In Vitro and In Vivo Evaluation of Antitumor Activity of Ligustrum robustum, A Chinese Herbal Tea.

Abstract

To examine the effect of the aqueous extract of Ligustrum robustum on tumor growth in vitro and in vivo and explore the possible molecular mechanisms. In in vitro study, cell viabilities of human cervical carcinoma cells (HeLa), human breast cancer cells (MCF-7), human prostate cancer cells (PC-3), human hepatoma cells (7721) and human colon carcinoma cells (SW480) were evaluated with cell counting kit-8. For L. robustum-treated Hela cells, early or late apoptosis were evaluated by annexin V/PI staining. Mitochondrial membrane potential was measured by staining cells with JC-1. Apoptosis was monitored by nuclear morphology based on chromatin condensation and fragmentation by 4',6-diamidino-2-phenylinole (DAPI) staining. Caspase-3 and -8 activity levels were measured by a colorimetric assay. In vivo, to evaluate the possible mechanism of L. robustum-mediated antitumor effect, nude mouse xenograft study was also conducted. In in vitro study, L. robustum was found to be toxic to HeLa, MCF-7, PC-3, 7721, SW480, with an half maximal inhibitory concentration value of 2-5 mg/mL (P<0.05). Moreover, externalization of phosphatidylserine, loss of mitochondrial membrane potential, DNA fragmentation and activation of caspase-3 and -8 were detected in L. robustum-treated Hela cells. Using a nude mouse model bearing Hela xenografts, we found that L. robustum reduced tumor volume and tumor weight (P<0.05), but had no effect on body weight and histological damage of important organs. Intraperitoneal injection of L. robustum caused a significant reduction in serum aspartate transaminase and alanine transaminase levels (P<0.05). Furthermore, cleaved caspase-3-positive and terminal nucleotidyl transferase-mediated nick end labeling (TUNEL)-positive cells were observed in L. robustum-treated tumor tissues. L. robustum inhibits tumor cell growth both in vitro and in vivo by inducing apoptosis in a caspase-dependent way without apparent hepatic toxicity and histological damage, which may offer partial scientific support for the ethnopharmacological claims of L. robustum as a herbal tea for its antitumor activity.