The Antitumor Effect And Monocyte Stimulated Activity Of Acanthopanax Gracilistylus Extract

Objective: study the antitumor effect and monocytic stimulated activity of Acanthopanax gracillus extract, isolate and purify its antitumor active component, produce the scientific evidence for developing and manufacturing new antitumor drugs.Methods: 1. using 3H-TdR incorporation method, we investigated the suppressive effect of acanthopanax gracilistylus extract on the proliferation of tumor cells such as MT-2, HL-60, Raji, TMK-1 and HSC-2 in vitro.2. Tumor bearing mouse modes were constructed by injecting tumor cell in subcutaneous, then were treated with Acanthopanax gracilistylus extract to observe its antitumor activity on tumor cells in vivo.3. The antitumor active components of acanthopanax gracilistylus extract were isolated and purified by chromatography, then confirmed its purifacation and molecular weight by SDS-PAGE. Furtherly its chemical components were investigated with protease E and NaIO4 treatment.4. The antitumor mechanism was measured by FACS and western-blot.5. Using 3H-TdR incorporation method, we analysed the effect of Acanthopanax gracilistylus extract on monocytic phagocytosis. The productions of TNF-αand IL-12 were measured by bio-chemical method and ELISA respectively. The expressions of TNF-αand IL-12(P40) were tested by RT-PCR.Results: 1. Acanthopanax gracillus extract(AGE) markedly inhibited the proliferation of tumor cells such as MT-2, HL-60, Raji, TMK-1, HSC-2 with dose-dependent manner.2. Treatment with AGE, the general condition of test mice was much better than that of control mice , with tumor growing slowly and life time prolonged.3. To study the chemical nature of the active component of AGE, crude AGE was chromatographed on a Sephacryl S-200 column. The activity of each fraction was assayed using MT-2 tumor cell. The active fractions were combined, lyophilized, dialyzed and rechromatogrophed on a Sephadex G-100 column. The active component was eluted as a single peak whose molecular weight was about 64kDa. This fraction showed a single band at 64kDa on SDS-PAGE. This fraction was sensitive to pronase E, but not to NaIO4 treatment suggesting that the effective molecule is a protein.4. Cell viability analysis showed that AGE inhibited the proliferation of tumor cells without affecting their viability. Treatment with AGE, the cell cycle was arrested at the G0/G1stage without inducing apoptosis or necrosis of tumor cells. Western-blot showed that AGE decreased Rb, CdK2 and CdK4 cell cycle relative enzyme and then inhibited the proliferation of tumor cells.5. Treatment with AGE, the productions of monocytic cytokine TNF-αand IL-12 were markedly enlarged with dose-dependent manner. The quantities of TNF-αproduced by monocyte cultured with LPS and AGE were much more than that produced by monocyte cultured only with LPS. This result indicated that AGE enhanced the stimulated effect of LPS on monocyte producing TNF-α.Obviously, AGE marked stimulated the monocytes to express TNF-αand IL-12 in vitro. Continuously treated by AGE, the contents of TNF-αand IL-12 in serum and spleen monocyte culture supernatant were improved, with the expression enhanced.Conclusion: Acanthopanax gracillus extract markedly inhibited the proliferation of many tumor cells in vitro, with dose-dependent manner; AGE markedly inhibited the proliferation of S180 in mice-mode, with life time prolonged. The antitumor active component of AGE is a 64kDa protein. This protein inhibited the proliferation of tumor cell by regulating the cell cyclin-protease. In addition, AGE markedly stimulated the proliferation of monocytes, not only enhancing its phagocytosis but also improved the production of expression of TNF-αand IL-12.