The immunomodulating effects of the Chinese herbal medicine Gan-Luh-Yin (GLY) on oral cancer cells：in vitro and in vivo

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Oral cancer is one of the major cancers in Taiwan. It was reported that the number of T and B cells and the levels of IL-2 in oral cancer patients are lower than normal subjects. However, the level of IL-4, IL-6 and TNF-α in oral cancer patients are higher. Gan-Luh-Yin (GLY) is usually used for promoting cure of Chinese herbal medicine in clinical patients. However, the effects of GLY anti-inflammation and immunomodulation are not clear in oral cancer. The purpose of this study is to investigate the mechanisms of GLY via anti- inflammation and immunomodulation. We measured the proliferation of T and B cells by Celltiter96 kit. And the stimulation of cytokines, the cell populations in peripheral blood mononuclear cell (PBMC), the activity of phagocytosis in macrophage and cytotoxic ability in nature killer (NK) cell were detected by Flow cytometer. The cytokines regulated proteins were determined by Western blotting and immunofluorescence staining. And the expression of nuclear factor kappa B (NF-κB) mRNA was measured by RT-PCR. We also used promoter assay and EMSA for determent the DNA binding ability in HNSCC cell line. In vitro, the results indicate that GLY in 200, 500 and 1000 μg/mL can promoted the proliferation of T and B cells under Con A and LPS stimulation, respectively. The result from CBA and FACS can examination indicated that: GLY can inhibit the secretion of TNF from CAL27, SCC-4, HSC-3 and TW206 cell. Beside, GLY suppressed TNF mRNA level by Real-time PCR. GLY also suppressed expression of proteins of the Akt phosphorylation, NF-κB p65 and ERK1/2. The translocation and DNA binding ability of NF-κB were decreased in CAL27 cell. In vivo, we treated BALB/c Nude mice and BALB/c mice with different concentrations of GLY. Our results showed that GLY were not effect on the body, liver and spleen weights of BALB/c mice. GLY could decrease the cytokines levels of TNF-α, and increase the levels of T cell and macrophage population in PBMC. And it also increased macrophage phagocytosic activity. In addition, GLY also increased the spleen cells proliferation when stimulated by Con A. GLY also reduced the volume of drinking water in BALB/c Nude mice orthotopic animal model. Taken together, we suggested GLY can promote anti-inflammatory in oral cancer may through effect on the levels of TNF-α. And the mechanisms of immune- respones were caused by increase the population of T cells and macrophage phagocytosic activity, then decrease the level of TNF-α in vivo.