NIRT Dye IR783 Conjugated With Anti-tumor Drug FTS For Targeting Breast Cancer Therapy And Its Molecular Mechanism

ObjectiveIR783,a near-infrared fluorescent dye,can target the tumor through the organic anion transporter(OATP)mediated.IR783 presents excellent fluorescence imaging capability with the emission wavelength at 700?900 nm.In addition,IR783 has good stability in the body.Therefore,using IR783 as a tumor targeting group linked with potential anti-tumor drugs to improve the tumor targeting of anti-tumor drugs will provide a new ideas for the development and improvement of anti-tumor drugs.Farnesylthiosalicylic acid(FTS),a Ras inhibitor,could effectively inhibit lung cancer,colon cancer and breast cancer,et al.However,because of the strong hydrophobicity,high dose in treatment and poor selectivity for tumors,FTS presents side effects on the normal tissues and cells,thus limiting the clinical application of FTS.In our previous study,we used FTS as a tool drug.FTS-IR783 was synthesized by FTS and IR783.Compared with FTS,FTS-IR783 characterized with a higher the water solubility,red fluorescence at 800 nm,and a more obvious anti-cancer activity on MCF7 and LTED breast cancer cells.Therefore,by establishing animal models and cell experiments,this study will systematically evaluate the tumor targeting ability,the uptake mechanism and intracellular distribution,the therapeutic effects and molecular mechanism of FTS-IR783 on breast cancer.Methods1.Tumor targeting evaluation of FTS-IR783Xegraft mice model was established by injecting at the right side of the thigh with 4T1-luc2 cells.When the tumor volume reached 800?1,000 mm³,mice were received 25 mg/kg of FTS-IR783(i.v.or i.p.)treatment.At 0,0.5,1,3,6,12,24,48,72 and 144 h after drug injection,the mice were photographed using Whole Body Imaging System.The distributions of FTS-IR783 in tissues were investigated using fluorescence imaging at 0,0.5,1,3,6,12,24,72,144 h after a single intraperitoneal injection of FTS-IR783.The tumor tissue homogenate was taken and the content of FTS-IR783 in tumor tissue was detected using UHPLC-MS/MS method.Meanwhile,MDA-MB-231,MCF7,4T1 and MCF10A cell models were used to detect FTS-IR783 uptake using laser scanning confocal microscopy and flow cytometry.2.Investigation on the uptake mechanism of FTS-IR783 in MDA-MB-231 cellsThe live cell real-time fluorescence detection was conducted to investigate the accumulation of FTS-IR783 in MDA-MB-231 cells.Flow cytometry was used to investigate the uptake of FTS-IR783 by MDA-MB-231 cells at different concentrations and time of administrations.The changes of FTS-IR783in MDA-MB-231 cells were investigated using OATP and endocytic inhibitors alone or in combination.3.The intracellular distribution of FTS-IR783 in MDA-MB-231 cellsA single cell sequence scanning technique was used to investigate the distribution of FTS-IR783 in MDA-MB-231 cells.Mitochondrial,endoplasmic reticulum and lysosome fluorescent dyes were used to investigate the distribution of FTS-IR783 in organelles.4.Anti-tumor activity evaluation of FTS-IR783 on breast cancer in vivoXegraft mice model was established by injected at the right armpit with MDA-MB-231-luc-D3H2LN cells.The mice were treated with FTS-IR783 for 21 days.Tumor volume and body weight were recorded every three days.At the end,the weights of the organ and tumor were recorded.At the same time,the 4T1-luc2passive transfer model was established by injected in tail vein of mice.Mice were recieved intraperitoneal injection with FTS-IR783 for 18 days,and the body weights were measured every three days.Imags of tumor in mice were pictured once a week.At the end,the weights of the organs were weighed.The lung tissue was photographed and the number of pulmonary nodules was counted.H&E staining were conducted to observe the pathological changes in tissues.5.The antitumor activity evaluation of FTS-IR783 in vitroThe cytotoxicities of FTS-IR783 to MDA-MB-231,MCF7,4T1 and MCF10A were investigated by MTT method,and the IC50 values were calculated.The anti-prolifiration effect of FTS-IR783 on MDA-MB-231,MCF7 and 4T1 cells was investigated by Ed U and plate clone tests.The effect of FTS-IR783 on apoptosis in MDA-MB-231,MCF7 and 4T1 cells was investigated using flow cytometry.The effect of FTS-IR783 on the EMT of MDA-MB-231 and 4T1 cells was investigated by scratch test.The effects of FTS-IR783 on proliferation,apoptosis and EMT related proteins were investigated by Western blotting6.Antitumor Molecular mechanism of FTS-IR783The antitumor-associated signal pathway of FTS-IR783 was investigated by Western blotting.The cell thermal transfer assay(SETSA)and Pull down assay were conducted to screen the target of FTS-IR783.Results1.FTS-IR783 could target on tumor both in vitro and in vivoThe results showed that the fluorescence was observed in the whole body after intravenous injection of FTS-IR783.The fluorescence value in tumor reached highest at 24 h.Even at 144 h,we could observe fluorescence in tumor.Meanwhile,fluorescence was observed in abdomen after intraperitoneal injection of FTS-IR783.The fluorescence was observed in tumor from 6 to 72h.FTS-IR783 could be detected in tumor tissues from 6 to 144 h after administration.At the same time,after 1 h treatment with FTS-IR783(50?M),the luminosity of FTS-IR783 in MDA-MB-231 and MCF7 cells was significantly higher than that in MCF10A cells.After 1 h treatment with FTS-IR783(5?M),the uptake of FTS-IR783 in MDA-MB-231 and MCF7 cells was more than that in MCF10A cells.2.The uptake mechnism of FTS-IR783 in cellsThe fluorescence value of cells were increased in time-dependent manner after treated with FTS-IR783(5?M)for 0,5,15,30,45,60 min.At the same time,the fluorescence values of cells were increased in dose-dependent manner after treated with different concentration of FTS-IR783.In addition,after pretreatment with the non-selective OATP inhibitor BSP,the fluorescent value of cells were significantly decreased compared with that of FTS-IR783 alone.When treated with OATP1B1 and OATP1B3 inhibitors E2G and CCK8,the fluorescent value of cells were also significantly decreased compared with that of FTS-IR783 alone.Similarly,after treated with non-selective endocytic inhibitor Sucrose,and selective inhibitors Chlorpromazine and Methyl-?-cyclodextrin,the fluorescence of the cells were also remarkably decreased compared with the single treatment of FTS-IR783.Conversely,macropinocytosis inhibitor Amiloride has no influence on FTS-IR783uptake.3.FTS-IR783 is mainly distributed in cytoplasmThe single cell sequence scanning results clearly showed that the cytoplasm of cells was filled with red fluorescence,while the nuclear area was not fluorescent.Fluorescent images and colocation analysis showed that nuclei(blue)did not overlap with FTS-IR783(red),while organic cells(green)including mitochondria,endoplasmic reticulum and lysosome were overlapped with FTS-IR783(red).4.FTS-IR783 inhibited breast cancer growth and metastasis in vivoFTS-IR783 could significantly inhibit the growth of tumor in Balb/C nude mice.Compared with the model group,the tumor inhibition rate of high-dose and low-dose FTS-IR783 were 15.24%and 20.45%,respectively.No bodyweight changes,no organ indexies changes and no pathological changes in liver,spleen and kidney were observed after FTS-IR783 treatments.At the same time,FTS-IR783 could inhibit the lung metastasis of 4T1-luc2cells in mice in dose-dependent manner.Compared with the model group,the metastasis inhibition rate of low-and high-dose of FTS-IR783 were 36.88%±2.89%and 50.16%±11.68%,respectively.No organ indexies and no pathological changes in liver,spleen and kidney were observed after FTS-IR783 treatments.5.FTS-IR783 suppressed breast cancer in vitroMTT results showed that the IC₅₀ values of FTS-IR783 on MDA-MB-231,MCF7and 4T1 were 4.36,12.68 and 16.11?M,respectively.Ed U assay results showed that FTS-IR783 dose-dependently inhibited cell proliferation in MDA-MB-231,MCF7 and 4T1 cells.In MCF7 cells,Ed U positive cells were reduced by 20.53%,56.75%and 62.59%,respectively,after 3,6 and12?M of FTS-IR783 treatments compared with control;in MDA-MB-231 cells,Ed U positive cells were reduced by 60.47%,71.68%and 74.35%,respectively,after 1,2 and 4?M of FTS-IR783 treatments compared with control;in 4T1cells,Ed U positive cells were reduced by 44.60%,60.10%and 64.91%,respectively,after 4,8 and 16?M of FTS-IR783 treatments compared with control.Cell colony assay showed that the colony inhibition rates were 24.62%,38.56%and 61.36%,respectively,after 3,6 and 12?M of FTS-IR783 treatments in MCF7 cells;27.57%,28.07%and 34.59%,respectively,after 1,2 and 4?M of FTS-IR783 treatments in MDA-MB-231 cells;28.03%,65.05%and 86.51%,respectively,after 4,8 and 16?M of FTS-IR783 treatments in 4T1 cells.Meanwhile,FTS-IR783 down-regulated the expression level of PCNA protein.Apoptosis detection results showed that 6?M of FTS-IR783 increased the percentage of apoptotic cells from 10.45%in control to 17.58%in MCF7 cells,4?M of FTS-IR783 increased the percentage of apoptotic cells from 11.93%in control to 20.93%in MDA-MB-231 cells,and 16?M of FTS-IR783 increased the percentage of apoptotic cells from 5.09%in control to 10.45%in 4T1 cells.Meanwhile,FTS-IR783 treatments significantly down-regulated Bcl-2expressions,and up-regulated Bax and Cleaved-caspase3 expressions in both MDA-MB-231,MCF7 and 4T1 cells.In addition,1,2 and 4?M of FTS-IR783 inhibited cell migration rates by 33.71%,37.60%and 64.00%,respectively,in MDA-MB-231 cells.8 and 16?M of FTS-IR783 inhibited cell migration rates by 47.24%and 80.51%,respectively,in 4T1 cells.FTS-IR783 treatments also reduced the expression levels of migration related proteins N-cadherin and Vimentin,and increased the expression levels of anti-migration protein E-cadherin.6 Antitumor Molecular mechanism of FTS-IR783FTS could down-regulated the expressions of Akt,p-Akt,ERK and p-ERK.FTS-IR783 had no significant effect on Akt and ERK protein expression,up-regulated the expression of p-Akt,and down-regulated the expression of p-ERK.FTS-IR783 more significantly decreased p-mTOR(S2448),p-mTOR(S2481),Raptor,G?L and p70S6K levels than FTS without influence on expression of mTOR.Moreover,mTOR inhibitor KU-0063794 increased the inhibition effects of FTS-IR783 on p-mTOR(S2448),p-mTOR(S2481)and p-p70S6K expressions,and enhanced the inhibitory effect of FTS-IR783 on breast cancer cells.To the contrary,mTOR activator MHY1485 reversed the inhibition effects of FTS-IR783 on p-mTOR(S2448),p-mTOR(S2481)and p-p70S6K expressions,and reversed the inhibitory effect of FTS-IR783 on breast cancer cells.CETSA and Pull down results showed that FTS-IR783 could combine with AMPK,and dose-dependently increase p-AMPK expression.ConclusionFTS-IR783 was uptake by cancer cells through dual mechnism of OATP mediated and endocytosis.Moreover,FTS-IR783 is mainly distributed at mitochondria endoplasmic reticulum and lysosomes in the cytoplasm of cells.In addition,FTS-IR783 could significantly inhibit the cell proliferation,induce apoptosis,and inhibit cell metastasis through inhibiting Ras/ERK and AMPK/mTOR signaling pathways.This study provides a theoretical basis for the anti-cancer mechanism study of FTS-IR783.At the same time,it provides a new idea for the modification of non-targeted antitumor drugs.