The Anti-tumor Activity Of Silk Fibroin Peptide On The Lung Cancer Cells And The Possible Mechanisms

Objective: Silkworm is one of the important industries in China,China Silkworm industry has enjoyed the reputation of the Silk Road in all ages.With the development of modern medicine in recent years,silkworm cocoons are widely used in medicine,food and medical equipment and so on,in which silk as raw material extracted silk protein is gradually applied to the research of slow-release drugs,hypoglycemic,anticancer drugs and antioxidant drugs.Therefore,the development of silk fibroin potential medicinal value has become a very meaningful research topic.Silk fibroin peptides was extracted from silk fibroin due to whose anticancer activity by the research group of zhaohongping in Biomechanics and Medical Engineering Institute of Tsinghua University.The main purpose of this study was to investigate the pharmacodynamics and mechanism of silk fibroin peptides around the following aspects: 1.Firstly,the safety evaluation of silk fibroin peptides was carried out to observe whether it has high safety.2.To observe the inhibitory effect of silk fibroin peptide on proliferation of lung cancer cells in vitro and in vivo.3.To explore the mechanism of silk protein peptide on inhibiting lung cancer cells growth.The results of this study will help to expand the role of silk fibroin peptide on against lung cancer medicine effect,which provides silk fibroin peptide a potential application basis for the clinical treatment of lung cancer.Methods: 1.To evaluate the safety and efficacy of silk fibroin peptides,a systematic toxicological experiment was carried out.BALB/c mice were randomly divided into normal saline group,low-dose silk fibroin peptide group(15000 mg/kg)and high-dose silk fibroin peptide group(30000 mg/kg).Intragastric administration was operated in each group for 14 days.The changes of body weight and the vital signs were recorded during the administration.At the end of the administration,the mice were dissected and the blood was taken to measure blood routine and blood biochemistry related indexes.2.To detect the inhibitory effect of silk fibroin peptide on malignant lung cancer in vitro and in vivo,we selected two typical lung cancer cell A549(lung adenocarcinoma cell)and H460(large cell lung cancer)and a normal lung epithelial cell Beas-2b.The inhibitory effect of silk fibroin peptide on the proliferation of lung cancer cells and normal cells was determined by MTT colorimetry and colony formation assay.H460 cells were subcutaneously inoculated in nude mice to establish subcutaneous tumor model of nude mice.The mice were randomly divided into normal saline group,cisplatin positive control group,silk fibroin peptide low dose group,silk fibroin peptide high dose group,and the mice were administered through two different routes of administration to observe the efficacy of silk protein peptide in vivo.3.To further study the mechanism of action of silk fibroin peptides on lung cancer cells,the effects of silk fibroin peptide on cell cycle,apoptosis and related proteins were detected.The effect of silk fibroin peptides on cell cycle and apoptosis was detected by flow cytometry,and then Western blot was used to detect the regulatory effect of silk fibroin peptide on the expression of Bax(apoptosis protein)and Bcl-2(anti-apoptotic protein).Results: 1.The results showed that the administration of mice did not significantly reduce body weigh through the system of acute toxicology experiments,mice did not appear abnormal vital signs,the organ color and size of mice after anatomy were normal,blood routine and blood biochemistry indicators were in a reasonable range of fluctuations,thus which demonstrated silk protein peptide has good safety in vivo;2.MTT assay showed that silk fibroin peptide had a broad-spectrum inhibitory effect on tumor cell proliferation and had little effect on normal cells.The silk fibroin peptide had long-term inhibitory effect on lung cancer cells and had a certain dose-effect relationship through colony formation assay.The results of experimental study on subcutaneous xenograft in nude mice showed that silk fibroin peptide could inhibit the growth of lung cancer cells subcutaneous xenograft in nude mice,which had good antitumor effect and low toxicity.3.The results of flow cytometry showed that the silk fibroin peptide could block the cell cycle of the lung cancer cells,arresting it in the S phase and inducing the apoptosis of the lung cancer cells,which had a obvious dose-effect relationship.The result of Western blot showed that silk fibroin peptide can regulate apoptosis-related important proteins,and it can up-regulate pro-apoptotic protein Bax and down-regulate anti-apoptotic protein Bcl-2.Conclusions: Silk protein peptide has good anti-tumor effect in vivo and in vitro and has low toxicity to the body,it is a natural anti-tumor extract of broad-spectrum and low toxicity.Based on the cell biology and molecular biology methods,the molecular mechanism of the action of silk fibroin peptide was elucidated.It was found that silk fibroin peptide could induce S phase arrest in tumor cells and induce apoptosis by regulating apoptosis-related protein.