Objective:

2

To characterize the molecular mechanism underlying the antineoplastic activity of

Celastrus orbiculatus

Thunb. extracts (COE).

Methods:

2

The human hepatocellular carcinoma HepG2 cells with mammalian target of rapamycin (mTOR) knockdown expressed (HepG2/mTOR-) were constructed using molecular biological technology.

In vitro

the HepG2/mTOR- cells were treated with COE at various concentrations (10

20

40

80

160 and 320 μg/mL). Cell viability was determined using 3-(4

5-dimethylthiazol-2-yl)-2

5-diphenyltetrazolium bromide (MTT) assays. According to the half-maximal inhibitory concentration (IC

50

) value (140 mg/L)

the concentrations of COE in the subsequent experiment was set to alleviate cytotoxicity. The HepG2/mTOR- cells were divided into 5 groups: negative control (untreated)

COE treatment groups (40

80

120 mg/L COE) and positive control group (cisplatin

DDP

2 mg/L)

respectively. Wild-type HepG2 cells were used as a blank control. The effects of COE on the cell apoptosis were analyzed by flow cytometry and transmission electronic microscopy (TEM)

respectively. The protein expression levels of mTOR signal pathways were determined by Western blotting.

In vivo

HepG2/mTOR- cells (2×10

6

cell/mice) were subcutaneously injected into the right flank of nude mice. Thirty-six female nude mice were randomly assigned to 6 groups according to body weight (6 mice per group) as follows: solvent vehicle control

Banmao Capsule treated group (BM

195 mg/kg)

Tegafur

Gimeracil and Oteracil Potassium Capsules (10 mg/kg) treated group

and different dosages of COE (10

20

40 mg/kg) groups. Tumor growth was monitored and immunohistochemical staining was used to examine the expression of apoptosis-related proteins in tumor tissues.

Results:

2

COE inhibited the proliferation significantly in a concentration-dependent manner in HepG2/mTOR

-

cells (

P

<

0.01). COE significantly induced the apoptosis of HepG2/mTOR

-

cells (

P

<

0.01)

and the apoptotic bodies can be observed under TEM. COE significantly inhibits the proteins expression of mTORrelated signal pathways.

In vivo

COE significantly inhibited tumor growth in nude mice (

P

<

0.01). Moreover

the results showed that COE down-regulated the expression of Bcl-2 and Bcl-xL

and up-regulated the levels of Bax and caspase-3 protein (

P

<

0.01).

Conclusion:

2

COE was a potential chemotherapeutic drug in HCC treatments via targeting mTOR signal pathway.