Pd(II) and Rh(III) Complexes with Isoquinoline Derivatives Induced Mitochondria-Mediated Apoptotic and Autophagic Cell Death in HepG2 Cells

{"full_text": " RESEARCH ARTICLE\n\n Received: June 10, 2020 | Accepted: July 27, 2020 | Published: Sept. 14, 2020\n\n\n\n\nPd(II) and Rh(III) Complexes with\nIsoquinoline Derivatives Induced\nMitochondria-Mediated Apoptotic\nand Autophagic Cell Death\nin HepG2 Cells\nNoor Shad Gul1\u2020, Taj-Malook Khan1\u2020, Yan-Cheng Liu1, Muhammad Iqbal Choudhary2, Zhen-Feng Chen1* & Hong Liang1*\n1\nState Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and\nPharmacy, Guangxi Normal University, Guilin 541004, 2International Center for Chemical and Biological Sciences,\nUniversity of Karachi, Karachi 74270\n\n*Corresponding authors: chenzf@gxnu.edu.cn; hliang@gxnu.edu.cn; \u2020N. S. Gul and T.-M. Khan contributed equally to this\nwork.\n\nCite this: CCS Chem. 2020, 2, 1626\u20131641\n\n\n\nNonplatinum metal complexes of [Pd(L1)Cl2] (C1), [Rh studies of C1 demonstrated higher safety than cisplat-\n(L1)Cl3(DMSO)] (C2), [Pd(L2)Cl2] (C3), and [Rh(L3) in and effective tumor growth inhibition. C1 is a po-\nCl3(DMSO)] (C4) with isoquinoline derivatives have tential anticancer drug candidate.\nbeen prepared and characterized. C1\u2013C4 exhibited\nhigher in vitro anticancer activity and lower toxicity\nthan the corresponding ligands and cisplatin against\nHepG2 cells. The mechanistic studies revealed that C1\narrested the cell cycle at S-phase by regulation of\ncyclin and cyclin-dependent kinases. C1 was accumu-\nlated in mitochondria, which increased the generation\nof reactive oxygen species (ROS) and endoplasmic\nreticulum (ER)-stress response through mitochondri-\nal dysfunction. Moreover, C1 stimulated Ca2+ release,\nactivated the caspase cascade, and triggered Keywords: palladium(II), rhodium(III), isoquinoline\nmitochondria-mediated apoptosis. The in vivo derivatives, anticancer, apoptosis, autophagy\n\n\n\n\n drugs with fewer side effects and better chemotherapeu-\nIntroduction tic ef\ufb01cacy than platinum-based drugs.5 During the past\nPlatinum drugs, including cisplatin and its analogs car- decade, nonplatinum metal complexes such as KP1019,\nboplatin (Paraplatin) and oxaliplatin (Eloxatin), have aurano\ufb01n, NAMI-A, and padelipor\ufb01n (TOOKAD) posses-\nbeen in clinical use to treat various solid tumors.1\u20133 De- sing promising anticancer activity against various kinds\nspite clinical success, drug resistance and severe side of cancers with different anticancer mechanisms are\neffects limit their wider use and effectiveness.4 Therefore, reported. Some of these complexes are now in different\nnumerous studies have been carried out to develop new phases of clinical trials.6,7 In addition, palladium(II),\n\n\nDOI: 10.31635/ccschem.020.202000363\nCitation: CCS Chem. 2020, 2, 1626\u20131641\nCitation denotes calendar and volume year of \ufb01rst online publication.\nIssue Assignment: Volume 3 (2021), Issue 6 1626\n\f RESEARCH ARTICLE\n\n\n\n\nrhodium(III), and copper(II) complexes are shown to (2015) for the treatment of soft tissue sarcoma.33 Also, we\nexhibit higher anticancer activity toward various human previously reported that gold complexes with bioactive\ncancer cells than platinum-based drugs.8\u201310 isoquinoline derivatives display high anticancer activity,\n In this regard, palladium(II) complexes presented differ- selectivity, and synergistic effects against different can-\nent anticancer properties against breast, lung, and pros- cer cell lines.34 Inspired by the anticancer activity of metal\ntate cancers than cisplatin.11,12 The palladium complexes complexes with isoquinolines, here, we synthesized pal-\nwith ethyl and benzyl amines induced apoptosis and DNA ladium(II) and rhodium(III) complexes with isoquinoline\ndamage by activating the p53 protein and causing cell derivatives and investigated their in vitro anticancer ac-\ncycle arrest.13\u201317 In addition, several palladium complexes tivity through the ROS/endoplasmic reticulum (ER)-\nwith triazole, pyrazole, and pyrrole as ligands induced stress-mediated and the autophagic cell death pathways.\nreactive oxygen species (ROS)-mediated apoptosis.18 In The in vivo anticancer activity of [Pd(L1)Cl2] (C1) was\nROS-mediated apoptosis, the intrinsic pathway is respon- also conducted on tumor xenograft model of mice\nsible for the release of cytochrome C and the activation of bearing HepG2.\ncaspases, which regulate the Bcl-2 family proteins and\nultimately lead to cell death.19,20 Many palladium com-\nplexes with imidazole and pyridine ligands produce high\n Experimental Methods\nlevels of ROS and reactive nitrogen species (RNS) that not Materials\nonly increase the risk of mutation and inhibit cell division\n The chemicals were purchased from Alfa Aesar (Ward Hill,\nbut also induce apoptosis if the stress persists.21\n MA) and Sigma-Aldrich (St. Louis, MO) and used without\n Similarly, rhodium(III) complexes with polyaromatic\n further puri\ufb01cation. Tris-buffered saline (TBS) solution\nligands have potent anticancer activities against various\n (50 mM NaCl, 5 mM Tris) of pH 7.4 was prepared in\nkinds of cancers, such as colon and breast cancers.22\n double-distilled water. For the in vitro cytotoxicity assays,\nMechanistic studies revealed that these complexes target\n 2 mM stock solution of metal complexes was prepared in\nDNA as well as induce mitochondrial damage that leads to\n dimethyl sulfoxide (DMSO). Similarly, 2 mM solution of\ncell apoptosis.23 Rhodium complexes with trichlorido-5,6-\n cisplatin was prepared in 0.9% NaCl saline. The working\ndimethyl phenanthroline ligands display high cytotoxicity\n solutions were prepared from stock solution by serial\ntoward Jurkat leukemia cells and induce apoptosis by\n dilution with TBS. Elemental analysis was used to deter-\noverproduction of ROS.24 Furthermore, cellular metabolic\n mine the purity of each complex, which were \u226595%.\nstudies of the rhodium complexes demonstrated that the\napoptotic cell death is time dependent with moderate\ninduction of ROS, which indicated the involvement of\n Instrumentation\nan activated intrinsic mitochondria-mediated pathway.25 A PerkinElmer 2400 Series II elemental analyzer was\nMoreover, rhodium complexes with phenylquinoline used for elemental analysis. Electrospray ionization mass\nligands are known as potent kinase C (PKC\u03b4) inhibitors, spectrometry (ESI-MS) spectra were recorded on a Bru-\nand kinase C is regarded as a critical regulator for various ker HCT Spectrometer. A Bruker AV\u2013600 nuclear mag-\ncellular functions.26 The racemic cyclometalated rhodium netic resonance (NMR) spectrometer was used for the\n(III) complexes are promising inhibitors of JAK2 kinase recording of 1H and 13C NMR spectra.\nactivity, which acts as the critical cell signaling mechanism\nfor cell proliferation and apoptosis. The abnormal activi- Synthesis and characterization\nties of these kinases could be correlated with irregular cell\ngrowth and survival.27 Ligands synthesis\n The formation of transition metal complexes with bio- 1-(2\u2032-Aminophenyl)-6,7-dimethoxy-isoquinoline (L1), 1-(2\u2032-\nlogically active ligands is an effective approach in the aminophenyl)-6-methoxy-3,4-dihydroisoquinoline (L2),\ndevelopment of anticancer drugs. However, one major and 1-(2\u2032-aminophenyl)-6-methoxy-isoquinoline (L3)\ndrawback of metal complexes is their instability in the were synthesized by the reported Bischler\u2013Napieralski\ncellular environment.28 Therefore, the selection of an reaction.35\nappropriate bidentate chelating ligand of oxygen and\nnitrogen, such as quinoline, bipyridine, phenanthroline,\n Spectroscopic data of L1\nand their derivatives, has been proposed to improve the\nstability and cytotoxic activity of metal complexes.29\u201331 Infrared (IR) (KBr, cm\u22121): 3328 (N\u2013H), 2927 (C\u2013H), 2852\nIsoquinoline is a prerogative heterocyclic compound (Ar\u2013H), 1623 (C=N), 1577, 1500 (C=C), 1247, 1115 (C\u2013C),\nexhibiting anticancer, anti-Alzheimer, anti-in\ufb02ammatory, 872, 756, 653. 1H NMR [500 MHz, (CD3)2SO, \u03b4]: 8.43 (d,\nanticonvulsant, and antimicrobial properties.32 Recently, J = 4.6 Hz, 1H), 7.68 (d, J = 4.6 Hz, 1H), 7.44 (s, 1H), 7.22 (m,\nthe tetrahydroisoquinoline derivative trabectedin was 2H), 7.18 (s, 1H), 6.91 (d, J = 6.3 Hz, 1H), 6.75 (td, J = 0.9,\napproved by the United States Food and Drug Adminis- 6.0 Hz, 1H), 5.11 (s, 2H) 3.98 (s, 3H), 3.75 (s, 3H). 13C NMR\ntration (USFDA (2007)) and the European Commission [100 MHz, (CD3)2SO, \u03b4]: 157.1, 157.0, 152.9, 150.1, 146.9,\n\nDOI: 10.31635/ccschem.020.202000363\nCitation: CCS Chem. 2020, 2, 1626\u20131641\nCitation denotes calendar and volume year of \ufb01rst online publication.\nIssue Assignment: Volume 3 (2021), Issue 6 1627\n\f RESEARCH ARTICLE\n\n\n\n\n141.1, 131.2, 129.5, 123.3, 122.7, 119.0, 116.4, 116.2, 105.9, 105.5, (C\u2013N), 2381 (C=N), 3429 (N\u2013H), 1614 (C=C), <400 (Pd\u2013Cl),\n56.2, 55.6 (Supporting Information Figures S1\u2013S3). 412 (Pd\u2013N). ESI-MS: m/z = 480.95 [M + Na]+ (Supporting\n Information Figures S10\u2013S13).\nSpectroscopic data of L2\n [Rh(L1)Cl3(DMSO)]\nIR (KBr, cm\u22121): 3409 (O\u2013H), 3293 (N\u2013H), 3184 (C\u2013H), 2966,\n2833 (Ar\u2013H), 1606 (C=N), 1556, 1486, 1450 (C=C), 1252, After formation of the complex via the general proce-\n1107 (C\u2013C), 833, 757, 682, 728. 1H NMR [500 MHz, dure, block yellow crystals of Rh(L1)Cl3(DMSO) (C2)\n(CD3)2\u00b7SO, \u03b4]: 2.72 (t, J = 7.2 Hz, 2H), 3.73 (t, J = 7.2 Hz, were harvested. A suitable crystal was selected for\n2H), 3.83 (s, 3H), 5.83 (s, 2H), 6.58 (t, J = 7.1 Hz, 1H), 6.79 single-crystal X-ray diffraction analysis. Yield: 65%. Ele-\n(d, J = 8.0 Hz, 1H), 6.85 (dd, J = 2.5, 8.6 Hz, 1H), 6.93 (d, J = mental analysis calcd for C19H22Cl3N2O3RhS (%): C,\n2.3 Hz, 1H), 7.04\u20137.14 (m, 3H). 13C NMR [125 MHz, 40.20; H, 3.91; N, 4.93; S, 5.65; found (%): C, 40.33; H,\n(CD3)2SO, \u03b4]: 26.7, 47.2, 55.9, 112.3, 113.4, 115.4, 116.5, 4.01; N, 5.04; S, 5.76. 1H NMR (600 MHz, DMSO-d6, \u03b4): 9.33\n121.1, 122.7, 129.8, 130.0, 131.0, 141.4, 148.4, 161.3, 166.4 (d, J = 6.0 Hz, 1H), 8.39 (d, J = 12.0 Hz, 1H), 7.92 (d, J =\n(Supporting Information Figures S4\u2013S6). 12.0 Hz, 1H), 7.73 (d, J = 6.0 Hz, 1H), 7.58 (s, 1H), 7.48 (s,\n 1H), 7.43 (dd, J = 12.0, 6.0 Hz, 1H), 7.34 (dd, J = 12.0,\n 6.0 Hz, 2H), 5.76\u20135.71 (m, 1H), 4.03 (s, 3H), 3.81 (s, 3H),\nSpectroscopic data of L3\n 3.53 (s, 3H), 3.43 (s, 3H). 13C NMR (150 MHz, DMSO-d6, \u03b4):\nIR (KBr, cm\u22121): 3461 (O\u2013H), 3329 (N\u2013H), 3202, 3048 (C\u2013H), 156.2, 154.6, 150.7, 145.8, 142.0, 135.6, 134.3, 131.8, 130.4,\n2922 (Ar\u2013H), 1617 (C=N), 1553, 1456, 1410 (C=C), 1256, 1116 125.5, 124.1, 123.1, 120.2, 107.4, 105.8, 56.7, 56.0. IR (KBr cm\n(C\u2013C), 833, 682, 628. 1H NMR [500 MHz, (CD3)2SO, \u03b4]: \u22121\n ): 2919 (\u2013OCH3), 3017 (C\u2013H), 1099 (C\u2013N), 2323 (C=N),\n8.50 (d, J = 4.7 Hz, 1H), 7.77 (m, 3H), 7.44 (d, J = 2.1 Hz, 1H), 3461 (N\u2013H), 1614 (C=C), 1425 (S=O), 626 (Rh\u2013S), <400\n7.26 (dd, J = 2.1, 8.0 Hz, 1H), 7.22 (m. 1H) 7.12 (dd, J = 1.1, (Rh\u2013Cl), 433 (Rh\u2013N). ESI-MS: m/z = 588.93 [M + Na]+\n6.2 Hz, 1H), 6.74 (m, 1H), 5.09 (s, 2H) 3.96 (s, 3H). 13C NMR (Supporting Information Figures S14\u2013S17).\n[100 MHz, (CD3)2SO, \u03b4]: 160.6, 159.1, 146.9, 142.9, 139.2,\n131.3, 129.6, 123.1, 122.6, 120.3, 119.5, 116.2, 116.1, 108.1, 105.4,\n [Pd(L2)Cl2]\n56.0 (Supporting Information Figures S7\u2013S9).\n After formation of the complex via the general proce-\n dure, block orange crystals of [Pd(L2)Cl2] (C3) were\nGeneral procedure for the formation of\n harvested. A suitable crystal was selected for\ncomplexes\n single-crystal X-ray diffraction analysis. Yield: 70%. Ele-\nMetal salts (1 mmol) and ligands (0.5 mM) were dissolved mental analysis calcd for C16H16Cl2N2OPd (%): C, 44.42; H,\nin an equal volume of dichloromethane and methanol, 3.95; N, 6.09; found (%): C, 44.53; H, 3.79; N, 6.18. 1H NMR\nand the total volume of 5 mL solvent was placed into a (600 MHz, DMSO-d6, \u03b4): 7.68 (d, J = 12.0 Hz, 1H), 7.61\u20137.58\n25 cm-long glass Pyrex glass tube. The mixture was (m, 1H), 7.50 (d, J = 12.0 Hz, 1H), 7.35\u20137.34 (m, 2H), 7.16 (d,\nfrozen by liquid nitrogen for 5 min, and the air was J = 6.0 Hz, 1H), 7.02 (s, 2H), 6.93\u20136.91 (m, 1H), 4.79 (d, J =\nremoved using a vacuum pump. The tube was sealed by 18.0 Hz, 1H), 3.87 (s, 3H), 3.46\u20133.42 (m, 1H), 2.82\u20132.79 (m,\n\ufb01re torch. After 72 h of constant heating at 80 \u00b0C, block 2H). 13C NMR (150 MHz, DMSO-d6, \u03b4): 166.9, 163.3, 141.9,\ncrystals were harvested. A suitable crystal was selected 139.7, 133.5, 133.1, 132.8, 131.9, 124.9, 121.2, 121.0, 113.4, 113.1,\nfor single-crystal X-ray diffraction analysis. All the re\ufb01ne- 56.2, 52.3. IR (KBr, cm\u22121): 2913 (\u2013OCH3), 3004 (C\u2013H), 1024\nment description and crystal data are listed in Supporting (C\u2013N), 2230 (C=N), 3425 (N\u2013H), 1634 (C=C), <400 (Pd\u2013\nInformation Table S2. Cl), 419 (Pd\u2013N). ESI-MS: m/z = 426.9 [M\u2013H]\u2013 (Supporting\n Information Figures S18\u2013S21).\n[Pd(L1)Cl2]\nAfter formation of the complex via the general proce- [Rh(L3)Cl3(DMSO)]\ndure, block orange crystals of C1 were harvested. A After formation of the complex via the general proce-\nsuitable crystal was selected for single-crystal X-ray dif- dure, block yellow crystals of Rh(L3)Cl3(DMSO) (C4)\nfraction analysis. Yield: 70%. Elemental analysis calcd for were harvested. A suitable crystal was selected for\nC17H16Cl2N2O2Pd (%): C, 44.62; H, 3.52; N, 6.12; found (%): single-crystal X-ray diffraction analysis. Yield: 60%. Ele-\nC, 44.71; H, 3.35; N, 6.23. 1H NMR (600 MHz, DMSO-d6, \u03b4): mental analysis calcd for C18H20Cl3N2O2RhS (%): C, 40.21;\n8.66 (d, J = 12.0 Hz, 1H), 7.88\u20137.86 (m, 1H), 7.63\u20137.60 (m, H, 3.75; N, 5.21; S, 5.96; found (%): C, 40.35; H, 3.71; N, 5.36;\n1H), 7.58 (s, 1H), 7.56 (d, J = 12.0 Hz, 1H), 7.47 (dd, J = S, 5.35 1H NMR (600 MHz, DMSO-d6, \u03b4): 9.39 (d, J = 6.0 Hz,\n18.0, 12.0 Hz, 2H), 7.38 (s, 3H), 4.00 (s, 3H), 3.82 (s, 3H). 13C 1H), 8.44 (s, 1H), 8.09 (d, J = 6.0 Hz, 1H), 7.97 (d, J = 12.0 Hz,\nNMR (150 MHz, DMSO-d6, \u03b4): 154.7, 153.1, 151.4, 144.0, 139.1, 1H), 7.57 (d, J = 6.0 Hz, 1H), 7.52 (d, J = 6.0 Hz, 1H),\n135.4, 133.2, 132.5, 132.1, 125.1, 122.4, 121.5, 106.5, 106.1, 56.8, 7.44\u20137.41 (m, 2H), 7.34\u20137.31 (m, 2H), 5.73 (s, 1H), 4.01\n56.1, 49.1. IR (KBr, cm\u20131): 2971 (\u2013OCH3), 3128 (C\u2013H), 1089 (s, 3H), 3.53 (s, 3H), 3.43 (s, 3H), 3.35 (s, 3H). 13C NMR\n\nDOI: 10.31635/ccschem.020.202000363\nCitation: CCS Chem. 2020, 2, 1626\u20131641\nCitation denotes calendar and volume year of \ufb01rst online publication.\nIssue Assignment: Volume 3 (2021), Issue 6 1628\n\f RESEARCH ARTICLE\n\n\n\n\n(150 MHz, DMSO-d6, \u03b4): 162.4, 158.7, 146.9, 142.1, 140.6, on the oscillator. Both phases of water and oil were col-\n134.9, 131.6, 131.4, 130.5, 125.4, 124.0, 122.6, 121.5, 120.6, lected and were gently separated and dried under vacu-\n105.3, 56.5, 42.1, 41.8. IR (KBr, cm\u22121): 3013 (\u2013OCH3), 3108 um after centrifugation at 5000g for 5 min. The substance\n(C\u2013H), 1019 (C\u2013N), 2350 (C=N), 3440 (N\u2013H), 1617 (C=C), obtained was dissolved in 300 \u03bcM HNO3 (65%) and diluted\n1411 (S=O), 679 (Rh\u2013S), <400 (Rh\u2013Cl), 413 (Rh\u2013N). ESI- by Milli-Q water containing 10 parts per billion indium, to 5\nMS: m/z = 558.92 [M + Na]+ (Supporting Information milliliters. The concentration of C1 (concentration of C1 in\nFigures S22\u2013S25). octanol (Co) and concentration of C1 in water (Cw)) was\n determined by ICP-MS using In as internal standard. The\n log Po/w values were calculated from Co/Cw.\nSingle-crystal X-ray crystallographic analysis\nA Bruker Smart APAX II was used at room temperature to Cell culturing and treatment\ncollect X-ray data of complexes C1\u2013C4. The monochro-\nmatic Mo-K\u03b1 radiation graphite source with wavelength Dulbecco\u2019s modi\ufb01ed eagle medium (DMEM) with fetal\nof (\u03bb = 0.71073 \u00c5) was used for analysis. The direct meth- bovine serum (10%) was used for culturing cells. The cells\nod for the solvation of crystal structure was applied by were held in a 5% CO2 humidi\ufb01ed environment at 37 \u00b0C.\nusing SHELXS-97.36 And for re\ufb01ning all nonhydrogen The stock solution of C1 (2 mM) was prepared in DMSO,\natoms, a method of full-matrix least-squares on F2 was and serial dilutions were performed to obtain the re-\napplied with the thermal parameters of anisotropy by quired working solutions in PBS.\nusing SHELXL-97.37 The atoms of hydrogen were isotro-\npically located at calculated positions. Assay of in vitro cytotoxicity\n The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-\nCellular uptake of complexes trazolium bromide) assay was used for the evaluation\n of the in vitro cytotoxicity. The cells with the density of\nThe cells were placed in a cell culture plate (100 mm) at\n 4 \u00d7 103 were cultivated in a \ufb02at-bottomed 96-well plate.\n37 \u00b0C in a 5% CO2 atmosphere for 24 h. Approximately\n The cells were treated with different concentrations of\n10 \u03bcM each of C1\u2013C4 complexes was added and incuba-\n the complexes, ligands, and cisplatin for 48 h. Cisplatin\ntion continued for 24 h. After 24 h the cells were washed,\n was dissolved in PBS and media containing <1% DMSO\ncollected, and digested with HNO3. Dilution was carried\n and used as the positive control. Negative control cells\nout by adding double-distilled water to 5% HNO3. Induc-\n were supplemented with DMEM. MTT solution was added\ntively coupled plasma mass spectrometry (ICP-MS) was\n after the treatment with drugs and incubated for another\nused for the measurement of metal content in the diluted\n 4 h. After adding DMSO to dissolve the formazan crystals\nsolution.38 The experiment was repeated three times and\n and discarding the supernatant, the absorbance at\nrepresented as mean \u00b1 SD.\n 490/650 nm was measured on a microplate reader.39\n For the determination of metal content in the mitochon-\n The cytotoxicity was assessed using the absorbance ratio\ndria, nucleus, and cytoplasm, the cells were placed in a\n of the treated cells and the control cells. Bliss method\n70 mm plate at 37 \u00b0C for 24 h in a 5% CO2 and 95%\n (n = 5) was used to measure the IC50 values, which show\nhumidi\ufb01ed atmosphere. The cells were treated and incu-\n the sensitivity of cells toward complexes and ligands.\nbated for 24 h with C1\u2013C4 at a concentration of 10 \u03bcM. After\nthe incubation, cells were washed three times with\nphosphate-buffered saline (PBS), and the Solarbio kit was Cell cycle determination\nused to extract mitochondria and nuclei, per the procedure Commercially available 70 mm plates were used for cell\ngiven by the manufacturer. All the samples were treated culturing. The cells were treated with C1 for 24 h. The cells\nwith concentrated HNO3 and dilution was done by adding were collected after 24 h, then washed with PBS, and\ndouble-distilled water up to 5% HNO3 concentration. By \ufb01xed in 70% ethanol for one night at \u201320 \u00b0C. After\nusing ICP-MS, the content of metal in the nucleus and resuspension in RNAs and propidium iodide (PI),\nmitochondria was measured. All these experiments were \ufb02uorescence-activated cell sorting (FACS) was used for\nrepeated three times and represented as mean \u00b1 SD. analysis of the cells. Mod Fit LT (version 3.3; Variety\n Software House, Topsham, Maine, USA) software was\nDetermination of lipophilicity used for cell cycle calculation.\n\nThe \ufb02ask-shaking method was used for the determina-\ntion of lipophilicity of C1\u2013C4. C1 is used as an example to\n Apoptosis assessment\ndemonstrate the procedure here. A mixture of an equal The cells were cultivated for 24 h in a six-well plate. After\nvolume of saturated stock solution of C1 in octanol (sat- treated with C1, the cells were collected, washed, and\nurated with NaCl of 0.9% W/V) and NaCl (0.9% W/V) incubated with PI (5 \u03bcL) and annexin V (5 \u03bcL) for 25 min at\naqueous (saturated with octanol) was shaken for 24 h 30 \u00b0C. The samples were protected from light during\n\nDOI: 10.31635/ccschem.020.202000363\nCitation: CCS Chem. 2020, 2, 1626\u20131641\nCitation denotes calendar and volume year of \ufb01rst online publication.\nIssue Assignment: Volume 3 (2021), Issue 6 1629\n\f RESEARCH ARTICLE\n\n\n\n\nincubation with dyes. FACS was used for the analysis of Western blot analysis\ncell apoptosis.\n The total protein was collected by incubating the cells\n in lysis buffer, and the BCA (the bicinchoninic acid assay)\nMitochondrial membrane potential analysis Protein Assay kit was used for the determination of\nA lipophilic \ufb02uorescent (JC-1) dye was used for measure- protein concentration. Equal amount (25 \u03bcg) of protein\nment of membrane potential of mitochondria. The cells was loaded in the lanes of sodium dodecyl\nwere exposed to C1 at different concentrations for 24 h. sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)\nAfter the exposure with C1, the cells were washed, col- gel (10%). The proteins were then transferred to poly\nlected, and incubated for 25 min with JC-1 dye. Flow (vinylidene di\ufb02uoride) (PVDF) membrane for 1 h at\ncytometry was used for the detection of membrane 120 V after the electrophoresis were done. The mem-\npotential of mitochondria. The damage to the mitochon- brane was placed in a solution containing the primary\ndrial membrane was shown by the orange \ufb02uorescence antibody at 4 \u00b0C for 8 h and incubated in nonfat milk\naccumulated in the mitochondria, whereas the dye mo- dissolved in TBST (Tris-Buffered Saline, 0.1% Tween) for\nnomeric form produced green \ufb02uorescence, which accu- 2.5 h at pH 7.4. The membrane was then incubated for 2 h\nmulated in the cytosol at the event of mitochondrial with a secondary antibody conjugated with horseradish\nmembrane damage. peroxidase at room temperature. TBST was used for\n washing the membrane, and signals were obtained by\n using the enhanced chemiluminescent kit (Western Blot-\nConfocal microscopic studies of ROS\n ting Kit of Peirce ECL).\ngeneration in ER\nHepG2 cells were seeded on the commercially available In vivo anticancer activity\ncoverslip of poly-1-lysine with the density of 2 \u00d7 106 in\n Nude mice from Peking Union Medical College (Beijing,\nsix-well plates with complete medium. Six hours after\n China) were used in this study. All the mice were housed\ntreatment with C1, the cells were washed and incubated\n at Guangxi Medical University facilitation center for\nfor 20 min with 25 mM of 2\u2032,7-dichlorodihydrouorescein\n experimental animals in a controlled environment of\ndiacetate (H2DCFDA) (488 nm/515 nm) at 37 \u00b0C.\n temperature, humidity, and 12 h light and dark cycle.\nH2DCFDA was replaced with prewarmed ER-Tracker Red\n Commercially available food was provided to the ani-\n(587 nm/615 nm) and the cells were incubated for 20 min.\n mals. The studies were performed per Guangxi Medical\nThe cells were washed with ultrapure water, and the\n University\u2019s Guide for the Caring and Use of Laboratory\ncoverslips were placed on the slides and immediately\n Animals. Tumor-bearing mice were divided into four\nanalyzed with confocal microscopy by using suitable ob-\n groups containing six mice in each group. The cisplatin\njective lenses and Zeiss FLUOWIEW viewer (Carl Zeiss\n was dissolved in 0.9% saline. Similarly, a saline solution of\n(Oberkochen, Germany)).\n 0.9% was used for vehicle control. When the diameter of\n the tumor reached 1.4 cm (day 0), the cisplatin or C1 was\nCaspases determination injected intraperitoneally. The cisplatin (2 mg/kg) and C1\nThe cells were treated with C1 for 24 h. Activated cas- (5 and 10 mg/kg) were administrated every 2 days. The\npases were measured by using CaspGLOW kit after the volume of tumor was calculated in mm3 by using the\ntreatment. The cells were then washed and incubated for given formula: volume of tumor = 0.5 \u00d7 (longest diame-\n20 min with 0.5 mL of FITC-LEHD-FMK in the dark and ter) \u00d7 (shortest diameter)2. The growth curves of tumors\nimmediately analyzed with FACS. The data were com- were drawn as the number of days after the \ufb01rst treat-\npared with the control. ment against the average tumor volume. The mice were\n sacri\ufb01ced after 14 days of treatment. The tumor was\nDetermination of intracellular Ca2+ levels collected and weighed, and formalin was applied for\n \ufb01xing in paraf\ufb01n embedding. The given formula was\nThe cells were treated with C1 and cisplatin for 24 h. used to calculate the tumor growth inhibition rate (IRT):\nThe intracellular level of Ca2+ was determined by using IRT = 100% \u2212 (mean tumor weight of the control group \u2212\nFluo-3Am (\ufb02uorescent dye). Afterwards, the cells were mean tumor weight of the experimental group)/mean\nwashed and incubated for 25 min with (5 \u03bcM) Fluo-3Am tumor weight of the control group.40\nin the dark and immediately analyzed with FACS at\n525 nm wavelength of excitation. The dye was broken\ndown into Am (Acetoxymethyl) and Fluo-3 by intracel-\n Statistics\nlular esterase when crossing the cell membrane. The SPSS (version 13.0, Armonk, New York, USA) was used\nFluo-3 bound with cellular Ca2+ resulting in a strong for data processing, including the Student\u2019s t test.\n\ufb02uorescence on excitation wavelength of 488 nm. p \u2264 0.05 was considered statistically signi\ufb01cant.\n\n\n\nDOI: 10.31635/ccschem.020.202000363\nCitation: CCS Chem. 2020, 2, 1626\u20131641\nCitation denotes calendar and volume year of \ufb01rst online publication.\nIssue Assignment: Volume 3 (2021), Issue 6 1630\n\f RESEARCH ARTICLE\n\n\n\n\n ESI-MS (Scheme 1). In addition, the stability of complexes\nResults and discussion C1\u2013C4 was determined under physiological conditions\n of pH 7.4 in TBS and Tris\u2013KCl\u2013HCl buffer by UV\u2013visible\nSynthesis and characterization of ligands\n spectroscopy and high-performance liquid chromatog-\nand complexes\n raphy (HPLC). The results indicated that complexes\nThree ligands of isoquinoline derivatives L1, L2, and L3 were C1\u2013C4 are stable under physiological conditions for\nsynthesized using the reported procedure. Ligand L1 48 h (Supporting Information).\nand L3 differ only in the number and position of the\nmethoxy group. L1 has two methoxy groups at position\n Crystal structure of complexes\n6 and 7, whereas L3 has only one methoxy group at\nposition 6. L2 is a dihydroisoquinoline, which has only one The crystal structures of the four complexes were\nmethoxy group at position 6.41 The ligands were charac- determined by single-crystal X-ray diffraction analysis\nterized by NMR spectroscopy and IR spectroscopy. (Figure 1). Selected bond angles and bond lengths are\n All the complexes, including C1, C2, C3, and C4 were reported in Supporting Information Table S1. C1 and C3\nprepared by mixing the ligand and corresponding metal showed distorted square planar geometry. In both com-\nsalt in a thick Pyrex glass tube containing equal volume of plexes, two atoms of chlorine and the heterocyclic nitro-\ndichloromethane and methanol as the solvent. The tube gen of bidentate isoquinoline ligand L1 and L2 are\nwas sealed by \ufb01re torch. After 72 h of constant heating coordinated with Pd(II). In C1, the bond lengths between\nat 80 \u00b0C, block crystals were harvested. The complexes palladium and nitrogen are Pd\u2013N1 = 2.031 \u00c5 and Pd\u2013N2 =\nwere characterized by elemental analysis, NMR, IR, and 2.036 \u00c5, whereas the bond lengths for palladium and\n\n\n\n\n MeO\n CH3OH:CH2Cl2 = 1:1 Cl\n N\n PdCl2\u00b72H2O MeO Pd Cl\n NH2\n MeO\n\n N\n MeO\n NH2\n C1\n MeO\n Cl Cl\n N\n CH3OH:CH2Cl2 = 1:1 MeO Rh Cl\n L1 NH2 DMSO\n RhCl3(DMSO)\n\n\n C2\n H\n H H H\n H H\n MeO MeO\n H H\n CH3OH:CH2Cl2 = 1:1 Cl\n N N\n PdCl2\u00b72H2O Pd Cl\n NH2 NH2\n\n\n\n\n L2 C3\n MeO MeO\n Cl Cl\n N CH3OH:CH2Cl2 = 1:1 N\n Rh Cl\n RhCl3(DMSO)\n NH2 NH2 DMSO\n\n\n\n\n L3 C4\n\nScheme 1 | Synthetic routes for complexes C1\u2013C4.\n\nDOI: 10.31635/ccschem.020.202000363\nCitation: CCS Chem. 2020, 2, 1626\u20131641\nCitation denotes calendar and volume year of \ufb01rst online publication.\nIssue Assignment: Volume 3 (2021), Issue 6 1631\n\f RESEARCH ARTICLE\n\n\n\n\n Figure 2 | Analysis of metal content in whole cells, mito-\n chondria, nucleus, and cytoplasm of HepG2 cells treated\n with C1\u2013C4 and cisplatin for 24 h performed by ICP-MS.\nFigure 1 | Crystal structures of C1\u2013C4.\n Mean \u00b1 SD: *p < 0.05 and **p < 0.01.\n\n\n In vitro cytotoxicity\nchlorine are Pd\u2013Cl1 = 2.282 \u00c5 and Pd\u2013Cl2 = 2.305 \u00c5. In C3,\nthe bond lengths between palladium and nitrogen are The cytotoxicity of C1\u2013C4 was investigated by MTT assay\nPd\u2013N1 = 2.035 \u00c5 and Pd\u2013N2 = 2.033 \u00c5, whereas the bond against seven cancer cell lines, including T-24, SKOV-3,\nlengths for palladium and chlorine are Pd\u2013Cl1 = 2.993 \u00c5 HepG2, MDA-MB216, MGC-803, A549, HeLa, and normal\nand Pd\u2013Cl2 = 2.984 \u00c5. The C2 and C4 complexes have human cell line WI-38, with cisplatin as a positive control.\nsix-coordinated distorted octahedral geometry in which The IC50 values obtained from the cell viability assay are\none bidentate L1 or L3, three chlorine atoms, and one S listed in Table 1. When treated with L1\u2013L3, no signi\ufb01cant\nfrom DMSO are coordinated with Rh(III). The Rh\u2013N2 effect was observed on cell viability in any cell line,\n(Rh1\u2013N2 and Rh2\u2013N2) of the bidentate ligands (L1 or whereas signi\ufb01cant impact was found in the case of\nL3) and Rh\u2013S1 (Rh1\u2013S1 and Rh2\u2013S1) are located perpen- complexes C1\u2013C4 with IC50 values ranging from 1.05 to\ndicular to each other, and the three chlorine atoms and 22.04 \u03bcM. Complexes C1\u2013C4 had higher cytotoxicity in\nRh\u2013N1 (Rh1\u2013N1 and Rh2\u2013N1) are positioned to the basal the cancer cells than the corresponding ligands and\npart of the square. The corresponding bond lengths are metal salts. Among the four complexes, C1 exhibited the\nas follows: Rh1\u2013N2 = 2.13 \u00c5, Rh2\u2013N2 = 2.09 \u00c5, Rh1\u2013S1 = highest in vitro anticancer activity against the tested cell\n2.27 \u00c5, and Rh2\u2013S1 = 2.25 \u00c5. lines, which is higher than that of cisplatin. Notably, C1\n\n\nTable 1 | IC50 (\u03bcM) Values of L1\u2013L3 and Complexes C1\u2013C4 Against Seven Cancer Cell Lines and One Normal Cell\n\nCompounds WI-38 T-24 SKOV-3 HepG2 MDA-MB216 MGC-803 A549 HeLa\n\nL1 >100 >100 >100 >100 >100 >100 >100 >100\nL2 >100 >100 >100 >100 >100 >100 >100 >100\nL3 >100 >100 >100 >100 >100 >100 >100 >100\nPdCl2\u00b72H2O >50 >50 >50 >50 >50 >50 >50 >50\nRhCl3(DMSO) >50 >50 >50 >50 >50 >50 >50 >50\nC1 29.04 \u00b1 0.3 5.08 \u00b1 0.4 6.07 \u00b1 0.5 1.05 \u00b1 0.4 4.05 \u00b1 0.3 2.34 \u00b1 0.9 3.04 \u00b1 0.3 5.04 \u00b1 0.3\nC2 38.02 \u00b1 0.2 11.03 \u00b1 0.7 14.03 \u00b1 0.6 9.03 \u00b1 0.1 8.02 \u00b1 0.4 13.03 \u00b1 0.4 17.08 \u00b1 0.4 9.03 \u00b1 0.5\nC3 48.02 \u00b1 0.7 25.04 \u00b1 0.6 27.02 \u00b1 0.6 22.04 \u00b1 0.6 34.05 \u00b1 0.6 29.04 \u00b1 0.5 35.06 \u00b1 0.5 28.08 \u00b1 0.1\nC4 41.03 \u00b1 0.1 16.02 \u00b1 0.5 19.06 \u00b1 0.2 14.02 \u00b1 0.5 17.04 \u00b1 0.6 21.35 \u00b1 0.4 25.07 \u00b1 0.1 19.04 \u00b1 0.2\nCisplatina 21.05 \u00b1 0.3 27.02 \u00b1 0.3 24.03 \u00b1 0.6 11.5 \u00b1 0.6 13.03 \u00b1 0.4 19.4 \u00b1 0.09 20.02 \u00b1 0.8 22.03 \u00b1 0.2\na\n The stock solution of cisplatin was made by dissolving 24 mg of powder in pure water (10 mL). The solution was placed at 4 \u00b0C. The\nrequired concentration was prepared in 0.9% NaCl solution. The quality of stock and working solution was checked for quality control\nbefore the performance of experiments.\n\n\nDOI: 10.31635/ccschem.020.202000363\nCitation: CCS Chem. 2020, 2, 1626\u20131641\nCitation denotes calendar and volume year of \ufb01rst online publication.\nIssue Assignment: Volume 3 (2021), Issue 6 1632\n\f RESEARCH ARTICLE\n\n\n\n\nshowed low cytotoxicity toward human normal cell line HepG2 cells were most sensitive to the complexes among\nWI-38. Compared with C3 and C4, complexes C1 and C2 the tested cancer cell lines. Therefore, ICP-MS was used\nexhibited lower IC50 values, which implies that the num- to examine the cellular uptake of C1\u2013C4 complexes in the\nber and position of the methoxy group on the isoquino- HepG2 cell line. The cells were treated with equal con-\nline scaffold affect the cytotoxicity of the complexes. centrations of complexes (10 \u03bcM) for 24 h, and the whole\nThis result is consistent with the fact that C1 and C2 have cell lysate was extracted. The determined uptake amounts\nhigher lipophilicity (log Po/w values) than C3 and C4 of C1\u2013C4 were in the order of C1 > C2 > C4 > C3, agreeing\n(Supporting Information). with their lipophilicity. Generally, the mitochondria and\n nucleus are typically considered the main targets of metal\nDetermination of cellular uptake complexes. Therefore, the distribution of these com-\n plexes in these two subcellular organelles as well as in\nThe cytotoxic activity of metallic anticancer drugs cor- cytoplasm was investigated. The amounts of metal in the\nrelates with their cellular uptake.42 Comparatively, the whole cells (106 cells) treated with C1\u2013C4 and cisplatin\n\n\n\n\nFigure 3 | Analysis of apoptosis in HepG2 cells treated with C1. (a) Effect of C1 and cisplatin on the apoptosis induction\nin HepG2 cells. (b) Histogram representing the percentage of cell population at late and early apoptosis. (c) The\nexpression level of apoptosis-related proteins by Western blot. (d) Western blot quanti\ufb01cation of bands by Image J.\nMean \u00b1 SD: *p < 0.05 and **p < 0.01.\n\nDOI: 10.31635/ccschem.020.202000363\nCitation: CCS Chem. 2020, 2, 1626\u20131641\nCitation denotes calendar and volume year of \ufb01rst online publication.\nIssue Assignment: Volume 3 (2021), Issue 6 1633\n\f RESEARCH ARTICLE\n\n\n\n\nwere 4.9, 2.6, 1.0, 1.7, and 1.5 nM, respectively. The amounts also showed a signi\ufb01cant correlation between the in vitro\nof C1\u2013C4 and cisplatin in mitochondria were determined cytotoxicity and cellular uptake of C1\u2013C4 in HepG2 cells,\nto be 1.6, 1.25, 0.4, 0.6, and 0.3 nM, respectively. The and the mitochondria may be the target of C1\u2013C4 in\nconcentration of C1\u2013C4 and cisplatin in the nucleus HepG2 cells (Figure 2). Since the C1 complex was the\n(106 cells) were determined to be 0.3, 0.2, 0.14, 0.16, and most effective against HepG2, we selected C1 to investi-\n0.3 nM, respectively. In addition, the concentration of gate its anticancer mechanism and in vivo anticancer\nthese complexes in the cytoplasm were 0.2, 0.1, 0.18, activity.\n0.12, and 0.2 nM, respectively. These results indicated that\nthe deposition of complexes in mitochondria was higher Apoptosis of HepG2 cells induced by C1\nthan that in the nucleus and cytoplasm, and the accumu-\nlation of C1 in mitochondria was higher than the other Mitochondrial dysfunction and ER-stress activate differ-\nthree complexes. The optimal lipophilicity associated ent pathways leading to cell death via apoptosis. For\nwith these complexes contributed to the interaction with example, cisplatin induced acute apoptosis via the gen-\nthe lipophilic mitochondrial inner membrane, which facil- eration of high levels of ROS and ER-stress, not by DNA\nitated the penetration into mitochondria. These \ufb01ndings damage.43,44 Therefore, we examined the apoptosis\n\n\n\n\nFigure 4 | Analysis of cell cycle in HepG2 cells treated with C1. (a) Flow cytometric representation of cell cycle arrest of\nHepG2 cells. (b) A graphical representation of different phases of cell cycle. (c) The expression level of proteins related\nto cell cycle by Western blot. (d) Western blot quanti\ufb01cation with Image J. Mean \u00b1 SD: *p < 0.05 and **p < 0.01.\n\nDOI: 10.31635/ccschem.020.202000363\nCitation: CCS Chem. 2020, 2, 1626\u20131641\nCitation denotes calendar and volume year of \ufb01rst online publication.\nIssue Assignment: Volume 3 (2021), Issue 6 1634\n\f RESEARCH ARTICLE\n\n\n\n\nFigure 5 | Analysis and graphical representation of mitochondrial membrane potential after exposure to C1 and\ncisplatin. Mean \u00b1 SD: *p < 0.05 and **p < 0.01.\n\n\n\ninduction ability of C1 by using PI and annexin V staining. Cell cycle arrest\nAfter the HepG2 cells were treated with different concen-\n The cell cycle comprises a series of sequential events\ntrations of C1 (1 and 2 \u03bcM) for 24 h, a dose-dependent\n that control cell division and growth. To investigate\nincrease of late apoptotic cell population was observed.\n whether C1 inhibited the growth of tumor cells through\nThe late apoptotic cell population increased from 0.7% in\n cell cycle arrest, HepG2 cells were treated with different\nthe control to 17.5% and 25.3%, respectively (Figures 3a\n concentrations of C1 and cisplatin and examined by \ufb02ow\nand 3b). Many chemotherapeutic agents induce apopto-\n cytometry using PI staining.47 The \ufb02ow cytometric data\nsis via mitochondrial-mediated pathways, which activate\n revealed that the percent population of cells in S-phase\napoptotic cascades in cancer cells.45 To examine the\n in the control was 20.56%, and it dose-dependently in-\nchanges of apoptosis-related proteins after the treatment\n creased to 50.07% and 62.28% with the treatment of 1\nof HepG2 cells with different concentrations of C1, West-\n and 2 \u03bcM C1, respectively. A comparatively less signi\ufb01cant\nern blotting analysis was carried out. The data indicated\nthe upregulation of proapoptotic proteins (Bak, Bax, cy-\ntochrome C, and Apaf-1) and downregulation of antia-\npoptotic proteins (Bcl-2 and Bcl-xl) (Figures 3c and 3d).\n Apoptosis follows different pathways; two pathways,\nthe intrinsic and extrinsic (death receptor-dependent),\nare most important. These pathways are attributed to the\nactivation of the caspase cascade, which results in the\nmorphological and biochemical alteration of cells.46\nTo explore the effect of C1 on the activation of these\npathways and the induction of mitochondrial-mediated\napoptosis, the primary caspases including caspase-3\n(executioner), caspase-8 (initiator), and caspase-9 were\ninvestigated. After the HepG2 cells were treated with C1\n(1 \u03bcM), the proteolytic activities of caspase-3 and cas-\npase-9 (mitochondrial-mediated) increased from 0.01%\nand 3.78% in the control to 45.2% and 50.2%, respective-\nly. Compared with these caspases, less change was ob-\nserved for caspase-8, which is Fas/TNF mediated. This\nresult showed that the apoptosis induced by C1 occurred Figure 6 | Cellular ATP level depletion with different\nvia the intrinsic mitochondrial pathway (Supporting concentrations of C1, recorded on microplate reader.\nInformation Figure S35). *p < 0.05, **p < 0.01, and ***p < 0.001.\n\nDOI: 10.31635/ccschem.020.202000363\nCitation: CCS Chem. 2020, 2, 1626\u20131641\nCitation denotes calendar and volume year of \ufb01rst online publication.\nIssue Assignment: Volume 3 (2021), Issue 6 1635\n\f RESEARCH ARTICLE\n\n\n\n\nFigure 7 | Flow cytometric analysis and graphical representation of ROS in HepG2 cells, after treatment with C1 and\ncisplatin. Mean \u00b1 SD: *p < 0.05 and **p < 0.01.\n\n\nincrease of cells in S-phase was observed after treatment for the detection of changes in adenosine 5\u2032-triphos-\nwith cisplatin (2 \u03bcM). The results demonstrated that cell phate (ATP) formation affected by C1. After HepG2 cells\ncycle arrest at S-phase may be another mechanism of were treated with different concentrations of C1, a\naction of C1 in HepG2 cells (Figures 4a and 4b). dose-dependent decrease in the intensity of ATP forma-\nCyclin-dependent kinase 2 (CDK2) plays a crucial role in tion was observed. For instance, an 82.5 \u00b1 0.5% decrease\nthe cell cycle G1\u2013S phase transition, which is activated by of ATP production was observed at the highest concen-\ncyclin-A and inhibited by p21.48 The oncogenic protein tration of C1 (8 \u03bcM). The results indicated that C1 inhibited\n(cdc25A) promotes the cell cycle from G1 to S phase. The the ATP formation and impaired the mitochondrial ener-\ninhibition of CDK2 by overexpressed p21, p53, and the gy metabolism of HepG2 cells. These results showed the\ndegradation of cdc25A collectively arrest the cells in involvement of mitochondrial damage in the apoptosis\nS phase.49 Western blotting analysis indicated that the induced by C1 (Figure 6).\nexpressions of p53 and p21 proteins were upregulated,\nwhereas the expressions of CDK2 and cdc25A were\ndownregulated in HepG2 cells when treated with\ndifferent concentrations of C1. These results provided\nstrong evidence that C1 regulated the cell cycle-related\nproteins, arrested cells in S phase, and induced apoptosis\n(Figures 4c and 4d).\n\n\nMeasurement of mitochondrial dysfunction\nMitochondria are recognized as a critical organelle inte-\ngrating the death signals from intrinsic and extrinsic\npathways. Therefore, mitochondrial damage and the re-\nlease of death factors contribute to the initiation of\napoptosis.50,51 To investigate whether C1 targeted mito-\nchondria, the \ufb02uctuation of mitochondrial membrane\npotential, which is an indicator of mitochondrial dysfunc-\ntion, was measured with a \ufb02uorescent dye probe JC-1.\nWhen the HepG2 cells were exposed to C1, the green\n\ufb02uorescence increased dose dependently as compared\nwith the control. This result indicated that the mito-\nchondrial membrane potential was affected by C1, sug-\ngesting mitochondrial membrane damage (Figure 5).\nThe mitochondrial damage results in the alteration of Figure 8 | Confocal microscopic studies of HepG2 cells\nvarious energy production pathways.52 Therefore, a after treatment with C1 [ROS generation indicated by\nluminescence-based assay (Cell Titer-Glo) was used green (DCFH) and ER-stress by ER-tracker red (Red)].\n\nDOI: 10.31635/ccschem.020.202000363\nCitation: CCS Chem. 2020, 2, 1626\u20131641\nCitation denotes calendar and volume year of \ufb01rst online publication.\nIssue Assignment: Volume 3 (2021), Issue 6 1636\n\f RESEARCH ARTICLE\n\n\n\n\nFigure 9 | The expression and quanti\ufb01cation of ER-stress related proteins in HepG2 cells after treatment with C1, using\nImage J for bands detection. Mean \u00b1 SD: *p < 0.05 and **p < 0.01.\n\n\nGeneration of ROS and ER stress colocalized and more defuse (Figure 8). These changes\n may result from the high permeability of the ER mem-\nMitochondria are a major source of ROS, and therefore\n brane, which could be correlated with the stress-induced\nany damage to the mitochondria can cause an increase in\n death by the ER. Western blotting was carried out to\nthe level of ROS, which leads to cellular dysfunction.53\n further investigate the ER-stress-related proteins, such\nThe ROS generation and induction of ER stress are\n as C/EBP homologous proteins (CHOP), PKR-like ER\nregarded as the potential action mechanisms of antican-\n kinase (PERK), and phosphorylated eukaryotic initiation\ncer drugs. Various cellular pathways are activated by the\n factor 2\u03b1 (elF2\u03b1), after HepG2 cells were treated with 1\u20132\nrelease of ROS.54 Our results showed that C1 induced\n \u03bcM of C1 for 24 h. The expression levels of CHOP, PERK,\ncytotoxicity via the targeting of mitochondria, thus the\n and eIF2\u03b1 were upregulated, which could be associated\neffect of C1 on the production of intracellular ROS was\n with ER-stress-induced apoptosis (Figure 9).\ninvestigated by \ufb02ow cytometry (Figure 7). In addition,\nconfocal microscopy was also used for the detection\nof ROS generation and ER stress. The detection of ROS Intracellular calcium production\nwas carried out by using a dye, H2DCFDA, which emits The level of intracellular Ca2+ is important for the main-\n\ufb02uorescence upon oxidation to 2\u2032,7\u2032-dichloro\ufb02uorescein tenance of mitochondrial membrane potential and the\n(DCF) by the cellular ROS. The ER was visualized by normal function of the ER. The ER is highly dependent on\nER-Tracker Red, which binds to the ER sulphonylurea the Ca2+ concentration for the regulation of homeostasis.\nreceptor. By means of confocal microscopy, the coloca- The imbalance of Ca2+ level contribute to the ER stress;\nlization of ER and ROS was clearly observed at the accumulation of unfolded proteins such as CHOP, PERK,\nconcentration of 1 \u03bcM C1. When the concentration of and eIF2\u03b1; and the depolarization of membrane potential\nC1 was increased to 2 \u03bcM, the ROS signals were less of mitochondria, which in turn induce apoptosis.55\n\n\n\n\nFigure 10 | Flow cytometric determination of the intracellular calcium ions (Ca2+) in HepG2 cells after treatment\nwith C1. Mean \u00b1 SD: *p < 0.05.\n\nDOI: 10.31635/ccschem.020.202000363\nCitation: CCS Chem. 2020, 2, 1626\u20131641\nCitation denotes calendar and volume year of \ufb01rst online publication.\nIssue Assignment: Volume 3 (2021), Issue 6 1637\n\f RESEARCH ARTICLE\n\n\n\n\n Therefore, we investigated the \ufb02uctuation of the Ca2+\n level by using a \ufb02uorescent dye (Fluo-3 Am), whose\n \ufb02uorescence intensity increases by 60\u201370 times when\n bound with Ca2+. HepG2 cells treated with C1 (1 \u03bcM)\n exhibited a high level of Ca2+ as shown by the strong\n green \ufb02uorescence, compared with the negative control\n and cisplatin-treated cells. These \ufb01ndings demonstrated\n the imbalance in the Ca2+ level caused by C1, which\n could cause the mitochondria and ER-stress-mediated\n apoptosis (Figure 10).\n\n\n Induction of autophagy\n Morphologically cell death can be described in three\n modes: necrosis, apoptosis, and autophagy.56 Autophagy\n is induced by stress signals, such as increased ROS,\n hypoxia, and mitochondrial damage.57 To investigate\n whether C1 induced cytotoxicity via autophagy, Western\n blotting was carried out to analyze some related pro-\n teins, such as LC3-I and LC3-II.58 The results showed that\n the expression of LC3-II increased, whereas the expres-\n sion of LC3-I decreased dose dependently. The increase\nFigure 11 | Expression levels of autophagy-related pro-\n of the LC3-II protein provided evidence of autophagy\nteins in HepG2 cells after treatment with C1, quanti\ufb01ed by\nImage J. Mean \u00b1 SD: *p < 0.05 and **p < 0.01.\n\n\n\n\nFigure 12 | HepG2 tumor xenograft mice model. (a) Effect of C1 and cisplatin on the growth of tumor volume. (b) Body\nweight of mice after treatment with C1 and cisplatin. (c) Effect of C1 and cisplatin on tumor weight expressed\nin percent inhibition values. (d). Photograph of tumors: negative control, C1-treated and cisplatin-treated group.\nMean \u00b1 SD: *p < 0.05 and **p < 0.01.\n\nDOI: 10.31635/ccschem.020.202000363\nCitation: CCS Chem. 2020, 2, 1626\u20131641\nCitation denotes calendar and volume year of \ufb01rst online publication.\nIssue Assignment: Volume 3 (2021), Issue 6 1638\n\f RESEARCH ARTICLE\n\n\n\n\ninduced by C1. The adaptor proteins p62 and sequesto- comparable anticancer activity but lower cytotoxicity\nsome 1 (SQSTM1), help the conjugation of LC3 in mis- with cisplatin. All these results suggested that C1 is a\nfolded proteins with ubiquitin moieties. Hence, the potential anticancer drug candidate.\nclearance of SQSTM1 and the ubiquitylated proteins me-\ndiated by the autophagic process further con\ufb01rmed the\ninduction of autophagy, as we found the downregulation\n Supporting Information\nof SQSTM1 in our experiment (Figure 11).59 Supporting Information is available.\n\n\nIn vivo anticancer activity of C1 Con\ufb02ict of Interest\nA tumor xenograft mice model bearing HepG2 cells was The authors declare no competing \ufb01nancial interest.\nused to investigate the in vivo anticancer activity of C1.\nThe mice were divided into four groups, negative\ncontrol, positive control, C1 (lower dose), and C1 Funding Information\n(higher dose), with six mice in each group. The positive This work was supported by the National Natural Science\ncontrol group was treated with cisplatin (2 \u03bcM), and the Foundation of China (grant no. 21431001), IRT_16R15, and\nnegative control group was dosed with saline (V/V) Natural Science Foundation of Guangxi Province of Chi-\ncontaining 5% DMSO. The C1-treated groups were na (grant nos. 2016GXNSFGA380005 and AD17129007)\ntreated with low (5 mg/kg) and high doses (10 mg/kg) as well as the \u201cBAGUI Scholar\u201d program of Guangxi\nof C1. The treatments were continued for 14 days, and Province of China.\nhalted when the mice were sacri\ufb01ced on day 14. The\nrates of tumor growth (IR) were measured for C1-low\nand C1-high dose, which were 32.5% and 43.2%, re- Acknowledgments\nspectively. Tumor growth inhibition was observed in a\n The authors thank Ming Chen, Ke-Bin Huang, and\ndose-dependent manner (Figure 12a). The tumor inhi-\n Chun-Zhi Ai for their helpful discussions regarding the\nbition rate of C1 at high dose was comparable with that\n experimental design.\nof cisplatin, which was 49.5% (Figure 12c). No apparent\nside effects and no substantial decrease in body\nweight were observed for C1, whereas a signi\ufb01cant References\nloss in bodyweight was observed for the group treated\n 1. Thayer, A. M. Platinum Drugs Take Their Toll. Chem. Eng.\nwith cisplatin (Figure 12b).The apparent sizes of tumor\n News 2010, 88, 24\u201328.\nwere provided after the fourteen days treatment in\n 2. Marques, M. P. M.; Gianolio, D.; Cibin, G.; Tomkinson, J.;\nFigure 12d. These \ufb01ndings suggested that C1 showed\n Parker, S. F.; Valero, R.; Lopes, R. P.; de Carvalho, L. A. B. B. A\nsimilar tumor inhibition as cisplatin with fewer side\n Molecular View of Cisplatin\u2019s Mode of Action: Interplay with\neffects. DNA Bases and Acquired Resistance. Phys. Chem. Chem.\n Phys. 2015, 17, 5155\u20135171.\n 3. 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