Mitochondrial targeted rhodium(III) complexes: Synthesis, characterized and antitumor mechanism investigation.

{"full_text": " Journal of Inorganic Biochemistry 218 (2021) 111400\n\n\n Contents lists available at ScienceDirect\n\n\n Journal of Inorganic Biochemistry\n journal homepage: www.elsevier.com/locate/jinorgbio\n\n\n\n\nMitochondrial targeted rhodium(III) complexes: Synthesis, characterized\nand antitumor mechanism investigation\nYan-Bo Peng a, Can Tao a, Cai-Ping Tan b, *, Ping Zhao a, *\na\n School of Chemistry and Chemical Engineering, Guangdong Pharmaceutical University, Education Mega Centre, No. 280, Waihuandong Road, Guangzhou 510006, PR\nChina\nb\n MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry, Sun Yat-Sen University, Guangzhou 510275, PR China\n\n\n\n\nA R T I C L E I N F O A B S T R A C T\n\nKeywords: Recently, rhodium complexes have received intensive attentions due to their tunable chemical and biological\nRhodium complexes properties as well as attractive antitumor activity. In this work, two imidazole triphenylamino rhodium com\u00ad\nMitochondrial plexes [Rh(ppy)2L1]PF6 (Rh1) and [Rh(ppy)2L2]PF6 (Rh2) (ppy = 2-phenylpyridine, L1 = 4-(1H-imidazo[4,5-\nROS\n f][1,10]phenanthrolin-2-yl)-N,N-diphenylaniline, L2 = N-(4-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)\nApoptosis\n phenyl)-4-methyl-N-(p-tolyl)aniline) have been synthesized and characterized. Both complexes display stronger\n anticancer activity against a various of cancer cells than cisplatin and they can effectively localize to mito\u00ad\n chondria. Further mechanism studies show that Rh1 induce caspase-dependent apoptosis through mitochondrial\n damage, down-regulate the expression of B-cell lymphoma-2 (Bcl-2)/Bcl2-associated x (Bax) and reactive oxygen\n species (ROS) elevation. Our work provides a strategy for the construction of highly effective anticancer agents\n targeting mitochondrial metabolism through rational modification of rhodium complexes.\n\n\n\n\n1. Introduction antitumor effects [18]. The rhodium(III) complex with benzimidazole\n designed by Fatemeh et al. shows a strong binding effect with DNA and\n Since the discovery of the anticancer properties and its successful protein, resulting in significant antitumor activity [14]. However, due to\nintroduction in the clinic in 1969, cisplatin and its analogues are the low fluorescence quantum yields of rhodium(III) complexes, they\nfrequently used for the treatment for many cancers, including ovarian, cannot be imaged in cells by optical imaging. To address this, imidazole-\nbladder, cervical, testicular, lung cancers as well as lymphomas, mye\u00ad based triphenylamino (TPA) derivatives have aroused the researcher\u2019s\nlomas and melanoma [1,2]. However, cisplatin has been severely attention because of their good fluorescent properties [19\u201321], and their\nlimited by the dose-limiting side-effects, including nephrotoxicity, intense interaction with cell biological molecules to produce antitumor\nneurotoxicity and nausea [3]. To address this, the development and effects [22,23]. Complexes containing imidazole triphenylamino ligands\nutilization of non\u2011platinum metal complexes with high antitumor ac\u00ad may enhance the fluorescence performance of rhodium(III) complexes\ntivities have been promoted [4,5]. Among them, rhodium(III) complexes significantly.\nhave recently attracted much interest due to their chemical and bio\u00ad Keeping this in mind, in this work, we synthesized and characterized\nlogical properties as well as various pharmacology properties [6\u201310]. two rhodium(III) complexes, Rh1 and Rh2, with different TPA\nRhodium(III) complexes have stable [11], diverse ligand structures substituted ligands (Fig. 1). The in vitro antiproliferative activities of\n[12,13], and can bind to biomolecules in cells [14\u201316], disrupt cell these two complexes were investigated against several cancer cell lines\nmetabolism and produce antitumor effects. Ma\u2019s group has reported a as well as a human normal cell line. The anticancer properties of the\nseries of cyclometalated rhodium complexes, which can inhibit the mitochondria-accumulated complex Rh1 which included mitochondrial\ndimerization of the signal transducer and activator of transcription 3 damage, cellular intracellular adenosine triphosphate (ATP) depletion,\n(STAT3) pathway, thereby inhibiting the proliferation of cancer cells ROS elevation and induction of apoptosis, were explored using a variety\n[17]. Chen et al. have reported some rhodium complexes containing of methods. Additionally, the possible anticancer mechanisms of Rh1\nisoquinolines, which target mitochondria and increase the level of mi\u00ad were elucidated by analysis of the expression of proteins related to the\ntochondria\u2019s reactive oxygen species (ROS) to produce significant mitochondrial death pathway.\n\n\n * Corresponding authors.\n E-mail addresses: tancaip@mail.sysu.edu.cn (C.-P. Tan), zhaoping666@163.com (P. Zhao).\n\nhttps://doi.org/10.1016/j.jinorgbio.2021.111400\nReceived 22 November 2020; Received in revised form 9 February 2021; Accepted 13 February 2021\nAvailable online 25 February 2021\n0162-0134/\u00a9 2021 Elsevier Inc. All rights reserved.\n\fY.-B. Peng et al. Journal of Inorganic Biochemistry 218 (2021) 111400\n\n\n2. Experimental section mmol) were placed in a 50 mL round bottomed flask with 30 mL of\n dichloromethane (CH2Cl2) and methanol (2: 1, v/v). The mixture was\n2.1. Materials heated at 50 \u25e6 C for 4 h under nitrogen. Then the mixture was cooled to\n room temperature, and the solvent was evaporated under reduced\n All reagents were used as received from commercial sources unless pressure. After that, the mixture was mixed with saturated aqueous\notherwise stated. 1,10-phenanthroline-5,6-dione, 4-(diphenylamino) NH4PF6, followed by stirring for 2 h. The crude product was purified by\nbenzaldehyde, 4-(di-p-tolylamino) -benzaldehyde, cisplatin, 3-(4,5- column chromatography on silica using CH2Cl2-methanol (100:1, v/v)\ndimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), as the eluent\uff0cgiving the product as a yellow powder (0.130 g, 62%).\n Bcl2-associated x (Bax), B-cell lymphoma-2 (Bcl-2) and Caspase-9, ESI-MS: 875.05 [M \u2212 PF6]+. 1H NMR (500 MHz, DMSO) \u03b4 14.12 (s, 1H),\ncytochrome C and \u03b2-actin antibay, 5,5\u2032 ,6,6\u2032 -tetrachloro-1,1\u2032 ,3,3\u2032 -tet\u00ad 9.19 (d, J = 7.2 Hz, 2H), 8.29 (d, J = 8.2 Hz, 2H), 8.17 (d, J = 7.3 Hz,\nraethyl benzimidazolylcarbocyanine iodide (JC-1), 2,7-dichlorodihydro 4H), 8.03 (t, J = 12.8 Hz, 4H), 7.96 (t, J = 7.8 Hz, 2H), 7.48 (d, J = 5.1\nfluorescein diacetate (H2DCFDA) and Annexin V-fluorescein Isothiocy\u00ad Hz, 2H), 7.39 (t, J = 7.8 Hz, 4H), 7.19\u20137.12 (m, 10H), 7.04 (dd, J = 16.0,\nanate (Annexin V-FITC) /Propidium Iodide (PI) apoptosis detection kit, 8.0 Hz, 4H), 6.31 (d, J = 7.6 Hz, 2H). 13C NMR (126 MHz, DMSO) \u03b4\nMitoTracker Deep Red (MTDR) were obtained from Beyotime 168.15, 167.89, 164.68, 153.26, 149.98, 149.55, 148.06, 144.45,\n(Shanghai, China). HeLa (human cervical cancer), A549 (human lung 144.34, 143.05, 139.27, 133.95, 132.77, 132.69, 130.79, 130.46,\ncancer), MDA-MB-231 (Human breast cancer) cells and MCF-10A 128.19, 127.00, 125.80, 125.34, 124.34, 123.64, 121.86, 120.67, 20.55.\n(human normal mammary epithelial cell lines) were obtained from Elemental analysis: calcd (%) for C53H37F6N7PRh\u2022H2O: C, 61.34; H,\nExperimental Animal Center of Sun Yat-Sen University (Guangzhou, 3.79; N, 9.45; found: C, 61.43; H, 3.73; N, 9.34.\nChina). Cells were incubated with Dulbecco\u2019s modifed Eagle medium\n(DMEM) (Gibco BRL) or Roswell Park Memorial Institute (RPMI) 1640 2.3.2. Synthesis of [Rh(ppy)2L2]PF6 (Rh2)\n(Gibco BRL) medium which containing 10% fetal bovine serum (FBS, This complex was synthesized according to a similar procedure with\nGibco BRL), 100 IU/mL streptomycin, 100 IU/mL penicillin at 37 \u25e6 C in Rh1, giving the product as a yellow powder (0.140 g, 67%). ESI-MS:\n5% CO2 and 95% air. 903.00 [M \u2212 PF6]+. 1H NMR (500 MHz, DMSO) \u03b4 14.08 (s, 1H),\n 9.26\u20139.09 (m, 2H), 8.29 (d, J = 8.1 Hz, 2H), 8.18 (d, J = 12.9 Hz, 2H),\n2.2. General instruments 8.14\u20138.09 (m, 2H), 8.03 (t, J = 14.9 Hz, 4H), 7.96 (t, J = 7.5 Hz, 2H),\n 7.48 (s, 2H), 7.19 (d, J = 8.1 Hz, 4H), 7.14 (t, J = 7.5 Hz, 2H), 7.04 (q, J\n Electrospray ionization mass spectra (ESI-MS) were detecting by an = 8.8 Hz, 10H), 6.32 (d, J = 7.6 Hz, 2H), 2.31 (s, 6H). 13C NMR (126\nLCQ system (Finnigan MAT, USA). 1H and 13C NMR spectra were MHz, DMSO) \u03b4 168.15, 167.89, 164.68, 153.26, 149.98, 149.55, 148.06,\ndetected by a Varian Mercury Plus 400 Nuclear Magnetic Resonance 144.45, 144.34, 143.05, 139.27, 133.95, 132.77, 132.69, 130.79,\nSpectrometer and 500 Nuclear Magnetic Resonance Spectrometer, 130.46, 128.19, 127.00, 125.80, 125.34, 124.34, 123.64, 121.86,\nrespectively. Microanalysis (C, H, and N) was carried out using an Ele\u00ad 120.67, 20.55. Elemental analysis: calcd (%) for C55H41F6N7PRh\u2022H2O:\nmentar Vario EL elemental analyzer. The electronic absorption spectra C, 61.98; H, 4.07; N, 9.20; found: C, 61.67; H, 4.10; N, 9.20.\nwere detected by a Perkin-Elmer Lambda 850 UV/Vis spectrometer. The\nemission spectra were detected by a Perkin-Elmer LS 55 luminescence 2.4. Cell cytotoxicity\nspectrometer. Inductively coupled plasma mass spectrometry (ICP-MS)\nwere detected by an iCAP RQ (Thermo Fisher) spectrometer. Confocal The cells were cultured in 96-well tissue culture plates at a density of\nmicroscopy images were detected by LSM 710 NLO (Zeiss) microscope. 10, 000 cells per well for 12 h. Then, the cells were incubated with varies\nFlow Cytometry was carried out on a BD FACS Canto II flow cytometer. concentrations of the compounds for 68 h. 20 \u03bcL of MTT (5 mg/mL) was\n then added to each well, and the plates were incubated for another 4 h.\n The media was carefully removed and 150 \u03bcL dimethyl sulfoxide\n2.3. Synthesis of ligands and its corresponding rhodium(III) complexes (DMSO) was added per well and incubated for 10 min with shaking. The\n absorbance was measured using a microplate reader. For the cytotox\u00ad\n The chloro-bridged dinuclear rhodium(III) precursor [Rh(ppy)2]2Cl2 icity assay in the presence of the inhibitors, HeLa cells were pre\u00ad\nwas synthesized according to the literature [13]. The ligand of 4-(1H- incubated with 10 mM N-acetyl-L-cysteine (NAC) or 50 \u03bcM Z-Val-Ala-\nimidazo[4,5-f][1,10]phenanthrolin-2-yl)-N,N-diphenylaniline (L1) was Asp(OMe)-FMK (z-VAD-FMK) for 1 h, and then treated with Rh1 at the\nsynthesized according to the literature [19]. The ligand N-(4-(1H-imi\u00ad indicated concentrations for 36 h. The cell viability was tested by\ndazo[4,5-f][1,10]phenanthrolin-2-yl)phenyl)-4-methyl-N-(p-tolyl)ani\u00ad measurement of the absorbance at 570 nm (Infinite F200, Tecan,\nline (L2) was synthesized according to the literature [24]. Switzerland).\n\n2.3.1. Synthesis of [Rh(ppy)2L1]PF6 (Rh1)\n The ligand L1 (0.093 g, 0.2 mmol) and [Rh(ppy)2Cl]2 (0.091 g, 0.1\n\n\n\n\n Fig. 1. Chemical structures of Rh(III) complexes.\n\n 2\n\fY.-B. Peng et al. Journal of Inorganic Biochemistry 218 (2021) 111400\n\n\n2.5. Lipophilicity measurement 2.11. ATP depletion assay\n\n The lipophilicities of Rh1 and Rh2 were tested according to the ATP quantification was detected using CellTiter-Glo\u00ae Luminescent\npreviously reported method [25]. Octanol-saturated water (W) and Cell Viability Assay kit (G7570, Promega, USA) according to the man\u00ad\nwater-saturated octanol (O) were acquired by mixing octanol and water. ufacturer\u2019s instructions. HeLa cells were plated at a density of 1.4 \u00d7 105\nAliquots of stock solutions of the rhodium complexes in W were added to cells/mL in 96 well plates and incubated for 24 h and then incubated\nequal volumes of O and were shaken at 1000 rpm for 72 h. Then the with Rh1 at the indicated concentrations for another 12 h. The culture\nsample was centrifuged at 8000 rpm for 3 min. The aqueous and octanol medium was extracted for 100 mL, and then 100 mL of cellTiter-Glo\nlayers were carefully separated into test tubes and measured by UV\u2013Vis reagent was added to each well, incubated for another 10 min at room\nspectroscopy. Partition coefficients of ruthenium complexes were temperature. The luminescence was measured employing a microplate\ncalculated using the eq. (1): reader (Infinite M200 Pro, Tecan, Switzerland).\nlogP = log([RhO]/[RhW] ) (1)\n 2.12. Hoechst 33342 staining\n\n2.6. Cell colocalization assay HeLa cells were cultured in confocal microscopy dishes for 12 h, and\n then treated with Rh1 at the indicated concentrations for 24 h. Cells\n HeLa cells were cultured in confocal microscopy dishes and incu\u00ad were washed twice with cold PBS and fixed with 4% paraformaldehyde\nbated for 12 h. Rhodium complexes were added to the dishes and at room temperature for 2 h. After the cells were labeled with 2\u2032 -(4-\nincubated with the cells at 37 \u25e6 C for 2 h. The cells were then incubated ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-1H,3\u2032 H-2,5\u2032 -bibenzimidazole\nwith MitoTracker Deep Red (MTDR) (100 nM) for 0.5 h. After that, the (Hoechst 33342, 5 \u03bcg/mL in water) for 30 min, they were analyzed\ncells were washed by phosphate buffer saline (PBS). The cells were immediately by confocal microscopy. \u03bbex = 405 nm, \u03bbem = 460 \u00b1 20 nm.\nimaged with a Zeiss LSM 710 NLO confocal microscope (63 \u00d7 oil). Rh1\nand Rh2: \u03bbex = 405 nm; \u03bbem = 600 \u00b1 20 nm. MTDR: \u03bbex = 633 nm, \u03bbem = 2.13. Annexin V/PI staining\n650 \u00b1 20 nm.\n HeLa cells were cultured in 6-wells plates for 12 h and treated with\n Rh1 at the indicated concentrations for 24 h. Cells were collected by\n2.7. ICP-MS analysis\n centrifugation and washed twice with cold PBS. The resuspended cells\n were incubated with Annexin V-FITC and PI for 30 min in the dark at\n HeLa cells were cultured in 10 cm culture plates for 12 h. Then, they\n 37 \u25e6 C. Data were collected by a flow cytometer. Annexin V (\u03bbex = 488\nwere incubated with the rhodium complexes (2 \u03bcM) at 37 \u25e6 C for 6 h. The\n nm, \u03bbem = 510 \u00b1 20 nm), PI (\u03bbex = 488 nm, \u03bbem = 610 \u00b1 20 nm)\ncytoplasmic, mitochondrial and nuclear were obtained using the com\u00ad\n (FACSCaliburTM, Becton Dickinson, Franklin Lakes, NJ, USA) and\nmercial kit (Beyotime, China). The rhodium concentration in the sub\u00ad\n analyzed with FlowJo 7.6 software (Tree Star, OR, USA).\ncellular compartments was determined using ICP-MS (Thermo\nElemental Co., Ltd). The data are reported as the mean \u00b1 standard de\u00ad\n 2.14. Western blot analysis\nviation (n = 3).\n HeLa cells were plated at 10 cm culture plates and treated with Rh1\n2.8. Mitochondrial membrane potential assay at indicated concentration for 24 h. The cells were collected and washed\n three times using PBS, then incubated with radioimmuno precipitation\n HeLa cells were cultured in 6-wells plates incubated for 12 h. Rh1 assay buffer (RIPA) at 4 \u25e6 C for 30 min, and the protein concentrations\nwith different concentrations were added to the wells and incubated were ascertained by UV. Equal amounts of cellular total proteins were\nwith the cells at 37 \u25e6 C for 12 h. The cells were then washed by fresh resolved by SDS-polyacrylamide gel electrophoresis and transferred to\nmedium three times. Then, the working buffer for JC-1 staining was nitrocellulose membranes. The membranes were blocked with 5%\nadded and the cells followed by incubating for 20 min. The fluorescence blocking buffer at room temperature for 2 h, and then incubated over\u00ad\nwas recorded by Flow cytometer. \u03bbex = 488 nm; \u03bbem = 580 \u00b1 20 nm night at 4 \u25e6 C with \u03b2-actin, Bcl-2, Bax, Caspase-9 and cytochrome C pri\u00ad\n(Red); \u03bbem = 510 \u00b1 20 nm (Green). mary antibodies, followed by incubation with HRP-conjugated\n secondary antibody for 2 h at room temperature. An enhanced chem\u00ad\n iluminescence kit was using for detection using Image Lab software (Bio-\n2.9. Intracellular ROS detection\n Rad Gel Doc XR+).\n\n HeLa cells were cultured in 6-wells plates incubated for 24 h. Then\n 2.15. Statistical analysis\nRh1 at the indicated concentrations were added and incubated for 12 h.\nThe cells were harvested and incubated with 10 \u03bcM H2DCFDA for 30 min\n All biological experiments were performed at least twice with trip\u00ad\nat 37 \u25e6 C in the dark. The cells were collected by centrifugation and\n licates in each experiment. Representative results are presented as the\nwashed twice with serum-free DMEM to remove the excess staining dye.\n means \u00b1 standard deviations.\nThe 2\u2032 ,7\u2032 -dichlorofluorescein (DCF) fluorescence intensity of cell was\ndetected by flow cytometry. \u03bbex = 488 nm; \u03bbem = 530 \u00b1 30 nm. The\n 3. Results and discussion\nmean fluorescence intensity (MFI) was analyzed using FlowJo 7.6\nsoftware.\n 3.1. Synthesis and characterization\n\n2.10. Colocalization assay of DCF and MTDR Rh1 and Rh2 were successfully synthesized using a facile method\n with high yields. The synthetic routes of the ligands and complexes are\n HeLa cells were cultured in confocal microscopy dishes incubated for elucidated in Scheme. S1. The complexes were characterized by ESI-MS,\n 1\n12 h and then treated with Rh1 (2 \u03bcM) for another 6 h. The colocali\u00ad H NMR, 13C NMR and elemental analyses (Fig. S1-S6). The UV\u2013Vis\nzation of the fluorescence of DCF and MTDR was obtained as previously absorption spectra of Rh1 and Rh2 were detected in CH2Cl2, acetonitrile\ndescribed [26]. MTDR: \u03bbex = 633 nm; \u03bbem = 650 \u00b1 20 nm. DCF: \u03bbex = (CH3CN) and PBS, respectively (Fig. 2A). The results clearly reveal that\n488 nm; \u03bbem = 510 \u00b1 20 nm. intense spin-allowed intragand transition (\u03c0 \u2192 \u03c0*) absorption bands at\n\n 3\n\fY.-B. Peng et al. Journal of Inorganic Biochemistry 218 (2021) 111400\n\n\n\n\nFig. 2. A) UV\u2013Vis spectra of Rh1 and Rh2 in CH2Cl2 (a), CH3CN (b) and PBS (c). B) Emission spectra of Rh1 and Rh2 (\u03bbexc = 405 nm) in CH2Cl2 (a), CH3CN (b), PBS\n(c) at 25 \u25e6 C.\n\n\napproximately 250\u2013340 nm in the UV region and less intense spin-\n Table 1\nallowed metal-to-ligand charge-transfer (MLCT) absorption bands at\n Cytotoxicity (IC50) of Rh1 and Rh2 towards cells in vitro.a\napproximately 350\u2013530 nm [27,28], respectively. These are the char\u00ad\nacteristic bands of rhodium complexes, suggesting the successful coor\u00ad Complex IC50 (\u03bcM)\ndination of the metal. In addition, no obvious change can be observed in HeLa A549 MDA-MB-231 MCF-10A\nthe time-dependent absorption spectra of Rh1-Rh2 (Fig. S7), indicating Rh1 1.13 \u00b1 0.03 1.77 \u00b1 0.04 0.74 \u00b1 0.02 4.57 \u00b1 0.10\nthe high stability of these two complexes. Rh2 2.10 \u00b1 0.20 2.97 \u00b1 0.20 1.58 \u00b1 0.04 8.78 \u00b1 0.31\n The emission spectra (Fig. 2B) and the phosphorescence lifetimes of Cisplatin 8.91 \u00b1 0.20 7.53 \u00b1 0.43 7.08 \u00b1 0.16 14.13 \u00b1 0.32\nRh1 and Rh2 in the indicated solutions were detected (Table S1).Upon a\n IC50 values are drug concentrations necessary for 50% inhibition of cell\nexcitation at 405 nm, Rh1 and Rh2 display yellow-red emission with an viability. Data are presented as means \u00b1 standard deviations obtained in at least\nemission maximum around 600 nm. The emission intensities of Rh1 and three independent experiments. Cells are treated with the complexes for 72 h.\nRh2 show a dependence on the polarity of the solvents, with no spectral\nshift observed. 3.3. Cellular uptake and localization\n Meanwhile, lipophilicity is commonly referred to as the partition\ncoefficient of the compound in n-octanol/water (Po/w). The lipophilicity Since both Rh1 and Rh2 emit phosphorescence at 600 nm\nof Rh1 and Rh2 was measured by the classical shake-flask method. The (Table S1), their cellular distribution was investigated by confocal\nlogPo/w values of Rh1 and Rh2 are 1.74 and 2.27, respectively. As ex\u00ad microscopic observation. Both the two complexes can be visualized in\npected, the lipophilicity of the complexes increases with adding a methyl the HeLa cells after 2 h incubation (Fig. 3). Colocalization experiments\ngroup on the benzene ring. This result is consistent with the conclusion with the mitochondrion-specific fluorescent probe MTDR show that Rh1\ndrawn by the previous reports [29]. and Rh2 can specifically localize to mitochondria (Fig. 3). The Pearson\u2019s\n colocalization coefficients obtained for Rh1 and Rh2 with MTDR are\n 0.90 and 0.88, respectively, indicating that both Rh1 and Rh2 can\n3.2. Cytotoxicity studies\n effectively target mitochondria.\n To further verify the distribution of Rh1 and Rh2 in different cellular\n The cytotoxicity of Rh1 and Rh2 were tested by MTT assay against\n compartments, the mitochondrial, cytosolic and nuclear fractions were\nHeLa, A549 MDA-MB-231 and MCF-10A cells. The IC50 (50% inhibition\n isolated from HeLa cells treated with complexes Rh1 and Rh2 (Fig. 4).\nof cell growth) values of Rh1 and Rh2 after an incubation with the\n As measured by ICP-MS, the contents of rhodium (III) in the in the\nindicated cells for 72 h are listed in Table 1 and the survival curves are\n mitochondria is much higher than that obtained in the cytosol and\ngiven in Fig. S8. Rh1 and Rh2 show higher cytotoxicities than cisplatin\n nuclei. These results collectively indicate that complexes Rh1 and Rh2\nagainst all the cell lines tested. Notably, Rh1 and Rh2 show a lower\n can specifically target mitochondria in HeLa cells. The results indicate\ncytotoxicity to human normal MCF-10A cells than to those cancer cells.\n that both Rh1 and Rh2 have much higher accumulated levels in mito\u00ad\nThe cytotoxicity potency of Rh(III) complexes are opposite to their lip\u00ad\n chondria compare with cytoplasm and nucleus.\nophilicity, which may result from the fact that the benzene substituents\nin Rh1 have stronger molecular interaction with biomolecular targets\nthan the methyl substituents in Rh2 [30,31]. Since Rh1 shows a higher 3.4. Mitochondrial damage\nanticancer potency than Rh2, we chose Rh1 for further investigation\nregarding the underlying mechanisms of anticancer activities. The cell uptake results indicate that the studied Rh1-Rh2 complexes\n\n 4\n\fY.-B. Peng et al. Journal of Inorganic Biochemistry 218 (2021) 111400\n\n\n\n\nFig. 3. Determination of intercellular colocalization of Rh1 and Rh2 with MTDR by confocal microscopy. HeLa cells were incubated with Rh1 and Rh2 (2 \u03bcM, 2 h),\nand then stained with MTDR (100 nM, 0.5 h) at 37 \u25e6 C. MTDR (\u03bbex = 633 nm, \u03bbem = 650 \u00b1 20 nm). Rh1-Rh2 (\u03bbex = 405 nm, \u03bbem = 600 \u00b1 20 nm).\n\n\n the mitochondrial membranes retain a high voltage. HeLa cells treated\n with complex Rh1 show a concentration-dependent red to green color\n shift in JC-1 fluorescence, and the percentage of cells with depolarized\n mitochondrial membranes increases from 8.3% to 54.4%. The results\n indicate that MMP of Rh1-treated cells are significantly decreased. To\n further investigate the effects of Rh1 on mitochondrial metabolic status,\n we detected the intracellular ATP level of the cancer cells treated with\n Rh(III) complex. The results show that Rh1 cause a significant dose-\n dependent decrease in ATP production as compared with the control\n cells (Fig. 5B). The ATP levels decrease to 78.7%, 64.7% and 54.5% for\n 1 \u03bcM, 2 \u03bcM and 4 \u03bcM, respectively, confirming the cellular mitochon\u00ad\n drial damage by the designed Rh(III) complexes.\n\n 3.5. Real-time tracking of mitochondrial morphology\n\n In living cells, mitochondrial integrity plays a vital role in main\u00ad\n taining cell function [35,36]. Mitochondrial morphology and functions\nFig. 4. Distribution of complexes Rh1-Rh2 in cellular compartments of HeLa are closely linked, and mitochondrial morphological changes are asso\u00ad\ncells measured by ICP-MS. Rh1 (2 \u03bcM) and Rh2 (2 \u03bcM) were with incubated\n ciated with many vital cellular functions [33]. Mitochondria changes in\nHeLa cells for 6 h at 37 \u25e6 C, and then were isolated from the cytoplasmic,\n number and morphology of mitochondria are often observed when they\nmitochondrial and nuclear using the corresponding commercial kits.\n are damaged. Thus, the therapeutic effect can be monitored by real-time\n tracking of the mitochondrial morphological changes in cells.\nare mainly localized in mitochondria. Mitochondrion is the energy\n Real-time tracking of the mitochondrial morphology change was\nproduction center of the cell, and it is also an essential component of\n performed by monitoring the emission of MTDR in HeLa cells treated\napoptotic signaling pathway in cell [32,33]. The pro-death factors are\n with Rh1 using confocal microscopy (Fig. 6). The MTDR phosphores\u00ad\nreleased from mitochondria, and initiate cell death signal when the\n cence presents a filamentous distribution at first, which is the normal\nmembrane integrity of mitochondria is damaged [34].\n mitochondrial morphology of cells. With the prolonged incubation with\n As complexes Rh1-Rh2 could be localized to mitochondria, their\n Rh1, mitochondria gradually become swelling, fragmentation and per\u00ad\nimpact on mitochondrial integrity was monitored by detecting the\n inuclear clustering in cells. This result indicates that the designed Rh1\nchanges in mitochondrial membrane potential (MMP). The red/green\n can damage the mitochondria of the cells, which may contribute to the\nfluorescence of JC-1, a mitochondria-selective aggregate dye, was\n anticancer activity of the rhodium complexes.\ndetected by flow cytometry (Fig. 5A). At low membrane potentials, JC-1\nexists in the form of the \u201cJ-monomer\u201d with green fluorescence. At high\nmembrane potentials, JC-1 forms \u201cJ-aggregates\u201d and displays red fluo\u00ad 3.6. Intracellular ROS detection\nrescence. The control cells show red fluorescence, which indicates that\n It is well known that mitochondria is not only a source of energy, but\n\n 5\n\fY.-B. Peng et al. Journal of Inorganic Biochemistry 218 (2021) 111400\n\n\n\n\nFig. 5. A) Effects of Rh1 on MMP analyzed by JC-1 staining and flow cytometry. HeLa cells were treated with Rh1 at the indicated concentrations for 12 h at 37 \u25e6 C.\nJC-1 monomers (Green): \u03bbex = 488 nm, \u03bbem = 510 \u00b1 20; JC-1 aggregates (Red): \u03bbex = 488 nm, \u03bbem = 580 \u00b1 20 nm. B) Intracellular ATP levels in HeLa cells. HeLa cells\nwere treated with Rh1 at the indicated concentrations for 12 h at 37 \u25e6 C. (For interpretation of the references to color in this figure legend, the reader is referred to the\nweb version of this article.)\n\n\n\n\nFig. 6. Real-time tracking of mitochondria in HeLa cells stained with Rh1 (2 \u03bcM) over a period of 32 h. MTDR were excited at 633 nm and the phosphorescence was\ncollected at 650 \u00b1 20 nm.\n\n\nalso a major source of ROS [37]. Elevated intracellular ROS levels and anticancer activity. As shown in Fig. 7C, the viability of HeLa cells\nmitochondrial damage are closely related to events in cell death [38,39]. pretreated with NAC was significantly reduced as compared with cells\nThe effects of Rh1 on intracellular ROS levels of HeLa cells were treated with Rh1 alone. These results indicate that Rh1 induces ROS-\ndetected by DCF assay using flow cytometry (Fig. 7A). After a 12 h dependent cell death.\ntreatment with Rh1, the MFI of DCF in HeLa cells exhibits a dramatic\nconcentration dependent ROS elevation, increasing to approximately\n 3.7. Induction of apoptosis\n9.77- fold at 4 \u03bcM of Rh1. Moreover, the overlap between the fluores\u00ad\ncence of DCF and MTDR shows that ROS are mainly generated in\n Mitochondria play a vital role in the adjusting of intrinsic pathway\nmitochondria (Fig. 7B). In addition, as an efficient quencher of ROS,\n for the apoptosis of cells [35,40], and regulate the cell apoptosis through\nNAC is frequently employed in the mechanism research of ROS-related\n the release of a different mitochondrial proteins where is\n\n 6\n\fY.-B. Peng et al. Journal of Inorganic Biochemistry 218 (2021) 111400\n\n\n\n\nFig. 7. A) Analysis of ROS levels by flow cytometry after HeLa cells were treated with Rh1 at the indicated concentrations for 12 h and stained with H2DCFDA. B)\nGeneration of mitochondrial ROS caused by Rh1 treatment. HeLa cells were treated with Rh1 at 2 \u03bcM for 6 h. Then the cells were co-stained with H2DCFDA and\nMTDR for confocal microscopic observation. DCF: \u03bbex = 488 nm; \u03bbem = 530 \u00b1 20 nm; MTDR: \u03bbex = 633 nm; \u03bbem = 650 \u00b1 20 nm. C) The impact of NAC on the\ncytotoxicity of Rh1. HeLa cells were treated with Rh1 for 36 h at the indicated concentrations in the absence or presence of NAC. Cell viability was measured by MTT\nassay. Data are represented as means \u00b1 SD of three independent experiments. **p < 0.02.\n\n\nintermembrane space, which leads to activation of caspases [41,42]. apoptotic phase (annexin V-positive) increase from 3.42% (control) to\nApoptosis is usually characterized by a series of prominent morpho\u00ad 10.98% (1 \u03bcM), 16.98% (2 \u03bcM), and 43.8% (4 \u03bcM) of the complex,\nlogical features, such as phosphatidylserine externalization, activation respectively, demonstrating that Rh1 can induce a dose-dependent\nof caspase family proteases, cytoplasmic shrinkage, plasma membrane apoptosis for cancer cells.\nblebbing, chromatin condensation or nuclear fragmentation [43,44]. Mitochondria control the intrinsic pathway of apoptosis by regu\u00ad\nChange in nuclear morphology treated with Rh1 was firstly investigated lating the translocation of pro-apoptotic proteins, e.g., cytochrome C,\nby Hoechst 33342 staining by confocal microscopy. from mitochondrial intermembrane space to cytosol [45,46]. The\n As shown in Fig. 8A, drug-treated control cells show normal overall release of cytochrome C proteins from mitochondria can activate death-\nmorphology and a homogeneous nuclear staining pattern. After treated driving proteolytic proteins known as caspases. The Bcl-2 family of\nwith Rh1 at indicated concentration for 24 h, the cells show distinct proteins [35,41,47], consisting of anti-apoptotic and proapoptotic\nmorphologic signals of apoptosis in a concentration-dependent manner. members, regulate MMP and mitochondrial perme-ability during\nMost of the cells display typical morphological changes that are char\u00ad apoptosis [47]. Therefore, we investigated the effect of Rh1-treatment\nacteristic of apoptosis, which including cell shrinkage, membrane on the expression of proteins related to the mitochondrial death\nbubbling, staining bright, condensed chromatin and fragmented nuclei. pathway using Western blotting. Cytochrome C, Caspase-9, Bcl-2, and\n During apoptosis, phosphatidylserine externalization is considered Bax, are known to play vital roles in the mitochondrial cell death\nto be a hall-mark of early apoptosis. Early apoptotic cells are annexin V- pathway. As shown in Fig. 8C, the results show that Rh1 can elevate the\npositive but PI-negative because phosphatidylserine is externalized expression level of Bax and Caspase-9, and promote the release of cy\u00ad\nwhile the plasmic membrane is integrated. Both late apoptotic and tochrome C proteins from mitochondria to cytosol. Moreover, Rh1 can\nnecrotic cells can be double stained by annexin V and PI. Flow cyto\u00ad reduce the expression level of Bcl-2. The down-regulated Bcl-2/Bax ratio\nmetric determined shows that treatment of HeLa cells with Rh1 leads to induced by Rh1 promotes the cellular apoptosis.\na dose-dependent increase in the percentage of apoptotic cells (Fig. 8B). To further explore the mechanism of cell apoptosis, z-VAD-FMK, a\nAfter treated with Rh1 for 24 h, the percentages of HeLa cells in well-accepted caspase inhibitor, is employed to pretreat the HeLa cells.\n\n 7\n\fY.-B. Peng et al. Journal of Inorganic Biochemistry 218 (2021) 111400\n\n\n\n\nFig. 8. A) Detection of apoptosis in HeLa cells stained with Hoechst 333342 after treatment with Rh1 for 24 h at the indicated concentrations by confocal mi\u00ad\ncroscopy. Hoechst 33342: \u03bbex = 405 nm; \u03bbem = 450 \u00b1 20 nm. B) Detection of apoptosis in HeLa cells stained with annexin V and PI after treatment with Rh1 for 24 h\nat the indicated concentrations by Flow cytometric. Annexin V (\u03bbex = 488 nm, \u03bbem = 510 \u00b1 20 nm), PI (\u03bbex = 488 nm, \u03bbem = 610 \u00b1 20 nm). C) Representative\nWestern blots of the effects of Rh1 on the protein expression levels of Bcl-2, Bax, Caspase-9 and cytochrome C, \u03b2-actin was assessed as a loading control. D) The\nimpact of z-VAD-FMK on the cytotoxicity of Rh1. HeLa cells were treated with Rh1 for 36 h at the indicated concentrations in the absence or presence of z-VAD-FMK.\nCell viability was measured by MTT assay. Data are represented as means \u00b1 SD of three independent experiments. **p < 0.02, compared with the cell viability of Rh1\ntreatment alone.\n\n\nCells pretreated with z-VAD-FMK (50 \u03bcM) show an outstanding increase mitochondria, damaged the integrity of mitochondrial membranes and\nin cell viability compared with cells treated with Rh1 alone (Fig. 8D). increased the level of ROS in cells. The immunobloting results indicated\nThis result confirmed that the cell death induced by Rh1 is mainly that cell death induced by Rh1 is executed through the caspase-\nexecuted through the caspase-dependent apoptotic pathway, and also dependent apoptotic pathway. Overall, we demonstrate that cyclo\u00ad\nshow that the damaged mitochondria by rhodium complexes initiates a metalated rhodium(III) complexes are potential leading compounds for\ncascade of events that can result in execution of cell death. further development as anticancer candidates acting through mecha\u00ad\n nisms different from those of cisplatin.\n4. Conclusion\n Authorship contributions\n Two cyclometalated rhodium(III) complexes containing the TPA\nmoiety were synthesized and characterized, and their in vitro antitumor None.\nactivities were tested. The TPA moiety endows the Rh(III) complexes\ngood fluorescence properties, and both Rh1 and Rh2 show higher anti- Declaration of Competing Interest\ntumor properties than cisplatin against a various of cancer cells.\nMechanism investigations indicated that Rh1 and Rh2 mainly located in None.\n\n 8\n\fY.-B. Peng et al. Journal of Inorganic Biochemistry 218 (2021) 111400\n\n\nAcknowledgment [19] H.-Q. Zheng, Y.-P. Guo, M.-C. Yin, Y.-T. Fan, Chem. Phys. Lett. 653 (2016) 17\u201323,\n https://doi.org/10.1016/j.cplett.2016.04.064.\n [20] R. Dheepika, R. Abhijnakrishna, P.M. Imran, S. Nagarajan, RSC Adv. 10 (2020)\n This work was supported by the Unique Feature and Innovation 13043\u201313049, https://doi.org/10.1039/d0ra00210k.\nProject of Guangdong Province, China (2017KTSCX103), the Social [21] S.B. Yadav, S.S. Sonvane, N. Sekar, Spectrochim. 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