Targeting Anticancer Effect Of Genistein Conjugate Mediated By Tissue Factor

Epidemiological data has showed that consumption of high soy—containing diet was associated with a low incidence of certain human cancers in east asia populations,such as breast,prostate and colon carcinomas,et al.There are 12 kinds of nature isoflavone component in soy.Genistein(5,7,4'—trihydroxyisoflavone,Gen),a major isoflavone components of soy,has been proved to have antitumor effects in vitro.Genistein is presumed to exert multiple effects related to the inhibition of cancer growth,including inhibition of protein tyrosine kinase(PTK) activity,topoisomeraseâ…¡inhibition,induction of differentiation,and inhibition of angiogenesis.Genistein also can inhibit cell proliferation, exert a specific G₂/M arrest and induce cell apoptosis at high concentrations(≥60μM).It has also been shown that genistein has hematology irradiation protective effect,which is integrating with radiotherapy.However,high plasma genistein concentrations are difficult to achieve and sustain because of its non—specific distribution in vivo and the weak estrogenic effect.Some of receptor is highly expressed on many types of tumor cells.As we known that ligands could bind to receptors specially,the concentration of genistein in tumor might be enhanced by using these ligands.Therefore side effects of genistein might be decreased.Tissue factor(TF) is a transmembrane receptor that forms an exceptionally strong and specific complex with its ligand,factorâ…¦(fâ…¦),as the initiation step of the blood coagulation cascade.It is reported recently that TF is highly expressed on endothelial cells of the tumor vasculature and on many types of tumor cells,such as colon,lung,prostatic and breast cancer.TF not only can enhance clotting of tumor patient,but also is closely related with tumor infiltration and metastasis.Because TF is normally not expressed on cells contacted with blood directly,this pattern made it possible that drugs could selectively targeting to both tumor vasculature and tumor cells through TF. Phage display peptide library was a powerful tool for binding site research between protein molecules and in vitro ligand selection.Foreign researchers have yielded a set of peptides from disulfide—constrained heptapeptide M13 library.These peptides demonstrated selective binding activity to the extracellular domain of TF and no coagulation activation.All of these peptides sequences contained ER motif(-ECLRSVVTC-) and may serve as a promisingly potential carrier for biotherapy or chemotherapy.Based on the questions and reports mentioned above we synthesized a kind of peptide, named FHS001(GECLRSVVTCGGKY),with solid phase peptide synthesis.The peptide could bind to TF without leading to blood coagulation.Furthermore,FHS001 was crosslinked with genistein using the hetero—bifunctional photoreactive cross—linking agent, sulfosuccinimidyl 6—(4'azido—2'—nitrophenybamino) hexanoate(Sulfo—SANPAH). The purpose of this study is to deliver genistein to tumor by using the FHS001 peptide.This, we hypothesized,will enhance the cytotoxicity of genistein against cancer cells and allow its chemotherapeutic properties to be achieved at pharmacologically relevant and achievable levels.There are four parts in our research.1.Survey of TF expression in a wide variety of malignant tissues.2.Preparation of FHS001—Gen conjugate.3.Study the biodistribution of FHS001—Gen in nude mice bearing human breast cancer.4.Study on anti—tumor effect of the FHS001—Gen conjugate.Main results are summarized as follow:1.TF antigen in 55 different cancer specimens was detected by immunohistochemistry method.In our survey,most of the cancer cells of epithelial cell origin stained brightly for TF antigen and TF expression could be demonstrated in each specimen within all subtypes colon cancer,lung cancer and gastric cancer.But TF isn't expressed on normal vasculature endothelial cells so it's a good targeting site for new anti—tumor conjugate.2.The FHS001(GECLRSVVTCGGKY) was achieved with solid phase peptide synthesis as designed and it's clotting activity was measured by prothrombin time(PT) assays.PT assays were done with both normal human plasma and fⅦ—depleted human plasma on an automated instrument used for patient sample.Then specific binding activity of FHS001 was detected by fluorescence microscopy using human breast cancer cells MCF —7 on which TF was highly expressed and human vasculature endothelial cells ECV—304 on which TF was not expressed.It had no influence on the PT values of normal human plasma or fⅦ—depleted human plasma even at high concentration.It could bind to TFpositive MCF—7 cells specially because the membrane of MCF—7 cells showed intense fluorescence in constrast to the TF—negative ECV—304 cells after adding the FITC-FHS001 into the culture medium.So FHS001 could be used as a targeting drug carrier.3.The conjugate,named FHS001—Gen,was achieved by photochemical conjugation and purified successfully from reaction supematant by high performance liquid chromatography(HPLC).The FHS001 conjugated to Gen with a two—step method:firstly attachment of the heterobifunctional photoreactive cross—linker sulfo—SANPAH to free amino groups on FHS001 to form a succinimidyl linkage;and then photolytic generation of a reactive singlet nitrene on the other terminus of the cross—linker in the presence of a 10—fold molar excess of Gen.This resulted in the attachment of Gen via its available C7hydroxyl group to lysine residues of FHS001.Reverse--phase HPLC using a WATERS 510 series was used for separation of FHS001--Gen.The purity of final products were＞95%.Mass spectrum analysis of FHS001--Gen showed a single Mw 1973.4 Da and revealed that each conjugate molecule was containing one FHS001 molecule and one Gen molecule.4.FHS001—Gen was labeled with ^99mTc and Genistein was labeled with Na¹²⁵I by using the Iodogen method.The specific activity of ^99mTc—FHS001—Gen and ¹²⁵I-Genistein were 932 KBq/μg and 336KBq/μg,and radiochemical purity of both were 96.5% and 98.7%respectively.Both of the products could be used in the biodistribution experiment.5.The radioactivity of tumor or main organs in nude mice bearing human breast cancer was detected at 10 min,30 min,60 min,120 min,240 min after intravenous injection and showed that the peak time of ^99mTc—FHS001—Gen concentrating in the tumor was about 10 min after injection and decreased slowly mainly due to elimination through kidneys.The radioactivity in tumor was higher than all detected tissues at 120min except kidneys,the peak ratio of T/NT value also attained at 120min after injection.Whereas the radioactivity of ¹²⁵I—Genistein in tumor was highest at about 30 min after injection and there was low level of radioactive distribution in the tumor.The radioactivity in tumor was lower than that in blood,liver and kidneys at all phases(P＜0.01).No speciality of distribution has been detected in Gen.The radioactivity in tumor of ^99mTC—FHS001—Gen were always higher than those of ¹²⁵I—Genistein.The results suggested biodistribution of Gen in the tumor had been enhanced by FHS001.6.Adding the FHS001—Gen conjugate into the culture medium of human breast cancer cell MCF—7,it's effects on the cell proliferation were studied by MTT analysis.In antiproliferation assays,the inhibition rates of treatment with 200μM FHS001—Gen and unconjugated Gen for 72 h were 83.1%and 65.4%respectively.The IC₅₀ for FHS001—Gen against MCF—7 cells were＞50—fold lower than those of Gen.The effect of FHS001-Gen was better than Gen(P＜0.05).On the other hand,MCF—7 xenograft tumor models in nude mice were made,the effects of FHS001—Gen conjugate on tumor growth and angiogenesis were studied.We also found the volume of tumor and MVD were significantly reduced in transplant tumor in nude mice after a low dose of FHS001—Gen administrating(74μmol/kg×5 times,i.v.).Attack against tumor vasculature may be an important role in anti—tumor activity of FHS001—Gen conjugate.Conclusion:1.TF antigen was overexpressed on both tumor vasculature and many kinds of cancer cells,so it's a good new target for anticancer research.2.The FHS001 peptide designed by us has no coagulation activity and has specific binding activity to TF.It's superior to other anticancer drug carriers.3.FHS001—Gen conjugate can be prepared using crosslinker.4.Using FHS001 to deliver Gen to TF,more cytotoxic agents had been entering cancer cells as compared to the unconjugated drug.In this manner,the selectivity of the treatment of the drug—carrier conjugate may be increased.5.FHS001—Gen conjugate has both the binding activity to TF and the cytotoxicity of genistein.The anticancer effect of genistein has been enhanced significantly after conjugated with the FHS001 peptide.