective control.

ELISA, enzyme-linked immunosorbent assay; GM-CSF, granulocyte monocyte-colony stimulating factor; IL, interleukin; TIMP, tissue inhibitor matrix metalloproteinases; TNF, tumor necrosis factor.

Figure 4.  Effect of Thuja occidentalis and TPS on pro-inflammatory gene expression. B16F-10 cells were incubated in the presence and absence of T. occidentalis or TPS for 4 h at 37°C in 5% CO₂ in serum free medium. Total RNA was extracted from B16F-10 cells and cDNA was synthesized and used for the amplification of pro-inflammatory cytokines, namely, IL-1β, IL-6, GM-CSF, and TNF-α genes. Mouse GAPDH (reduced glyceraldehyde phosphate dehydrogenase) was used as housekeeping gene. Polymerase chain reaction products were analyzed by agarose gel electrophoresis (n = 3).