Synthesis Of Genistein-IR783 And FTS-IR783 Conjugates And The Anti-tumor Function For Breast Cancer Therapy

ObjectiveBreast cancer is the most common type of cancer in women.The incidence rates of Chinese women diagnosed with breast cancer continue to increase every year.The chemotherapeutic agents for breast cancer treatment are usually cytotoxic and kill faster growing cancer cells because they are not specifically delivered to cancer cells resulting in undesirable side effects by killing nomraml cells.In addition,cancer cells unavoidably develop resistance to the therapeutic agents sooner or later.Thus there lacks effective treatment for relapsing breast cancer.For these reasons,there is an urgent need on finding of agents with high efficacy and low toxicity for breast cancer treatment.Current studies use heptamethine cyanine dye IR783 as a carrier which has high propensity for cancer cells and accumulate with time,Genistein and farnesylthiosalicylic acid(FTS)are two anti-cancer agent currently under intense research investigation.These two anti-cancer agents we choose as starting compounds to explore a novel approach of tumor targeted therapy because there promising pharmacological profi le.Genistein is an isoflavone existing in a variety of p]ants,especially soyabean and fava beans.It has a broad spectrum of biological activi ty such as antioxidant,anti cancer,anti-inflammatory.Neverthc less,i t still is an attractive compound because of its natural availability and low toxieity profile which makes genistein a focus of research in pharmacological and nutritional fields worldwide.Another drug,the Ras inhibi tor S-trans-trans-famesylsalicylic acid(FTS,is a promising anti-cancer agent with moderate potency and low toxicity,currently undergoing clinical trials as a chemotherapeutic agent.FTS has displayed its potential against a variety of cancers including endocrine resistant breast cancer.However,the poor pharmacokinetic profile due to its high hydrophobicity is a major hindrance for its continued advancement in clinic.Small molecules of the near-infrared fluorescent cyanine dye have high affinity to the organic anion transfer peptide(OATPs)which is highly expressed in cancer cells.It has been reported that near-infrared fluorescent cyanine dyes are readily to gather in cancer cells.Therefore,we chose IR 783,a well-defined heptamethine cyanine dye,as a carrier molecule to conjugate with Genistein and FTS as it can be conjugated with the drug molecule as ease with fairly straightforward and mild chemical reaction.Our studies sought to exploit whether the near-infrared fluorescent IR 783 conjugation with Genistein and FTS can enhance cancer cell targeted delivery and improve the dissolution of Genistein and FTS.We synthesized and investigated the anti-cancer properties of these new compounds,Genistein-IR783 and FTS-IR783 conjugates,for their antitumor activities in vitro and targeted tumor delivery in vivo.Methods1.Chemical synthesis of Genistein-IR783 and FTS-IR783 conjugatesUsing heptamethine cyanine dye IR 783 as a carrier to conjugate Genistein and FTS.Both Genistein-IR783 and FTS-IR 783 conjugates were chemically synthesized and purified by column chromatography over silica gel and thoroughly characterized by spectroscopic analysis such as HPLC?NMR,UV-VIS near infrared fluorescence and MS.Prior to biological studies the purity and homogeneity of Genistein-IR 783 and FTS IR 783 conjugates were confirmed by analytical HPLC.2.Cell viability studies of Genistein-IR783 and FTS-IR783 conjugatesCell viability studies in response to these compounds were evaluated in human breast cancer cells MCF 7,normal breast cells MCF 10A and endocrine therapy resistant LTED cells.The cell lines were treated with Genistein(5,10,25,50 and 75?M),IR-783(5,10,25,50 and 75?M)and Genistein-IR783 conjugate for 72 hrs(5,10,25,50 and 75?M).The cell lines also were treated with FTS(5,10,25,50 and 75?M)?IR-783(5,10,25,50 and 75?M)and FTS-IR783 conjugate for 72 hrs(5,10,25,50 and 75?M).Nuclei were counted using a Coulter counter for cell growth and viability assays.The apoptotic effects of FTS-IR783 conjugate at different concentrations were performed with an ELISA kit.3.In vitro cell uptake studies of Genistein-IR783 and FTS-IR783 conjugatesUptake experiments were carried out in MCF 7 breast cancer cells,benign breast epithelial cells,MCF 10A and LTED cells.Specific uptake of Genistein-IR783(5,10,25,50 and 75?M)and FTS-IR783(5,10,25,50 and 75?M)conjugates was monitored by intracellular fluorescent intensity using LI-COR scanner.Intra-celluar distribution of Genistein-IR783 conjugate was determined by confocal microscopy in MCF 7 and MCF 10A.The time-dependent of cellular uptake of Genistein-IR783 conjugate and potential mechanistic pathway involved were studied by BD FACS Caliber flow cytometer.The video of cellular uptake of Genistein-IR783 conjugate was captured by confocal microscopy in MCF 7 cell.In addition,LC/MS/MS analysis of MCF 7 cells lysates allowed us to confirm and quantify Genistein-IR783 conjugate inside the cells.4.Cellular colocalization of Genistein-IR783 conjugateCellular colocalization of Genistein-IR783 conjugate in MCF 7 cells was observed by intracellular fluorescent intensity using confocal microscopy.5.In vivo NIRF imaging of mice bearing breast tumorUptake and organ distribution of Genistein-IR783 and FTS-IR783 conjugates in nude mice bearing MCF 7 tumors were monitored by near infrared fluorescence emission scans with IVIS imager.The uptake of Genistc in-IR783 conjugate was also confirmed in tumor tissue sections by confocal microscopy.Results1.Chemical synthesis of Genistein-IR783 and FTS-IR783 conjugatesThe synthesis of Genistein-IR783 and FTS-IR783 conjugates have been successful.2.Cell viability studies of Genistein-IR783 and FTS-IR783 conjugatesThe in vitro cell viability assays of the Genistein-IR783 conjugates were performed in MCF 10A?MCF 7 and LTED cell lines.The results indicated that Genistcin-IR783 conjugate inhibit growth of MCF 7 cells and with less inhibition on MCF 10A and LTEI)cells.The results showed improved potency of Genistein-IR783 conjugate(IC50?10.4±1.0?M)as compared with both parent Genistein(IC50?24.8±0.5?M)and carrier IR783(IC50?25.7±0.7?M).Especially carrier IR783 is less toxic to normal cells as compared with Genistein.The results indicated that FTS-IR783 conjugate inhibit growth of MCF 7 cells and LTED cells prominently and with less inhibition on MCF 10A cells.FTS-IR 783 conjugate(25 ?M)induced LTED cell death whereas unconjugated FTS and IR-783 at same concentration.FTS-IR 783 conjugate also induced death of MCF 7 cell.3.In vitro cell uptake studies of Genistein-IR783 and FTS-IR 783 conjugatesComparative cancer cell specific uptake of genistein-IR 783 conjugate was tested in human breast cancer MCF 7 and human mammary gland MCF 10A(normal)cells.The NIRF signal intensity was measured as characteristics of conjugate uptake at 800 nm with Li-COR,and the fluorescence was also observed by eonfocal microscopy.The data showed a concentration-dependent uptake by MCF 7 cells and to a less extent by MCF 10A cells.It was clear that uptake by MCF 7 cells was significantly higher than MCF 10A cells at all concentrations tested.BSP,a classical OATP inhibitor,was used for mechanistic studies and inhibit in Genistein-IR783 conjugate accumulation in cells.OATP inhibitor resulted in about 40%reduction in genistein-IR 783 uptake in MCF 7,and pre-incubation with BSP caused more than 15%decrease of genistein-IR 783 conjugate upake in MCF 10A cells.In addition to OATPs,Chlorpromazine,Methyl-?-cyclodextrin and amiloride based mechanism might also be involved.Pre-incubation with amiloride caused about 40%decrease of genistein-IR 783 conjugate uptake.Cellular uptake was decreased by 20~30%when the cells were pretreated with Methyl-?-cyclodextrin or chlorpromazine.The results suggest that macropinocytosis and OATPs played a more important role in Genistein-IR 783 conjugate uptake as a mechanistic pathway in the cells.LC/MS/MS results of cell lysates showed that MCF 7 cells have higher rate of accumulation of conjugate with time(as high as~450 nM with 20 ?M drug dose).The result suggest that the cellular uptake of Genistein-IR 783 conjugate is dose-dependent in MCF 7cells.4.In vitro cell uptake studies of Genistein-IR783 and FTS-IR783 conjugatesGenistein-IR 783 conjugate was also observed to be entrapped in endochylema of MCF 7 cells.5.In vivo NIRF imaging of mice bearing breast tumorThe In vivo optical imaging in the near infrared fluorescence region was performed on a xenogen IVIS Spectrum instrument with excitation wavelength at 745 nm and emission at 820 nm.Fluorescent intensity in the tumors were significantly higher than in surrounding normal tissues in the mice receiving Genistein-IR 783 conjugate.Similarly,higher fluorescent intensities were found in tumor tissues in the mice receiving FTS-IR 783 conjugate.These results suggest that IR-783 could lead a tumor specific delivery.ConelusionConjugation of IR 783 with Genistein and FTS resulted in two new compounds,Genistein-IR 783 and FTS-IR 783.These two new compounds exhibited better anti-cancer properties compared with their parent molecules.These improved anticancer characteristics could result from tumor specific targeting of these new compounds.The results of this study prove our hypothesis that pharmacological profiles of potential anticancer agents can be significantly improved by conjugating with IR-783.This improvement in targeted profile of known candidate drug molecules will make a significant method to clinic medice in the future.