Study On ¹³¹I-Labeled CAERIN Peptide In Thyroid Cancer

Background and ObjectiveCaerin peptide is a tumor therapeutic peptide?Caerin peptide has applied for an international patent?identified by our research team from the skin gland secretions of Australia tree frogs?Litoria genus?in the coastal rainforest area of Australia,which can inhibit the proliferation and growth of a variety of tumor cells.Thyroid cancer is the most common malignancy in the endocrine system.Among them,differentiated thyroid cancer has a higher incidence.Radioiodine-refractory DTC and undifferentiated thyroid cancer are currently clinical difficult problems.Therefore,we investigated whether Caerin peptide has cytotoxic effects on two human thyroid cancer cell lines?CAL-62 cell lines and B-CPAP cell lines?,whether it can be taken up by cells,and whether radionuclide ¹³¹I can be stably labeled in Caerin peptide and biological evaluation of ¹³¹I-Caerin in thyroid cancer in vivo and in vitro.We preliminarily investigated the therapeutic value of ¹³¹I-Caerin peptide in thyroid cancer.MethodsPart 1:The cellular level study of Caerin peptide in human thyroid cancer.The Cell Counting kit-8?CCK-8?test was used to detect the cytotoxicity effect of Caerin peptide on CAL-62 and B-CPAP cells,and the50 inhibiting concentration(IC₅₀).Using Laser scanning confocal microscope imaging,we observed the possible positions of Caerin peptide on CAL-62 and B-CPAP cells and whether they can be taken up by the cells.Part 2:The preparation of ¹³¹I-Caerin and detection of its basic property identification.Caerin peptide was labeled with radionuclide ¹³¹I by Chloramine-T direct labeling method to prepare ¹³¹I-Caerin,and the labeling rate was determined by paper chromatography.After the labeling is completed,the basic physical and chemical properties of ¹³¹I-Caerin are determined,including pH,specific activity,Octanol-Water partition coefficient,and in vitro stability.Part 3:The cellular level study of ¹³¹I-Caerin in human thyroid cancer.The biological evaluation of ¹³¹I-Caerin includes studies at the cellular and animal levels.At the cellular level,the uptake and elution of ¹³¹I-Caerin in CAL-62 and B-CPAP cells were studied by cell uptake and elution experiments.The cytotoxicity effect of ¹³¹I-Caerin on CAL-62 and B-CPAP cells were detected by CCK-8 method.At the animal level,CAL-62 tumor bearing nude mice were prepared.¹³¹I-Caerin were injected into the tumor to observe the body weight and tumor volume changes of the nude mice in each group.After the treatment,the tumor tissues were completely dissected to be weighed and compared,and the HE staining of tumor tissue was observed.ResultsPart 1:The cellular level study of Caerin peptide in human thyroid cancer.CCK-8 test results showed that Caerin peptide had obvious cytotoxicity effect on CAL-62 and B-CPAP cells,IC₅₀ was 9.856?g/mL and 4.038?g/mL,respectively,and the cytotoxicity effect was gradually increase with the increasing of peptide concentration.Laser scanning microscope confocal imaging showed that Caerin peptide can be taken up by CAL-62 and B-CPAP cells,and was mainly concentrated in the cytoplasm,and further play a toxic role.Part 2:The preparation of ¹³¹I-Caerin and detection of its basic property identification.By Chloramine-T method,Caerin peptide can be directly labeled with radioactive 131 iodine,which was easy to operate and had short labeling time.The ¹³¹I-Caerin had a high labeling rate of about 92.25%?98.25%,and the specific activity was about 9.25×10⁵KBq/mg.It was a colorless,transparent and clear solution with a pH of about 7.5.The Log P of ¹³¹I-Caerin was0.827±0.036,indicated that it was weakly lipid soluble.¹³¹I-Caerin,whether stored at room temperature?25??,37?,or placed at room temperature?25??,37?fetal bovine serum and normal saline for 72 hours,had good in vitro stability.Part 3:The cellular level study of ¹³¹I-Caerin in human thyroid cancer.¹³¹I-Caerin can be taken up by CAL-62 and B-CPAP cells,and the binding rate gradually increases with time,and has better binding capacity and longer residence time.CCK-8 test results showed that ¹³¹I-Caerin had cytotoxic killing effect on CAL-62 and B-CPAP cells,and it is more cytotoxic than Caerin peptide and Na¹³¹I.CAL-62 tumor bearing nude mice tumor model was successfully prepared.Afrer injecting each group of drugs into the tumor,the body weight of tumor bearing nude mice in each group increased gradually with the extension of treatment time,and there was no significant difference between the groups?P>0.05?.The results of tumor growth of tumor bearing nude mice showed that,compared with the PBS group or Na¹³¹I group,the tumor volume of the tumor bearing nude mice in the ¹³¹I-Caerin and Caerin group gradually decreased,the difference was statistically significant?P<0.05?,and the tumor volume in the ¹³¹I-Caerin group decreased significantly,while the tumor volume in PBS group or Na¹³¹I group increased slowly.After treatment,the weight of tumor tissue showed that compared with the PBS group or the Na¹³¹I group,the tumor weight of the ¹³¹I-Caerin group and the Caerin group was lighter.The difference was not statistically significant?P>0.05?.It may be due to the short observation time.However,the results of HE staining results showed that the degree of necrosis of the tumor tissue in the ¹³¹I-Caerin group was obvious,and it was more obvious than that in the Caerin group,indicating that the therapeutic effect of ¹³¹I-Caerin on the tumor tissues was more significant.ConclusionCaerin peptide has cytotoxic killing effect on two thyroid cancer cells,CAL-62 and B-CPAP cells,and can be taken up and enriched in the cytoplasm.It can successfully obtain ¹³¹I-Caerin with higher labeling rate and better stability.¹³¹I-Caerin can be taken up by CAL-62 and B-CPAP cells,has good cell binding ability,has cytotoxic effect,and can inhibit tumor growth of CAL-62 tumor bearing mice in vivo.¹³¹I-Caerin has the potential to become a new type of radiotherapy for thyroid tumors,especially undifferentiated thyroid cancer,and it also expands new ideas for thr treatment of RAIR-DTC.