Anti-Tumor Effect And Mechanism Of TT-1 On TT Cells And HepG2 Cells Combined With IFN-?

Human cancer remains a leading cause of mortality worldwide despite recent advances in therapeutic methods. Incidences of thyroid cancer have steadily increased, and the disease has become the most prevalent endocrine malignancy. Thyroid cancer is estimated to occur in 9.1 per 100,000 females and 2.9 per 100,000 males in developed countries. Chemotherapy and biochemotherapy have also been used in the treatment of advanced melanoma; however, they lack sufficient efficacy, and their toxicity and significant side effects greatly restrict their clinical application. Therefore, it is necessary to find an efficient and low-toxic anticancer drug for patients with thyroid cancer.Hepatocellular carcinoma(HCC) is one of the most common malignancies worldwide and a leading cause of death among cirrhotic patients. The treatments for HCC include surgical resection, liver transplantation and ablation, and chemotherapy, which has not been effective as expected.In recent years, the biological immune therapy methods have been emerged, such as cancer vaccines, monoclonal antibodies, immune suicide gene or gene therapy and etc, which have been in clinical or preclinical trials and have achieved certain effect, but the effect has not been ideal. Therefore, we need to find a more effective treatment of medullary thyroid cancer and human hepatocellular carcinoma. Recent studies have demonstrated that antimicrobial peptides(AMPs) can selectively kill cancer cells because the membrane proteins of cancer cells are negatively charged due to glycosylation. However, few studies have focused on the therapeutic effects on medullary thyroid cancer and human hepatocellular carcinoma.In this study, we choose Melittin,which have obvious antitumor effect in AMPs and rearranged the structure of melittin to obtain a novel peptide TT-1. In vitro and in vivo experiments were conducted to demonstrate the antitumor efficacy of TT-1 peptides to medullary thyroid cancer and human hepatocellular carcinoma. The results were displayed as follows:1 TT-1 has obvious antitumor activity in vivo and in vitro to TT cells of human MTC line(1) TT-1 could significantly inhibit the growth of TT cells in vitro,but for Nthy-ori3-1 cells,the growth inhibition was low. TT-1 had selective inhibitory effect of the growth of TT cells with a dose-dependent and time-dependent manner. AV-PI apoptosis double staining experiments showed that TT-1 polypeptide was capable of inducing apoptosis TT cells in TT-1 at the concentration of 2,4,8?g/ml, TT cell apoptosis rate was 12.31 %, 18.62 % and 31.07 %, respectively. Investigation of mechanism displayed TT-1 could up-regulated Bax and down-regulated Bal-2 protein expression and mRNA expression level in the TT cell;TT-1 could up-regulated caspase-3 and caspase-9 both protein expression and mRNA expression level in the TT cell.(2) TT-1 could significantly inhibit the growth of MTC line TT xenograft tumors in vivo. Model group xenografts volume increased with time, the tumor volume of the polypeptide TT-1 high, medium and low-dose group(0.04 mg/kg?0.20 mg/kg?1 mg/kg) was significantly smaller compared with the model group. Compared with model group, Tumor weight dose-dependent of polypeptide TT-1 medication groups were down-regulated, TT-1 0.04 mg/kg?0.20 mg/kg?1 mg/kg tumor inhibiting rates was 5.8%,48.6% and 56.8%, respectively. In conclusion, TT-1 can significantly inhibit the growth of TT cells xenograft tumors and have distinct antineoplastic activity in vivo.2 TT-1 demonstrated potent anti-tumor effect on hepatocellular carcinoma cell line HepG2 in vivo and in vitro,and it can play a great synergistic therapeutic effect in the combination of IFN-?.(1) Different doses(0,0.5,1,2,4,8,16,32?g/ml) of TT-1 and TT cells cellular effected 72 h to detect cell viability, which displayed TT-1 significantly inhibited the growth of TT cells with a dosage-dependent manner. IC50 was obtained according to the statistics, which is 3.762 � 0.285 ?g/m L. When the concentration of TT-1 was 32 ?g/ml, the cell viability was only 14.8%.(2) After demonstration of well antitumor efficacy of TT-1 polypeptide on TT cell line, we combined the peptide with IFN-? for immunotherapy of HepG2 xenograft tumor model. The combination of TT-1 and IFN-? could enhance the antitumor activity significantly compared to TT-1. Compared with the model group, the tumor inhibitory rate of TT-1 + IFN-? was approximate 80%. Moreover, investigation of mechanism suggested that NK cells played a key role in the immune response. The results above demonstrated that the antitumor efficacy of TT-1 + IFN-? was mediated by NK cells via MICA-NKG2 D pathway specifically.In conclusion, this study first investigated the antineoplastic activity of cationic antimicrobial peptides TT-1 which was mutated by Melittin for MTC line TT cells. We expounded mechanism of TT-1 to anti-MTC and confirmed its effects through the experiments in vivo and in vitro. Moreover, we demonstrated excellent anti-tumor efficacy on HepG2 cell lines and combined IFN-? with TT-1 for immunotherapy of HepG2 xenograft tumor model. The mechanism was well understood and established by corresponding strategies, which was mediated by NK cells via MICA-NKG2 D pathway specifically. The study was fully evidenced and provides a new strategy for medullary thyroid cancer and human hepatocellular carcinoma therapies.