Internal Radiation Therapy With The Iodine-131-Labeled Caerin 1.1 Peptide For Human Anaplastic Thyroid Cancer In Nude Mice

Background:（1）Anaplastic thyroid cancer（ATC）is a highly malignant endocrine tumor with strong invasion and high mortality.It is reported that ATC mostly adopts total thyroidectomy plus high-dose radiotherapy and/or systemic chemotherapy,patients treated with surgery experienced a median overall survival of 6.6 months,which increased to 9.6 months with adjuvant therapy,but those who treated with primary radiation therapy only,chemotherapy only,palliative therapy,and without treatment survived a median of 3.0 months.However,the survival rate or survival time of ATC patients is still not optimistic,so a more effective ATC treatment method is urgently needed clinically.（2）Caerin 1.1 peptide is a kind of natural host defense peptides.Recent studies have confirmed that Caerin 1.1 peptide can inhibit and kill a variety of tumors,change tumor microenvironment（TME）and recruit more anti-tumor immune cells to tumor sites.（3）Our previous studies in vitro confirmed that the iodine-131(¹³¹I)-labeled Caerin 1.1 peptide has better antitumor effect on CAL-62 cell than Caerin 1.1peptide only and ¹³¹I only.Objective:（1）The effect of Caerin 1.1 peptide on inhibiting the proliferation,growth and migration of CAL-62 cell was verified.The mechanism of Caerin 1.1 peptide acting on CAL-62 cell was also explored.（2）Objective to prepare ¹³¹I-Caerin 1.1 peptide and establish nude mice model of ATC,and to investigate the therapeutic effect of the internal radiation therapy（IRT）with ¹³¹I-caerin 1.1 on CAL-62 tumor in nude mice.Methods:（1）Antitumor activity of Caerin 1.1 peptide was determined by MTT assay,plate colony formation and cell wound scratch assays,and the mechanism of the inhibition of Carein 1.1 peptide on the growth of CAL-62 cell was identified by Fluorescence Activating Cell Sorter（FACS）and Western Blot（WB）.（2）¹³¹I-Cerin 1.1 peptide was prepared by chloramine-T method,and the labeling rate was measured by paper chromatography.We successfully established the tumor-bearing mouse model of CAL-62 and they were randomly divided into four groups tha t received one of the following treatments:PBS,Caerin 1.1 only,¹³¹I-Caerin 1.1,¹³¹I only.The mice were injected intratumorally with PBS,Caerin 1.1 peptide（8.0μg）,¹³¹I（7.4MBq）or¹³¹I-Caerin 1.1（7.4MBq）every 3 days for a total of three times.Then,3 days after the final treatment,the mice were sacrificed,and the tumors were isolated and weighed.H&E and TUNEL staining was performed to detect the apoptotic cells and necrotic area in the tumor tissue sections.Results:（1）MTT assay showed that Caerin 1.1 peptide inhibited the growth of CAL-62 cell and was dose-dependent;When the concentration was less than15.0μg/ml,there was no significant cytotoxic effect on Nthy-ori 3-1 cell.We determined the half-maximal inhibitory concentration(IC₅₀)values for the Caerin 1.1 peptide in CAL-62 and Nthy-ori 3-1 cell and the IC₅₀ values were8.7μg/ml and 18.5μg/ml.（2）Plate colony formation assay showed that with the increased concentration of Caerin 1.1 peptide,CAL-62 cell decreased significantly.When the concentration of Caerin 1.1 peptide reached 5.0μg/ml,the clonal proliferation level of CAL-62 cell was significantly decreased（P<0.05）.（3）Wound scratch assays showed that with the increased concentration of Caerin 1.1 peptide,the migration ability of CAL-62 cell decreased significantly.When the concentration of Caerin 1.1 peptide reached 5.0μg/ml,there was significant difference in wound healing area among the treatment groups and control group.（4）Cell cycle showed that compared with the 0μg/ml group,the G1 phase decreased and the S phase increased,with significant statistical difference（P<0.001）.Compared with the 30.0μg/ml group,the G1 phase of the40.0μg/ml group was significantly decreased（P<0.0001）,increased significantly in S phase（P<0.0001）.（5）Western Blot showed that compared with blank control group,the expression of p-AKT was significantly decreased by different concentrations of Caerin 1.1 peptide（P<0.05）.（6）Three days after the final treatment,the volumes of PBS,Caerin 1.1 peptide,¹³¹I,¹³¹I-Caerin 1.1 peptide were（114.8±20.8）mm³,（116.5±13.1）mm³,（113.2±7.1）mm³,（67.0±16.3）mm³.There was a significant difference of the tumor volume in the ¹³¹I-Caerin 1.1 peptide group compared to the other treatment groups in vivo（P<0.001）.After the treatment,the tumors were isolated and weighed,the weights of PBS,Caerin 1.1 peptide,¹³¹I,¹³¹I-Caerin1.1 peptide were（122.6±20.0）mg,（136.5±15.2）mg,（112.0±26.3）mg,（49.5±15.5）mg.The weight of ¹³¹I-Caerin 1.1 peptide group significantly reduced than any other treatment groups（P<0.001）.H&E staining showed the most obvious necrosis of tumor cells in the ¹³¹I-Caerin 1.1 peptide group compared with the PBS group（P<0.05）.The TUNEL staining results showed that the apoptotic staining degree of ¹³¹I-Caerin 1.1 peptide group was significantly higher than the other groups（P<0.05）.Conclusions:（1）Caerin 1.1 peptide could significantly inhibit the growth and invasion,and might block the cell circle and induce cell apoptosis.（2）¹³¹I-Caerin 1.1 peptide inhibited the growth of CAL-62 tumors in vivo,providing an option for ATC treatment.