Studies On Anti-Tumor Activities Of Pseudolaric Acid-Band Its Mechanism Of Action

During the past ten years more attentions have been given to uncovering the benefits of the natural products in relation to cancers. Pseudolaric acid-B (PLAB), a novel diterpene acid, isolated from Pseudolarix kaempferi Gorden and identified as the main antifungal constituent, possesses various pharmacological activities. Recently, PLAB has been noted for its notable cytotoxic activities against P-388 lymphocytic leukemia, KB carcinoma of the nasopharynx, HT-1080 fibrosarcom, human breast cancer, human melanoma, human lung cancer and human colon cancer cell lines in vitro. In order to explore the possibility of developing it as a plant-derived, low toxic and effective anticancer drug, the mechanisms underlying the tumor cytotoxicity of PLAB and the inhibition effect on transplanted mice tumor cell growth in vivo, which were poorly defined before, were studied. Main results could be summarized as follows. 1 Effects of PLAB on the proliferation of cancer cells In a series of experiments, eleven tumorigenic human cells, including nasopharyngeal carcinoma cell (KB), human colorectal carcinoma cell (HCT-8), human colorectal adenocarcinoma cell (HT-29), human lung adenocacinoma cell (A549), human kidney carcinoma cell (KETR-3), human cervical cancer cell (HELA), human breast adenocarcinoma cell (MCF-7), human bladder carcinoma cell (EJ), human ovarian carcinoma cell (SKOV3, A2780), human stomach carcinoma cell (BGC-823) and one normal human kidney proximal tubular epithelial cell (HKC) were chosen to determine the cytotoxic activity of PLAB. After incubating for 96h, increasing concentrations of PLAB (0.125～1.0μmol/L) led to a gradual decrease of viable cells fraction. MTT assay showed that IC50 was 0.17μmol/L (KB) to 5.20μmol/L (A-549). Because of these variations, the cytotoxic activity of PLAB had been determined against a panel of human tumor cells of different origins. Results of SRB assay showed anticancer activity by the cytostatic effect when it was incubated in the low concentration, the GI50 of PLAB to HELA, SKOV3 was 1.12μmol/L, 0.49μmol/L respectively, but PLAB showed anti-cancer effect through the cytotoxic effect when it was incubated in the high concentration, the LC50 of PLAB to the above two cell lines was 11.35μmol/L, 16.73μmol/L respectively. Further more, the results of cell growth curve and colony formation of cancer cells matched with the above results. 2 Inhibition of tumor growth in vivo by PLAB The effect of PLAB on tumor growth was studied in KunMing and C57BL/6 mice using transplanted models with Lewis lung cancer and Hepatocarcinoma 22 (H22) respectively.Intraperitoneal injection of PLAB (30mg/kg and 60mg/kg) was performed every day, followed with tumor cells inoculation. Mouse H22 hepatocarcinoma was sensitive to PLAB, 30mg/kg and 60mg/kg PLAB treatment resulted in 14.4% and 40.1% (P<0.01) inhibition of tumor growth compared with untreated control. Lewis lung cancer was more sensitive with inhibitory rate of 39.1% at 30mg/kg PLAB and 47.0% (P<0.01) at 60mg/kg PLAB. Neither death nor altered weight modification of the host occurred, PLAB seemed little toxicity in our experimental conditions. Effects of PLAB in vivo showed that daily injection of PLAB at less than toxic doses reduced the tumor weights. 3 Causing cell cycle arrest by PLAB To evaluate the possible role of cell cycle arrest in PLAB-caused growth inhibition, HELA, SKOV3 cells were treated with PLAB. Cell cycle distribution was evaluated by flow cytometric analysis after staining of cellular DNA with propidium iodide at the different concentrations, which indicated that PLAB induced an accumulation in G2+M phase of cell cycle. After treatment with 0.25~4.0μmol/L of PLAB for 24h, the number of cells in G2+M phase was higher than that in untreated cells. The percentage of cells arrested in G2+M phase increased with the increasing of concentration. 4 Inducing apoptosis by PLAB PLAB induced morphological changes by Giemsa and Hoechest staining, which were characteristics of apoptosis.Contrast cells displayed excellent growth...