Mechanism Of Small Molecular Nanoparticles Compound CLT-003 Inhibiting Proliferation, Invasion And Migration Of Pancreatic Cancer

BackgroundPancreatic ductal adenocarcinoma（PDAC）is the most difficult malignant gastrointestinal cancer worldwide.More than 350,000 people are diagnosed and340,000 die from this disease every year.Its 5-year survival rate was less than 8%.Compared with other malignant tumors,PDAC is a highly invasive tumor with early metastasis potential and is not sensitive to chemotherapy or targeted therapy,and surgical treatment is the most effective treatment.In addition to the special biological behavior of pancreatic cancer,it may also be closely related to its abundant interstitial composition and highly hypoxic and hypoxic tumor microenvironment.Tumor tissue hypoxia is a typical feature of PDAC and is associated with poor prognosis.Several basic studies have found that high expression of hypoxia-inducibility factor hif-1 can promote cancer cell proliferation,apoptosis resistance,epithelial-mesenchymal transformation（EMT）,invasion and metastasis,as well as resistance to radiochemotherapy.Previous studies in our laboratory also confirmed that high expression of hif-1 can promote proliferation and apoptosis resistance of pancreatic cancer cells,as well as promote invasion and migration of pancreatic cancer.Therefore,inhibitors targeting the function of hif-1 have been widely developed in basic and preclinical studies of anti-tumor.Physi CAL on pancreatic cancer for years spent oxygen microenvironment done in-depth research,small molecule CLT-003nanoparticles compound is our cooperation experimental unit,the Oklahoma Dr Chen Danyang laboratory synthesis of benzene peptide analogues of imide,by nanotechnology wrapped in poly lactic acid glycolic acid copolymer nanoparticles in the formation of the compound,medicine effective component is imide benzene peptide analogues.CLT-003 can significantly inhibit the proliferation of vascular endothelial cells and the activation of hif-1α,which has been applied for the United States patent for the synthesis of drugs for the treatment of diabetic retinopathy.In addition,Dr.Chen also found that CLT-003 can inhibit the activity of pancreatic cancer cells and induce G2/M cell cycle arrest.Therefore,in view of the two roles of CLT-003,we hypothesized whether CLT-003 could play an anti-tumor role by inhibiting the function of hif-1 in pancreatic cancer.Based on this,we design and carry out this research project.Method1.4-nitrobenzene in acetic acid and 2,6-dimethyl anhydride diisopropyl aniline coupling reaction,generate（2,6 diisopropyl phenyl）-5-nitro-1-hydrogen-different indole-1,3-diketone,the intermediate product of nitrocellulose by transfer hydrogenation reduction,generated a purity of 99.2%active pharmaceutical ingredient,namely CLT-003 compounds of active pharmaceutical ingredients.It is then coated with polylactic acid hydroxyacetic acid copolymer nanoparticles by professional nanotechnology.2.A variety of tumor cell lines including pancreatic cancer,lung cancer,cervical cancer,colon cancer and breast cancer,as well as normal breast epithelial cells were selected.The cytotoxicity of CLT-003 on these cell lines was detected by CCK8experiment,and the range of IC50 value of each cell was determined.Cell cycle assay and apoptosis assay were used to verify the effect of CLT-003 with different concentration gradient on the cell cycle and apoptosis of pancreatic cancer cells.Cell scratch assay was used to verify the effect of clt-003 on cell migration,and transwell assay was used to detect the effect of clt-003 on cell invasion ability.Finally,the effect of clt-003 on the proliferation of pancreatic cancer cells in two-dimensional and three-dimensional space was verified again by plate cloning and soft gare cloning experiments.3.A subcutaneous xenograft tumor model of pancreatic cancer SW1990 cells（BABL/C）in nude mice and a xenograft tumor model of pancreatic cancer in situ in PANC02 cells were established.CLT-003 was given intravenously at low doses（50mg/kg）and high doses（100 mg/kg）.The effect of CLT-003 on the growth of pancreatic cancer was observed using small animal in vivo imaging technology.Meanwhile,the effect of CLT-003 on the proliferation and apoptosis of pancreatic cancer was evaluated by IHC technique at the tissue specimen level.4.The effects of CLT-003 on HIF-1αwere evaluated by qPCR,wetern blot and immunofluorescence.QPCR,wetern blot and ELISA were used to detect the effect of CLT-003 on the expression of hif-1 downstream target genes.The effect of CLT-003 on the transcriptional activity of hif-1αwas evaluated by double luciferin assay.5.The effect of CLT-003 on PI3K/AKT/mTOR signaling pathway was detected by wetern blot.Protein degradation assay and ubiquitination assay were used to evaluate the effect of CLT-003 on the stability of hif-1α.The effect of CLT-003 on nucleation of hif-1αprotein cells was evaluated by protein nucleoplasmic separation assay and immunofluorescence assayResults1.Small molecule CLT-003 nanoparticle compound,molecular formula isC₂₀H₂₂N₂O₂,molecular weight is 322.4 g/moL,and its effective component is theanalogue of phenylpeptimide.Its main function is to treat the angiogenesis of diabetic retinopathy.In addition,CLT-003 can inhibit the activation of hypoxia-induciblefactor HIF-1αin vascular endothelial cells and reduce the expression level of vascular endothelial growth factor VEGF.2.After 72 hours of treatment with CLT-003,CCK8 assay was used to detect that the IC50 of CLT-003 for the above mentioned cells was between 2-8μM/L,while the IC50 of MCF-10A for normal breast epithelial cells was 16.9±0.8μM/L,and its cytotoxicity was significantly reduced.CLT-003 at concentrations of 0,2.5,5 and 10μM/L was used to treat 4 types of pancreatic cancer cells for 18-24 hours,and significant G2/M phase block was observed in cell cycle progression.The higher the concentration was,the higher the proportion of G2/M phase block was,with statistical difference（P<0.01）.Similarly,24h after the treatment of pancreatic cancer cells with clt-003,apoptosis was significantly increased,and the apoptosis ratio increased with the increase of CLT-003 concentration,showing a statistical difference（P<0.01）.Clt-003 was found to inhibit the proliferation of tumor cells in two-dimensional and three-dimensional space through plate cloning and soft agar cloning experiments,and the difference between each concentration groups was statistically significant（P<0.01）.In addition,compared with the blank group,CLT-003 significantly inhibited the planar mobility and invasion ability of pancreatic cells after 24-36h of treatment,and the difference between concentrations was also statistically significant（P<0.01）.3.Compared with the normal saline group,the growth of subcutaneous and orthotopic pancreatic transplantation tumor was significantly inhibited in the CLT-00350 mg/kg and 100 mg/kg treatment groups,and the inhibition rate was the most significant in the CLT-003 100mg/kg group（P<0.01）,but there was no significant difference in body weight between the normal saline group and the low-dose and high-dose CLT-003 treatment groups.In addition,CLT-003 treatment also extended the survival period of pancreatic orthotopic transplanted tumor mice,and the difference between groups was statistically significant（P<0.01）.4.Under normoxic and hypoxic conditions,2.5,5 and 10μM/L CLT-003 can lower the mRNA and protein expressions of VEGF,TCF-αand GLUT1,the downstream target genes of HIF-1,compared with the blank treatment group.CLT-003Inhibits the transcriptional activity of HIF-1.In addition,CLT-003 can significantly inhibit the expression level of HIF-1αprotein by acting on CFPAC-1 cells of pancreatic cancer for 24 h and 48 h,and this inhibitory effect is time-dependent and concentration-dependent,but it does not inhibit the mRNA of HIF-1α.In addition,CLT-003 inhibites the expression of HIF-1αprotein in lung cancer,breast cancer and cervical cancer cells.5.Under normoxic and hypoxic conditions,2.5,5 and 10μM/L CLT-003 acted on4 types of pancreatic cancer cells for 6-12 h compared with the blank treatment group.Western blot showed that CLT-003 significantly inhibited the activation of PI3K/AKT/mTOR signaling pathway.Compared with the blank group,CLT-003 had no significant effect on the degradation rate and ubiquitination level of HIF-1αprotein.Under hypoxic conditions,western blot and immunofluorescence results showed that CLT-003 could significantly inhibit the nucleation process of HIF-1αprotein.Conclusions1.CLT-003 nanoparticle compound is a small molecule compound formed by encapsulating the analogues of phenylpeptimide in poly（lactic acid hydroxyacetic acid）copolymer nanoparticles through nanotechnology.The main role of CLT-003 is to inhibit the angiogenesis of diabetic retinal,which may play a role by inhibiting the activation of hypoxia-inducible factor HIF-1 in vascular endothelial cells and the expression level of vascular growth factor VEGF.2.CLT-003 can inhibit the cell activity of a variety of tumor cells,and has anti-tumor universality,but less cytotoxicity for normal epithelial cells;CLT-003 induces G2/M cell cycle arrest in pancreatic cancer cells,promotes apoptosis,and inhibits cell proliferation.In addition,CLT-003 can inhibit the invasion and migration of pancreatic cancer cells.3.CLT-003 can significantly inhibit the growth of pancreatic cancer tumor and prolong the survival period in animals,without obvious toxic and side effects;4.CLT-003 can inhibit the expression of HIF-1αprotein in pancreatic cancer tissues and cells,reduce the transcriptional activity of HIF-1α,and further inhibit the expression of VEGF、TCF-α、and GLUT1,the downstream target genes of HIF-1α.5.CLT-003 can inhibit the activation of PI3K/AKT/mTOR pathway in pancreatic cancer cells,thereby affecting the translation and synthesis of HIF-1αprotein.CLT-003also inhibits the nucleation of HIF-1αprotein,further inhibiting the transcription of HIF-1.