| Thyroid cancer is the most common malignant tumor of the endocrine system, of which about 5%-10% is medullary thyroid cancer(MTC) that originates from the parafollicular cells(thyroid C cells).MTC has a high malignant degree,a high risk of disease progression and a high rate of postoperative recurrence, the malignant degree is between differentiated and anaplastic thyroid carcinoma. According to statistics, metastatic cervical adenopathy is present in about 50-80% of patients at initial presentation, and up to 20% of patients harbor distant metastases at the time of diagnosis.Currently, surgery has been the preferred treatment for clinical MTC yet, but the recurrence rate and long-term survival of patients have not been greatly improved. The majority of patients at the time of recurrence with severe condition, having lost surgery opportunity. In addition, radioactive iodine(RAI) has no place in the treatment of MTC, the effect of radiotherapy and chemotherapy is poor. 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.Antimicrobial peptides(AMPs) widely exist in the microorganisms, plants, and animals, which are considered as an essential components in innate immunity. AMPs have a broad spectrum of bioactivity such as antibacterial, antifungal, antiviral and etc. The latest researches have showed that cationic antimicrobial peptides could kill cancer cells by choice but harmless to normal mammal cells, which makes it the focus of recent research. Howerer, the effect of AMPs against thyroid cancer has not been reported.This study selectes bovine myeloid antimicrobial peptide 28(BMAP-28) and Melittin,which have obvious antitumor effect in AMPs. We achieved the following results by in vitro and in vivo experiments to detect the effects of anti-tumor for MTC:1 BMAP- 28 has obvious antitumor activity in vivo and in vitro to TT cells of human MTC line1.1 BMAP-28 could significantly inhibit the growth of TT cells in vitro, with a dose-dependent and time-dependent manner. AV-PI apoptosis double staining experiments showed that BMAP-28 polypeptide was capable of inducing apoptosis TT cells in BMAP-28 at the concentration of 1,2,4 μM, TT cell apoptosis rate was 6.51%,14.63, and15.32%, respectively. Investigation of mechanism displayed BMAP-28 could up-regulated caspase-3 and caspase-9 protein expression and increased both m RNA expression level in the TT cell. In addition, after BMAP-28 effected MMP-3 and MMP-9 in TT cells which was related to promote the tumor invasion and metastasis showed BMAP-28 inhibited MMP-3 and MMP-9 protein transcription and translation.1.2 BMAP-28 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 BMAP-28 high, medium and low-dose group(1mg / kg, 0.50 mg / kg, 0.25 mg / kg) was significantly smaller compared with the model group. Compared with model group, Tumor weight dose-dependent of polypeptide BMAP-28 medication groups were down-regulated, BMAP-28 0.25,0.5,1mg / kg tumor inhibiting rates was 30%, 34.29%, and55.71%, respectively. BMAP-28 can significantly inhibit the growth of TT cells xenograft tumors and have distinct antineoplastic activity in vivo.2 Melittin vitro significantly inhibits the growth of TT cells. Compared with Melittin, transforming the body TT-1 saves cost enormously, improves stability, reduces toxicity.2.1 Different doses(0,0.5,1,2,4,8,16,32μM)of Melittin and TT cells cellular effected 48 h to detect cell viability, which displayed Melittin significantly inhibited the growth of TT cells. Compared with BMAP-28 group, the Melittin suppress the growth of TT cell is more obvious.2.2 AMPs have low yields, high costs and poor stabilities, limiting its further application. Therefore, we transformed Melittin which was resisting TT cell and had a good activity. We utilized bioinformatics, based on structural characteristics, to design a new AMPs TT-1 with 11 amino acids. It infinitely saved costs compared to the parent peptide. Body transformation of TT-1 was not only retaining the active site Melittin amino-terminal region, but replaced the original unstable amino acid proline with positively charged lysine. Hydrophobic and hydrophobic of TT-1 increasing which prompts its activity increased, stability improved; TT-1 charge ratio Melittin decreases, meaning that toxicity will be reduced.3 TT-1 has evident antineoplastic activity of human MTC line TT cells in vitro and vivo3.1 TT-1 has no evident cytotoxic effect in for human normal thyroid cells Nthy-ori3-1, but it is capable of killing anti-TT cells specifically. AV-PI apoptosis double staining experiments results showed that TT-1 induced TT apoptosis in vitro. After TT-1 effected, we detected expression of caspase-3, caspase-9, Bax and Bcl-2 transcriptional and translational levels, which was related to apoptosis in TT cells. The results indicated that TT-1 activated pro-apoptotic protein caspase-3, caspase-9, and Bax and down-regulated anti-apoptosis protein Bcl-2.3.2 TT-1 significantly inhibited the growth of MTC line TT xenograft tumors. Model group xenografts volume increased with time, the tumor volume of TT-1 high, medium dose groups(1mg / kg, 0.2 mg / kg)are more obvious smaller compared with the model group. The tumor volume of TT-1 low-dose group(0.04 mg / kg) was almost the same as the model group. The tumor mass of polypeptide TT-1 high dose group(1mg / kg, 0.2 mg / kg) was smaller compared with the model group significantly(** P <0.01).The tumor inhibiting rates of TT-1 three doses 1mg / kg, 0.2 mg / kg, 0.04 mg / kg were 56.8%, 48.6%, and5.8%, respectively.In conclusion, this study first investigated the antineoplastic activity of cationic antimicrobial peptides BMAP-28 and Melittin for MTC line TT cells. We expounded mechanism of BMAP-28 to anti-MTC and confirmed its effects through the experiments in vivo and in vitro. Meanwhile, we got a novel cationic antimicrobial peptide TT-1 by transformed Melittin to attain the similar effect to resist the activity of TT cells. It is not only saving costs, improving stability, but also reducing the toxicity. Therefore, it opens a new door for MTC treatment strategy. |
PDF Full Text Request