Study On The Relationship Between Anticancer Activity Of Anthraquinone Derivative H And Its Regulation Of Cell Energy Metabolism

ObjectivesThe aim of this research was to study the killing activity of anthraquinone derivative H on EGFR overexpressed nasopharyngeal carcinoma CNE1 cells and breast cancer MDA-MB231 cells,and to explore the relationship between anthraquinone derivative H killing CNE1 and MDA-MB231 cells and regulating energy metabolism.Methods1.The distribution of anthraquinone derivative H in CNE1 cells and MDA-MB231 cells was observed under laser scanning confocal microscope.2.MTT assay was used to detect the proliferative effect by anthraquinone derivative H alone and combinating anthraquinone derivative H with chemotherapeutic drugs cisplatin（or adriamycin）and gefitinib on the CNE1 and MDA-MB231 cells.3.Flow cytometry was used to detect the apoptosis of CNE1 cells and MDA-MB231 cells,respectively when those cells were treated with anthraquinone derivative H.4.The ultrastructure of CNE1 cells and MDA-MB231 cells after treatment with different concentrations of anthraquinone derivative H were observed by transmission electron microscopy.5.The expression of EGFR,GLUT1,HIF-1α,LC3,p62 and caspase-3proteins in CNE1cells and MDA-MB231 cells after treated with anthraquinone derivative H was detected by Western Blotting.6.ATP generation detection of anthraquinone derivative H on CNE1 cells,MDA-MB231 cells and normal human breast MCF-10A cells were achieved by ATP detection kit.7.Rhodamine 123 was used to detect the effect of anthraquinone derivative H on the mitochondrial membrane potential of CNE1 cells and MDA-MB231cells.8.BCECF AM was used to detect the effect of anthraquinone derivative H on the intracellular pH（pHi）of CNE1 cells and MDA-MB231 cells.9.Immunofluorescence was used to investigate the integrity of the microfilament F-actin and microtubuleβ-tubulin of CNE1 cells and MDA-MB231 cells under administration of anthraquinone derivative H.10.Molecular docking softwares were used to analyze the binding mode of anthraquinone derivative H to key proteins of energy metabolism GLUT1 and HK2.Results1.The results of laser scanning confocal microscope observation showed that anthraquinone derivative H mainly distributed in cytoplasm accompaning withperinuclear accumulation,but no observation of nuclear distrubition.2.MTT results showed that anthraquinone derivative H significantly inhibited the proliferation of CNE1 cells and MDA-MB231 cells as illustrating with the IC₅₀（1.14±0.32）and（2.30±0.42）μmol/L,respectively.The proliferative inhibition of anthraquinone derivative H was significantly better than cisplatin and gefitinib on CNE1 cells,and better than that of adriamycin and gefitinib on MDA-MB231 cells.Anthraquinone derivative H without cytotoxicity concentration could effectively increase the sensitivity of CNE1 cells and MDA-MB231 cells to gefitinib,but did not increase the sensitivity of CNE1cells to cisplatin and MDA-MB231 cells to adriamycin.Compared with gefitinib and adriamycin,anthraquinone derivative H showed lower cytotoxicity to normal breast cells MCF-10A.3.Flow cytometry results showed that anthraquinone derivative H promoted the apoptosis of CNE1 cells and MDA-MB231 cells at the concentration of 1,2,4μmol/L,the apoptosis rate was（35.56±2.21）,（61.46±2.46）and（89.96±1.92）on CNE1 cells,（40.80±3.31）,（56.71±1.43）and（83.28±2.86）on MDA-MB231cells respectively.The apoptosis rate was higher than that of cisplatin,adriamycin and gefitinib in CNE1 cells and MDA-MB231 cells.4.The ultrastructure of CNE1 cells and MDA-MB231 cells were observed by transmission electron microscope after CNE1 cells and MDA-MB231 cells were treated with anthraquinone derivative H at 0.5 and 2μmol/L for 48 h.At the concentration of 0.5μmol/L,the structure of mitochondria,endoplasmic reticulum and Golgi apparatus were damaged in varying degrees.When the concentration reached 2μmol/L,the organelle was seriously damaged,the mitochondria swelled,the cristae disappeared,the mitochondria vacuolated,the nucleus wrinkled,the chromatin gathered,and a large number of vacuoles appeared in the cytoplasm.It can be seen that lysosome phagocytosis damaged mitochondria and other morphological changes.Especially a certain number of lipid droplets were observed in CNE1 cells.5.The results of Western Blotting showed that anthraquinone derivative H decreased the expression of EGFR in CNE1 cells and MDA-MB231 cells,inhibited the expression of energy metabolism related proteins GLUT1 and HIF-1α,whereas promoted the transformation of LC3-I to LC3-II and up-regulated the expression of p62.Anthraquinone derivative H activated the cleaved-caspase-3 activity in CNE1 cells,up-regulates caspase-3 in MDA-MB231 cells but did not activate the activity of cleaved-caspase-3.6.The results of ATP content determination showed that anthraquinone derivative H had a concentration-dependent inhibitory effect on ATP content in CNE1 and MDA-MB231 cells.Compared with control group,the relative percentage of ATP in CNE1 cells treated with 2 and 4μmol/L anthraquinone derivative H was（84.21±2.48）,（74.34±4.16）and（67.28±6.04）,respectively.The relative percentage of ATP in MDA-MB231 cells was（78.24±4.25）,（61.18±3.24）,（54.13±5.15）.However,there was no significant effect on the content of ATP in MCF-10A cells,and the relative percentage of ATP was 96.08±2.15),（104.24±5.21）,（95.94±4.22）,respectively.7.The results of mitochondrial membrane potential detection showed that with the increase of time and the concentration of anthraquinone derivative H,the mitochondrial membrane potential of CNE1 cells and MDA-MB231 cells decreased at first and then increased.8.The results of pHi showed that for CNE-1 cells,the pHi of control group was（7.53±0.34）,1,2,4μmol/L derivative H treatment group was（6.24±0.29）,（6.17±0.17）and（6.10±0.32）,respectively.For MDA-MB231 cells,pHi of control group was（7.59±0.44）,1,2,4μmol/L derivative H treatment group was（6.36±0.21）,（6.25±0.19）and（6.02±0.36）,respectively.Anthraquinone derivative H down-regulated the pHi of CNE1 cells and MDA-MB231 cells in a concentration-dependent manner.However,the chemotherapeutic drugs cisplatin and adriamycin at the concentration of 4μmol/L had no effect on the pHi of the cells.9.Under confocal microscope,the nucleus of CNE1 and MDA-MB231cells treated with anthraquinone derivativeH was deformed,the microfilaments in the cytoplasm decreased greatly,and a small number of green nodes appeared next to the nucleus.There were only a few microfilaments near the cell membrane.On the other hand,the number of microtubules decreased and gathered in a ring around the nucleus.10.Computational molecular dock was conducted focusing on the each targets,GLUT1 and HK2,with anthraquinone derivative H,its anthracene ring and its quinazoline part,individually as well as Gefitinib.The London dG of anthraquinonederivative H,the anthracene ring and the quinazoline part and gefitinib docking with GLUT1 scored at-19.0851,-11.8720,-11.0016 and-13.4712（kcal/mol）,respectively;meanwhile those molecule and precursors docking with HK2 scored at-15.3239,-13.2169,-12.7377 and-13.8193（kcal/mol）,respectively.Conclusions1.Anthraquinone derivative H has strong killing activity on nasopharyngeal carcinoma CNE1 cells and breast cancer MDA-MB231 cells with high expression of EGFR.2.Anthraquinone derivative H can induce apoptosis of nasopharyngeal carcinoma CNE1 cells and breast cancer MDA-MB231 cells by inhibiting energy metabolism,damaging mitochondria,changingthe microenvironment of hypoxia,internal alkali and external acid,and destroying the microfilament microtubule structure of cells.3.Molecular docking analysis show that anthraquinone derivative H probably form a stable complex with the energy metabolism related protein GLUT1 or HK2.4.Anthraquinone derivative H has a dual role of simultaneously inhibiting the activity of EGFR and energy metabolism pathway,which is expected to start a novel model of targeted drug therapy.