The Pro-apoptotic Effect And Migration And Invasion Inhibitory Effect Of Momordin Ic On HepG2Cells And The Functional Mechanisms

Tumorigenesis results from disbalance of cell proliferation, differentiation, metabolism andgrowth. Cell apoptosis, which is mediated by series of genes and proteins, is a self-regulatedcontrol of growth and development and response to exogenous stress to clear abnormal,excessive and damaged cells. Thus, apoptosis plays a significant role in malignant tumorprevention and treatment. Cancer cells possess the ability to invasion and migration, formingmetastatic tumor. Therefore, it is of great importance to explore functional factors that inhibitcancer cell metastasis in addition to apoptosis induction in terms of cancer therapy.Momordin Ic (oleanolic acid-3-O-β-D-xylopyranose(1�'3)β-D-Pyranoid glucose) is anatural triterpene saponin derived from the dried fruit of Fructus Kochiae. It has been shownto inhibit ethanol-induced gastric mucosal lesionsin and CCl4-induced hepatic injury, preventglucose-induced blood sugar increase and function as antianaphylaxis and antipruritic.However, there is little evidence about the anti-tumor characteristics of Momordin Icinternationally. The aim of the present research was to evaluate the anti-tumor merit ofMomordin Ic in aspects of apoptosis induction and metastasis inhibition. Furthermore, thepossible mechanisms of Momordin Ic-induced cell apoptosis and metastasis inhibition werealso elucidated. The major contents and results are as follow.(1) We first analysed the effect of Momordin Ic on human hepatoma carcinoma cellsgrowth. The results showed Momordin Ic effectively inhibited HepG2, Huh7and Hep3B cellsgrowth in a time and concentration-dependent manner. However, less than20μM ofMomordin Ic had little effect on normal liver cells (HL7702, BRL-3A). HepG2cells werefurther used to evaluate the morphologic change and apoptosis phenomenon induced byMomordin Ic. It was suggested that cells exposed to Momordin Ic shrinked and turned smaller,round and displayed condensed chromatin, demonstrating the typical apoptoticcharacterization. The increase of cleaved caspase-3and PARP fragmentation was alsodetected. Thus it was suggested that the suppressive effect of Momordin Ic on cancer cellgrowth was stimulation of cell apoptosis.(2) The possible mechanism of Momordin Ic-induced HepG2cell apoptosis wasdemonstrated. The results indicated that Momordin Ic blocked Bcl-2expression, promoted Bax expression, decreased MMP and induced cytochrome c release, implying that MomordinIc induced apoptosis via mitochondrion-mediated (endogenous) pathway.Momordin Ic inhibited Akt and Erk1/2while activated JNK and p38MAPK. Theapplication of PI3K/Akt inhibitor (LY294002) and activator (Insulin), MAPKs inhibitors(SB203580、SP600125and U0126) further demonstrated that Momordin Ic regulated Bcl-2family members (Bcl-2, Bax) via PI3K/Akt and MAPK pathways, resulting in mitochondriondysfunction which induced cell apoptosis. There was also an interaction between PI3K/Aktand MAPK pathways.HepG2cells treated with Momordin Ic displayed decrease of HO-1and increase of iNOSprotein expression as well as ROS production. NAC, an anti-oxidant, inhibited MomordinIc-induced cytochrome c release, HO-1protein expression decrease and PI3K/Akt and MAPKpathways regulation. The level of NO was not altered by Momordin Ic. These results showedthat Momordin Ic-induced apoptosis was initiated by oxidative stress.Momordin Ic down-regulated COX-2expression and up-regulated PPARγ, PGC-1α andFOXO4expression. Inhibition or activation of PI3K/Akt and MAPK pathways intervenes theeffect of Momordin Ic on these proteins. In brief, Momordin Ic triggered ROS productionfollowed by mitochondrion dysfunction mediated by PI3K/Akt and MAPK pathways, leadingto cytochrome c release into the cytoplasm, caspase-3activation and PARP fragmentation thatrepresent apoptosis.(3) We explored the effect of Momordin on HepG2cell (a typical human epithelialcarcinoma cell) cell-matrix adhesion, invasion and migration.The results showed thatMomordin Ic inhibited fibrosis of deformation, homozygous intercellular attachment,simulative extracellular matrix adhesion and wounding-healing ability. Transwell systemassay also suggested that cell migration and invasion were suppressed. The above resultsdemonstrated that Momordin Ic could inhibited HepG2cell matrix adhesion, migration andinvasion.(4) The possible mechanism of the inhibitory effect of Momordin Ic on cell matrix adhesion,migration and invasion was elucidated further. We found that the protein level of VCAM-1,ICAM-1, MMP-9was reduced while E-cadherin expression was induced. Momordin Icinhibited cell matrix adhesion, migration and invasion via PI3K/Akt and MAPK pathwaysthat regulated the expression of VCAM-1, ICAM-1, MMP-9and E-cadherin.Momordin Ic suppressed COX-2expression and promoted PPARγ and PGC-1α expression.COX-2inhibition enhanced the effect of Momordin Ic on E-cadherin expression induction;the effect of Momordin Ic on ICAM-1and MMP-9inhibition as well as E-cadherin expressionstimulation was weakened by PPARγ inhibition. In conclusion, Momordin Ic inhibited VCAM-1, ICAM-1, MMP-9and induced E-cadherin expression via PI3K/Akt and MAPKpathways as well as COX-2and PPARγ regulation, preventing cell matrix adhesion, migrationand invasion.