目的 制备局部黏着斑激酶(focal adhesion kinase,FAK)抑制剂defactinib的微晶制剂,利用正电子发射型计算机断层显像检测仪(positron emission computed tomography,PET)对免疫缺陷小鼠肝细胞癌(hepatocellular carcinoma,HCC)肿瘤模型进行PET检测,确定defactinib微晶制剂体内抑制HCC细胞摄取葡萄糖的作用,及其抗肿瘤活性。方法 利用球磨法制备defactinib微晶制剂,利用光学显微镜观察晶型特征。在不同HCC细胞系中检测FAK的蛋白表达水平,选取FAK最高表达的HCC细胞系接种于BalB/c免疫缺陷小鼠(裸鼠)皮下,形成皮下肿瘤。在皮下肿瘤中注射defactinib微晶制剂,在固定时间点收集动物血液或肿瘤组织标本,通过检测标本中defactinib的分布确定defactinib微晶制剂的缓释特性。在此基础上,使用defactinib微晶制剂在皮下肿瘤组织中进行注射。在固定时间点进行PET检测:动物行尾静脉注射300 μCi(11.1 MBq)的核素探针18F-FDG,约50 min后进行PET检测,使用盖革计数器检测动物皮下肿瘤组织与血液的放射性强度比值,进行定量分析。结果 在所选细胞系中,MHCC97-H细胞中FAK的表达水平显著高于其他细胞系。defactinib微晶制剂具有缓释特性,能够长效抑制MHCC97-H皮下肿瘤组织对18F-FDG的摄取,单次给予defactinib微晶制剂就能够实现。结论 本实验成功制备了FAK抑制剂defactinib的微晶制剂,建立了利用PET检测defactinib微晶制剂活性的方法。
Abstract
OBJECTIVE To prepare the microcrystal of defactinib and identify the in vivo activity of defactinib-microcrystal on hepatocellular carcinoma (HCC) cells using PET (positron emission computed tomography) methods. METHODS The protein level of focal adhesion kinase (FAK) in HCC cell lines was examined by Western blot. The solubilizing solution or microcrystal of defactinib was prepared. MHCC97-H cells, which express highest level of FAK, were injected to nude mice to form the subcutaneous tumor. The solubilizing solution or microcrystal of defactinib was injected into tumor tissues. The clearance curve or anti-tumor efficiency of solubilizing solution or microcrystal of defactinib was identified by LC-MS/MS or PET/CT methods. Mice were injected with 300 μCi(11.1 MBq) 18F-FDG and analyzed by PET after 50 min. RESULTS The solubilizing solution or microcrystal of defactinib was successfully prepared. MHCC97-H expresses highest level of FAK than HCC other cell lines. defactinib slowly released by defactinib-microcrystal. Treatment of defactinib-microcrystal sustainably attenuated the absorbing of 18F-FDG in MHCC97-H cells. CONCLUSION The solubilizing solution or microcrystal of defactinib is successfully prepared. A method to identify the in vivo activity of FAK inhibitor is also established.
关键词
局部黏着斑激酶 /
肝细胞癌 /
正电子发射型计算机断层显像 /
defactinib /
药物微晶制剂
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Key words
positron emission computed tomography /
hepatocellular carcinoma /
focal adhesion kinase /
defactinib /
microcrystal
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参考文献
[1] WANG F S, FAN J G, ZHANG Z, et al. The global burden of liver disease:the major impact of China [J]. Hepatology,2014,60(6):2099-2108.
[2] YAN Z, ZENG J, YU Y,et al. HBVcircle: a novel tool to investigate hepatitis B virus covalently closed circular DNA [J]. J Hepatol,2017,66(6):1149-1157.
[3] CHAN A W, CHONG C C, MO F K,et al. Applicability of albumin-bilirubin-based Japan integrated staging score in hepatitis B-associated hepatocellular carcinoma [J]. J Gastroenterol Hepatol,2016,31(10):1766-1772.
[4] JIA H, YANG Q, WANG T,et al. Rhamnetin induces sensitization of hepatocellular carcinoma cells to a small molecular kinase inhibitor or chemotherapeutic agents [J]. Biochim Biophys Acta,2016,1860(7):1417-1430.
[5] XIE H,YU H,TIAN S,et al. What is the best combination treatment with transarterial chemoembolization of unresectable hepatocellular carcinoma a systematic review and network meta-analysis[J]. Oncotarget, 2017, 8(59):100508-100523. doi:10.18632/oncotarget.20119.
[6] ZHAO J X,YUAN Y W,CAI C F,et al. Aldose reductase interacts with AKT1 to augment hepatic AKT/mTOR signaling and promote hepatocarcinogenesis [J]. Oncotarget,2017,8(40):66987-67000.
[7] BAI Y C, HSIA Y C, LIN Y T, et al. Effect of tumor microenvironment on selective uptake of boric acid in HepG2 human hepatoma cells [J]. Anticancer Res,2017,37(11):6347-6353.
[8] YIP C W, LAM C Y, POON T C W, et al. Granulin-epithelin precursor interacts with 78-kDa glucose-regulated protein in hepatocellular carcinoma [J]. BMC Cancer,2017,17(1):409. doi:10.1186/s12885-017-3399-x.
[9] XU D,JIN J,YU H,et al. Chrysin inhibited tumor glycolysis and induced apoptosis in hepatocellular carcinoma by targeting hexokinase-2 [J]. J Exp Clin Cancer Res,2017,36(1):44. doi:10.1186/s13046-017-0514-4.
[10] LI L, CHE L, WANG C, et al. [(11)C]acetate PET imaging is not always associated with increased lipogenesis in hepatocellular carcinoma in mice [J]. Mol Imaging Biol,2016,18(3):360-367.
[11] HSU C C, WU L C, HSIA C Y,et al. Energy metabolism determines the sensitivity of human hepatocellular carcinoma cells to mitochondrial inhibitors and biguanide drugs [J]. Oncol Rep,2015,34(3):1620-1628.
[12] LI L, KANG L, ZHAO W, et al. miR-30a-5p suppresses breast tumor growth and metastasis through inhibition of LDHA-mediated Warburg effect [J]. Cancer Lett,2017,400:89-98. doi:10.1016/j.canlet.2017.04.034.
[13] ZHAO J, HUANG X, XU Z, et al. LDHA promotes tumor metastasis by facilitating epithelial mesenchymal transition in renal cell carcinoma[J]. Mol Med Rep,2017,16(6):8335-8344.
[14] CHOUDHARY K S, ROHATGI N, HALLDORSSON S,et al. EGFR signal-network reconstruction demonstrates metabolic crosstalk in EMT [J]. PLoS Comput Biol,2016,12(6):e1004924.
[15] YU M, ZOU Q, WU X,et al. Connexin 32 affects doxorubicin resistance in hepatocellular carcinoma cells mediated by Src/FAK signaling pathway [J]. Biomed Pharmacother,2017,95:1844-1852. doi:10.1016/j.biopha.2017.09.065.
[16] ZHAO X K, YU L, CHENG M L, et al. Focal adhesion kinase regulates hepatic stellate cell activation and liver fibrosis [J]. Sci Rep,2017,7(1):4032. doi:10.1038/s41598-017-04317-0.
[17] RIMASSA L,PRESSIANI T,PERSONENI N,et al. Regorafenib for the treatment of unresectable hepatocellular carcinoma [J]. Expert Rev Anticancer Ther,2017,17(7):567-576.
[18] KIM D W, TALATI C, KIM R. Hepatocellular carcinoma (HCC):beyond sorafenib-chemotherapy [J]. J Gastrointest Oncol,2017,8(2):256-265.
[19] CHEN Y, FENG F, GAO X, et al. MiRNA153 reduces effects of chemotherapeutic agents or small molecular kinase inhibitor in HCC Cells [J]. Curr Cancer Drug Targets,2015,15(3):176-187.
[20] BEST J,SCHOTTEN C,LOHMANN G,et al. Tivantinib for the treatment of hepatocellular carcinoma [J]. Expert Opin Pharmacother, 2017, 18(7):727-733.
[21] RAOUL J L, GILABERT M, ADHOUTE X, et al. An in-depth review of chemical angiogenesis inhibitors for treating hepatocellular carcinoma [J]. Expert Opin Pharmacother,2017,18(14):1467-1476.
[22] LIN X M, HU L, GU J, et al. Choline kinase α mediates interactions between the epidermal growth factor receptor and mechanistic target of rapamycin complex 2 in hepatocellular carcinoma cells to promote drug resistance and xenograft tumor progression [J]. Gastroenterology,2017,152(5):1187-1202.
[23] NIU L, LIU L, YANG S, et al. New insights into sorafenib resistance in hepatocellular carcinoma:responsible mechanisms and promising strategies [J]. Biochim Biophys Acta,2017,1868(2):564-570.
[24] SHIMIZU T, FUKUOKA K, TAKEDA M, et al. A first-in-Asian phase 1 study to evaluate safety, pharmacokinetics and clinical activity of VS-6063, a focal adhesion kinase (FAK) inhibitor in Japanese patients with advanced solid tumors [J]. Cancer Chemother Pharmacol,2016,77(5):997-1003.
[25] XU X, FAN Z, KANG L, et al. Hepatitis B virus X protein represses miRNA-148a to enhance tumorigenesis [J]. J Clin Invest,2013,123(2):630-645.
[26] XU X J, FAN Z Y, LIANG C Y, et al. A signature motif in LIM proteins mediates binding to checkpoint proteins and increases tumour radiosensitivity [J]. Nat Commun,2017,8:14059.
[27] ASGHARZADEH M R, BARAR J, POURSEIF M M, et al. Molecular machineries of pH dysregulation in tumor microenvironment:potential targets for cancer therapy [J]. Bioimpacts,2017,7(2):115-133.
[28] PEERLINGS J, VAN DE VOORDE L, MITEA C, et al. Hypoxia and hypoxia response-associated molecular markers in esophageal cancer: a systematic review [J]. Methods,2017,130:51-62.
[29] KOU W, WU X A. Research progress in transporter and tumor therapy and prognosis [J]. Chin Pharm J(中国药学杂志),2017,52(19):1657-1663.
[30] YUEN C A,ASUTHKAR S,GUDA M R,et al. Cancer stem cell molecular reprogramming of the Warburg effect in glioblastomas:a new target gleaned from an old concept [J]. CNS Oncol,2016,5(2):101-108.
[31] VALVONA C J,FILLMORE H L,NUNN P B, et al. The regulation and function of lactate dehydrogenase A:therapeutic potential in brain tumor [J]. Brain Pathol,2016,26(1):3-17.
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脚注
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基金
国家自然科学基金项目资助(81601377)
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