Lipid-soluble EGCG Inhibits Hepatoma Cells In Vitro And In Vivo

ObjectiveHepatoma is one of the dangerous malignancies,55% of global hepatoma patients are from China. Morbidity in Jiangsu, Zhejiang and Shanghai is increasing rapidly. Surgery is the favorite therapeutic scheme, which is however accompanied with high recurrences. Hepatoma patients usually suffer from rapid progression and many complications such as liver cirrhosis, ascites and jaundice. While traditional chemotherapeutic drugs may result in serious side effects, it is necessary to search new natural compounds to cure hepatoma, alleviate terminal patients’ suffering and reduce the side effects.Flavonoid is the natural polyphenol compound widely distributed in plants. This compound includes flavonoid, flavanol, isoflavone, flavonol, etc. Flavonoid has anti-inflammatory, antineoplastic, anti-bacteria and anti-radiation effects. Besides, it also works well in curing cardiovascular disease, improving immunity and regulating endocrine. The pharmacological mechanism mainly lies in the ability to scavenge free radical, resist oxidation or affect some enzymes. In a word, flavonoid has been studied in-depth since its excellent pharmacological potency, slight side effects and easy access to.Tea polyphenol is one of the common used antioxidant flavonoid phytochemicals. A great deal of epidemiology studies indicate that people who consume green tea have lower risk of cancer. Abundant experiments both in vitro and in vivo also demonstrate tea polyphenols are able to suppress a series of tumor. Nevertheless, normal tea polyphenols are water soluble and are so unstable with bad absorbtivity to reach an effective concentration in the body, resulting in limitation to clinical application. EGCG is the main as well as most effective composition of tea polyphenols. EGCG can be esterified to link ester groups of different molecular weight. The esterified EGCG is lipid-soluble with higher absorbtivity and stability. We aim to identify the inhibiting effect of lipid-soluble EGCG (L-EGCG) on hepatoma cells in vitro and in vivo.Materials and methodsH22 and HepG2 cells were treated with L-EGCG for 24h. The morphological changes were observed under light microscope. To verify the toxicity of L-EGCG to this two hepatoma cells, CCK8 cell counting kit and MTT were used to detected cell viability. Hoechst/PI double staining was used to detect cell apoptosis. Apoptosis and autophagy related proteins were tested by western blot.The most appropriate injection cell number to establish subaxillary tumor-bearing model in ICR mice was explored and this model was used to detect the effects of lipid-soluble EGCG on the ICR mice subaxillary xenograft. 25 mg/kg CTX was administrated through intraperitoneal injection while 40 mg/kg water-soluble EGCG (H-EGCG), corn oil,25 mg/kg,50 mg/kg and 100 mg/kg L-EGCG were administrated by gavage the day after the injection. The daily administration continued ten days and then the mice were cervical dislocated to dissect the solid tumor and compare the inhibitory effect of L-EGCG with the control group.Model of nude mice bearing HepG2 cell orthotopic xenograph were established after the surgical incision, appropriate cell amount as well as injection position were confirmed. Next day,25 mg/kg CTX was administrated through intraperitoneal injection while 40 mg/kg H-EGCG, corn oil,25 mg/kg,50 mg/kg and 100 mg/kg L-EGCG were successively administrated by gavage. The administration was carried out five times a week and continued thirty days or ninety days, respectively. For the thirty days model mice, liver was dissected to separate solid tumor after cervical dislocation. Tumor weight was used to compare the inhibition rate between L-EGCG groups and the control group. Ninety days model mice were used to observe survival time. At the end point, liver was dissected and the left lope was fixed in neutral formaldehyde, HE and masson staining was carried out to demonstrate hepatic architecture and fibrosis changes with L-EGCG administration.ResultsUnder the microscope, both H22 and HepG2 cells were wrinkled with low refractive index and cell fragment were also observed with L-EGCG treatment. CCK 8 cell counting and MTT tests showed that L-EGCG inhibited cell viability of both H22 and HepG2 cells in concentration and time dependent manner. Hoechst/PI double staining indicated L-EGCG administration induced apoptosis in H22 as well as HepG2 cells. Moreover, L-EGCG increased PARP cleavage. Meanwhile, the autophagic protein p62 and LC3Ⅱ were increased as well. These results indicated that L-EGCG induced apoptosis and inhibited autophagy at the same time.It was found that 106 of H22 cells in 100μl per mouse are necessary to build stable subaxillary xenograph model in ICR mice. Administration of 25 mg/kg and 50 mg/kg L-EGCG for 10 days could significantly inhibit the solid xenograph (p<0.05), compared with corn oil group.The appropriate HepG2 cell concentration, injection bulk, injection position on the left liver lope, and the spilling or bleeding prevention were necessary to establish the stable HepG2 nude mice xenograph model. Administration with L-EGCG for one month, especially at doses of 25mg/kg and 50mg/kg, could significantly decrease weight of the solid xenograph, compared with the corn oil group. At the end of three month, half of the mice (3/6) in corn oil group survived. In CTX treated group although the tumor mass were much smaller than that in the control group, the survival rate was not increased. In L-EGCG treated groups, however,5/6,6/6, and 4/6 tumor bearing mice survived in 25mg/kg,50mg/kg, and 100mg/kg groups, respectively, although the difference was not significant by survival analysis. A reasonable explanation is the number of animals in each group was not enough to reach the statistical significant. HE and masson staining showed that L-EGCG could accelerate solid tumor necrosis and alleviate hepatoma fibrosis.Conclusion1. L-EGCG could inhibit both mouse (H22) and human (HepG2) liver cancer cells in vitro, probably through promoting cell apoptosis as well as inhibiting later stage of autophagy.2. L-EGCG could inhibit both subaxillary xenograph in ICR and orthotopic xenograph in nude mice. Besides, L-EGCG intends to prolong the survival ratio of xenograph model mice.3. L-EGCG could accelerate solid tumor necrosis and alleviate hepatoma fibrosis.