Rational Design And Development Of Multi-targeting Metal Anticancer Complexes

In this study,we focused on designing and developing new ligands.These ligands were then coordinated with transition metals i.e.Cu???,Bi???and In???to develop new metal multitargeting anticancer complexes.These ligands and their metal complexes were characterized by infrared spectroscopy?IR?,mass spectrometry,X-ray crystallography,and nuclear magnetic resonance?NMR?.The cytotoxicity of these ligands and complexes toward several human cancer cell lines was carried out using MTT assay.Intracellular metal complexes were measured using inductively coupled plasma mass spectrometry?ICP-MS?.Tumor cells morphology,3D cell sphere and metastasis in tumor cells were examined using wound healing assay and 3D cell sphere technology.Anticancer mechanisms,including cell cycle distribution,cell apoptosis,mitochondrial depolarizations,intracellular reactive oxygen species?ROS?,intracellular Ca²⁺and cellular caspase of the tested tumor cells were carried out using flow cytometry.Cell cycle and apoptosis-related proteins and enzymes were analyzed using Western blot techniques.Inhibitory study of topoisomerase I and DNA cleavage and the binding study was carried out using gel electrophoresis procedure.In the results,some new metal complexes with significant antitumor activities were obtained.???In this study,we designed,developed and synthesized a ligand?i?2-naphthalenol,1-[[?2-pyridinylmethyl?imino]methyl]?HL?,then coordinated to Cu²⁺to obtained Cu???compounds?Cu?HL?Cl₂,C1?and modified with co-ligand pyridine?Cu?L??py?Br,C2?.This Cu???compounds are then studied for structural activity relationships and anticancer mechanisms.?ii?2-acetyl-3-ethylpyrazine and four different N-4 position modified derivatives of thiosemicarbazides were used to design four novels Bi???complexes,i.e,C1(C₁₈H₂₆Cl₆Bi₂N₁₀S₂),C2(C₄₀H₅₆Cl₆Bi₂N₂₀S₄),C3(C₃₀H₃₂Cl₆Bi₂N₁₀S₂),and C4(C₂₆H₃₆Cl₆Bi₂N₁₀S₂),and Ligands?iii?2,6-diacetylpyridine Thiosemicarbazide and their N-4position modified derivatives used to design four novels In???complexes,i.e.C1(C₁₁H₁₄Cl₂InN₇S₂),C2(C₁₃H₁₇Cl₂InN₇S₂),C3(C₂₃H₂₅ClInN₇S₂),and C4(C₁₅H₂₃Cl₂InN₇S₂).The C1-C4 of Bi???and C1-C4 In???complexes were synthesized by stirring protocol and crystallized by an evaporation method.The crystal structures were identified by x-ray crystallography,infrared spectroscopy?IR?,mass spectrometry,and NMR.In Cu???complexes,the molecular structures show the arrangement of the Cu???metal center.The Cu???metal center in C1 is pentacoordinate by two nitrogen atoms and an oxygen atom of the ligand and two terminal chlorine atoms.The coordination geometry in C2 was approximately the same as in C1.Cu???in C2 was pentacoordinate with two nitrogen atoms and one oxygen atom of the ligand,one nitrogen atom of the pyridine substituent,and one terminal bromine atom.C1-C4 of the Bi???complexes have binuclear structures.The two Bi atoms of the dimer are separated and making a double bridge.One Bi atom of complexes C1 and C4 are hepta-coordinated with one Ligand-1 or Ligand-2 vis two N atoms and one S atom of the Schiff-base ligand and four Cl atoms.In C2 and C3,each of Bi???atoms is hexacoordinated by one S atom and two N atoms of Schiff-base ligand and three Cl atoms.In In???complexes C1-C4,the central In atom of each of the ligand is surrounded by coordination polyhedron.In C1 and C2,the central In atom of the dimer are hepta-coordinated,binding to three N-atoms,two S-atom of the ligands,and two terminal chlorine atoms of the complex,while in C3 and C4,the central In atom is coordinated with three N-atoms,two S-atoms,and one Cl atom.???The cytotoxicity of the ligand and complexes were measured using MTT assay.The Cu???complexes,C1,and C2 exhibited lower IC₅₀0 value against A549 cells of?1.06±0.01??M and?0.7±0.013??M,respectively than that of Cisplatin?17.36±0.25??M,but showed lower cytotoxicity against HL-7702 cells?11.02±0.15 and 10.05±0.12?respectively.The cytotoxicities of ligands L1-L4 and Bi???complexes against T-24,Hela,A549,MGC-803,and Wi38 were examined using MTT assay.The treated T24 cells with C1-C4,remarkable cell viability was observed,which was ranging from 2.38?M to 35.89?M.Among the Bi???complexes,C4 showed more potent cytotoxicity against tumor cells,which was several folds more cytotoxic than cisplatin but showed less toxicity against non-tumor cells.Similarly,the cytotoxicity of four N4-modified thiosemicarbazide ligands?L1-L4?,four In???thiosemicarbazone complexes?C1-C4?and cisplatin was determined separately by using MTT assay.To conduct the cytotoxicity assay,five tumor cell lines H460,SKOV3,MGC-803,Hela and T24,and nontumor cells HL-7702 were selected.In???complexes particularly C4showed considerable cytotoxicity against T24 cells?9.10±0.55?M?.the as designed metal complexes were found to be more selective towards tumor cells vs normal cells and more potentially inhibited the tumor cells than Cisplatin.To investigate and measure the total intracellular metal complexes concentration,the treated cells were analyzed using ICP-MS.The total intracellular contents of?i?Cu and Pt in C1,C2,and Cisplatin in A549 cells were measured as 18nmol,21nmol,and 15nmol,which constituted33%,38%,and 27%respectively.The co-ligand pyridine bound to the C2 could influence the concentration of complex matter being absorbed into the cells and could enhance the cytotoxicity of the Cu²⁺complexes.?ii?Similarly,the total intracellular contents of bismuth?Bi?measured in treated T24 cell for C1-C4,and Cisplatin were 0.99nmol,1.27nmol,1.88nmol,4.05nmol,and0.434nmol or 11.46%,14.76%,21.81%,46.93%,and 5.02%respectively,and?iii?In???complexes C1-C4 total intracellular contents of indium and Cisplatin was 1.02nmol,1.40nmol,1.69nmol,3.09nmol,and 0.548 nmol which were 13%,18%,21.8%,39.8%,and 7.05%respectively.The ICP-MS results suggesting that the intracellular absorptions of the as-synthesized complexes were structural dependent,and were higher than that of Cisplatin.???.Flow cytometry was used to analyze the cell cycle distribution and apoptosis in tumor cells.The 2`,7`-dichlorofluorescein diacetate?DCF-DA?assay kit was used to determine the ROS level in tumor cells.The fluorescence intensity was investigated by flow cytometry.This Cu???complexes exerted the chemotherapeutic effect through generation ROS in A549 cells,showing that C1 and C2 complexes generated ROS in A-549 cells.Furthermore,the Cu???complexes arrested the cell cycle at the G₀/G₁ phase,while the Bi???complex C4 and In???complex C4 arrested the cell cycle at S-phase.The flow cytometry analysis data revealed that the percentage of untreated A-549 cells in the G₀/G₁phase was 52.82%.When treated with Cu???complex C1?1.4?M?,it was increased to 57.09%.However,when it was treated with Cu???complex C2?1.4?M?,it raised further to 70.30%.Similarly,the percentage of untreated T-24 cells in the S-phase was 38.48%and,while treated with Bi???complex C4?2.5?M and 5?M?,it was increased to 44.32%and 54.80%respectively.Besides,treated the T24 cell with In???complex C4?5?M and 10?M?,increased the accumulation of T24 cells in S-phase to 31.12%and 39.19%respectively compared to the untreated cells that were 29.33%,suggesting that the C4 arrested the cell cycle in S-phases.Besides,Annexin-V/PI double staining assay was used to measure the occurrence of apoptosis in tumor cells.The rate of apoptosis in A549 cells treated with Cu???complex C1?1.4?M?was 28.4%,and this was increased to 33.3%while treated with Cu???complex C2?1.4?M?.The rate of apoptosis in T24 cells treated with Bi???complex C4?2.5?M and 5?m?12.8%and 29.2%respectively.Similarly,treated the T24 with In???complex C4?5?M and 10?M?the occurrence of apoptosis was increased to 34%and 47%respectivelyWestern blot was used to detect the effect of complexes on the cell cycle-related proteins,the results show that the Cu???complexes C1 and C2 remarkably inhibited the expression of Cyclin E,cyclin-dependent protein kinase-2?CDK2?,while the Bi???complex C4 and In???complex C4 inhibited the expression of Cyclin A and CDK2 in varying degrees.The Bi???complex C4 and In???complex C4 significantly inhibited the cell cycle regulatory factor Cdc25A and up-regulated the expression of p21 and p27 in a concentration-dependent manner.To determine the mitochondrial??m,the JC-1 mitochondrial potential assay kit was used.The A549 cell was treated with Cu???complexes C1 and C2,the??m decreased to 27.9%,and39.1%respectively.Similarly,after treatment of T24 cells with Bi???complex C4,and In???complex C4,the??m decreased to a varying degree compared to untreated T24 cells.Western blot data quantify the Bax,Bcl-2,Bcl-xl,and cytochrome-c,which are the mitochondrial-mediated apoptosis regulatory proteins.The tumor cells were treated with metal complexes separately.The expression of Bax and cytochrome-c were upregulated,where Bcl-2and Bcl-xl were downregulated.Besides,the caspase-9 and caspase-3 were observed to be cleaved in tumor cells,indicating the activation of mitochondrial apoptosis in cancer cells.The function of the ER is Ca⁺⁺dependent;imbalance the Ca⁺⁺level in ER resulting in ionic or redox state.Oxidation stress occurs in the ER lead to the accumulation of unfolded proteins.ER-stress was analyzed by using ER-tracker red to bind to Sulfonylurea receptors in the ER.The treated A549 cells with Cu???complexes C1 and C2,and T24 cells were treated with Bi???complex C4 and In???complex C4 separately,cleared colonization was observed in the treated tumor cells.The intracellular Ca⁺⁺were analyzed by flow cytometry.The T24 cell was treated with Bi???complex C4 and In???complex C4 separately,significant deviated peaks were observed,indicating the imbalance of Ca⁺⁺concentration in T24 cells.A wound-healing assay was applied to measure the metastasis in a tumor cell.T24 cells were treated with Bi???complex C4 and In???complex C4.Compared with untreated cells,the treated cells showed a minor increase or no increase in migration toward the wounded side,while the untreated cells migrated to the wounded side,showing inhibition of tumor cells growth by the metal complexes.Similarly,transwell assay was applied to measure the invasion of T24cells after treated with Bi???complex C4 and In???complex C4,which was significantly inhibited,resulting in the inhibitions of metastasis in tumor cells.Apoptosis signaling-regulated protein kinase?ASK-1?is a MAP-kinase family protein,also called mitogen-activated protein kinase?MAPK?.Proteins related to the MAPK pathway were examined by western blot technique,resulting in the activation of MAPK-mediated apoptosis in tumor cells.Ultraviolet-visible spectroscopy experiments showed that the absorption peaks of the complexes were reduced with different degrees by adding CT-DNA to the solutions of Cu???complexes C1 and C2,which confirmed that the complexes were bound to the CT-DNA helix by insertion.Fluorescence spectroscopy experiments show that the Cu???complexes C1 and C2could compete and substituted EB in EB-DNA to cause a decrease in the fluorescence intensity of the solution.The results of agarose gel electrophoresis show that the Cu???complexes C1 and C2 have a stronger effect on DNA.DNA-topoisomerases represented an essential family of DNA-processing enzymes,and some topoisomerase inhibitors are used clinically for the treatment of numerous human cancers.The gel electrophoresis method was used to determine the topo-I cleavage in vitro.It was observed that the Cu²⁺compounds significantly inhibited the cleavage of the topo-I enzyme by using a concentration-dependent manner,leading to the inhibition of DNA replication and cell apoptosis.