Rhenium-guanidine complex as photosensitizer: trigger HeLa cell apoptosis through death receptor-mediated, mitochondria-mediated, and cell cycle arrest pathways.

{"full_text": " Metallomics, 14, 2022, mfac008\n https://doi.org/10.1093/mtomcs/mfac008\n Advance access publication date: 12 February 2022\n Paper\n\n\n\nRhenium\u2013guanidine complex as photosensitizer: trigger\nHeLa cell apoptosis through death receptor-mediated,\nmitochondria-mediated, and cell cycle arrest pathways\nShu-Fen He1 ,2 ,\u2021 , Jia-Xin Liao1 ,\u2021 , Min-Ying Huang1 , Yu-Qing Zhang1 , Yi-Min Zou1 , Ci-Ling Wu1 , Wen-Yuan Lin1 , Jia-Xi Chen1 , \u2217\nand Jing Sun1 , \u2217\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/5/mfac008/6527583 by guest on 12 May 2026\n1\n School of Pharmacy, Guangdong Medical University, Dongguan 523808, China and 2 Department of Pharmacy, Dongguan People\u2019s Hospital, Dongguan, 523059,\nChina\n\u2217\n Correspondence: Xincheng Road, Guangdong Medical University, Dongguang 523808, Guangdong, China. Tel.: +86-769-2289-6322; E-mail: jiaxi@gdmu.edu.cn\n(Jia-Xi Chen); sunjing@gdmu.edu.cn (Jing Sun).\n\u2021\n Contributed equally to this work.\n\n\n\nAbstract\nThe growing evidence over the past few decades has indicated that the photodynamic antitumor activity of transition metal com-\nplexes, and Re(I) compounds are potential candidates for photodynamic therapy. This study reports the synthesis, characterization,\nand anti-tumor activity of three new Re(I)\u2013guadinium complexes. Cytotoxicity tests reveal that complex Re1 increased cytotoxicity\nby 145-fold from IC50 > 180 \u03bcM in the dark to 1.3 \u00b1 0.7 \u03bcM following 10 min of light irradiation (425 nm) in HeLa cells. Further, the\nmechanism by which Re1 induces apoptosis in the presence or absence of light irradiation was investigated, and results indicate that\ncell death was caused through different pathways. Upon irradiation, Re1 first accumulates on the cell membrane and interacts with\ndeath receptors to activate the extrinsic death receptor-mediated signaling pathway, and then is transported into the cell cytoplasm.\nMost of the intracellular Re1 locates within mitochondria, improving the reactive oxygen species level, and decreasing mitochondrial\nmembrane potential and ATP levels, and inducing the activation of caspase-9 and, thus, apoptosis. Subsequently, the residual Re1 can\ntranslocate into the cell nucleus, and activates the p53 pathway, causing cell cycle arrest and eventually cell death.\n\nKeywords: Re(I) complex, cytotoxicity, phototoxicity, membrane receptors, apoptosis, mechanism\n\n\nGraphical abstract\n\n\n\n\nRhenium complexes act as photosensitizer to induce apoptosis in HeLa cells.\n\n\n\nIntroduction safe cervical cancer treatment and drugs to enhance patients\u2019 sur-\n vival rate and quality of life. Among these treatments, photody-\nDespite the extensive progress in disease prevention by vaccines,\n namic therapy (PDT) has broad prospects, owing to lower trauma,\nscreening, early detection, and treatment, cervical cancer remains\n less toxicity, and shorter time of the treatment.4\u20136\nthe second most common cancer faced by women worldwide.1\u20132\n Compared with traditional photosensitizers (porphyrins, ph-\nTo date, the traditional treatments for cervical cancer include\n thalocyanines, etc.) and material compounds, the phosphores-\nsurgery, radiotherapy, and chemotherapy, but these are hindered\n cence properties of ruthenium,7 iridium,8 and rhenium9 have\nby the disadvantages of strong invasion, poor targeting, and high\n attracted much research attention. Some of these compounds,\nrecurrence rate.3 Therefore, it is urgent to develop an efficient and\n such as TLD1433, have entered clinical trials.10 Tricarbonyl Re(I)\n\nReceived: October 30, 2021. Accepted: January 27, 2022\n\u00a9 The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com\n\f2 | Metallomics\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/5/mfac008/6527583 by guest on 12 May 2026\nFig. 1 Chemical structures of Re(I) complexes Re1\u2013Re3.\n\n\nis easy to modify and coordinate, and most Re(I) complexes tection of argon for 4 h in the dark. Crude products were\nhave the potential to be used as cell imaging agents, anti-tumor purified by recrystallization with CH3 CN/CH3 CH2 OCH2 CH3 . Dif-\ndrugs, and photosensitizers.11\u201320 Previous studies report that ferent monodentate ligands, namely pyridine (for Re2) and\nmitochondria-targeted tricarbonyl Re(I) complexes lead to tumor imidazole (for Re3), and Re1 were refluxed in methanol or tetrahy-\ncell death through irreversible oxidative stress and disruption drofuran under argon for 24 h to obtain complexes Re2 and Re3.\nof glutathione metabolism.21 DNA photolysis studies show that The chemical structures of the three complexes were completely\nRe\u2013NLS and Re\u2013Bomesin compounds can accumulate in the nu- characterized with mass spectrometry, 1 H NMR spectroscopy, and\ncleus and significantly increase cytotoxicity under light irradia- elemental analysis (Figs. S1\u2013S6). Regarding the data of the mass\ntion, damaging DNA through singlet oxygen and thus leading to spectrometry, 1 H NMR spectroscopy, and elemental analysis, it\ncell apoptosis.22 Although the photophysical properties and anti- was verified the molecular structure of three complexes accord\ntumor mechanism of Re(I) complexes have been extensively ex- with theoretical values. The UV\u2013Vis absorption spectra of com-\nplored, limited studies have investigated these complexes on the plexes Re1\u2013Re3 in PBS, CH3 CN, and CH2 Cl2 were also obtained\ncellular level and in animals. Moreover, the mechanism of the using a UV\u2013Vis spectrometer. The relatively strong absorption\nphotodynamic effect is still unclear. band at about 250\u2013330 nm in the ultraviolet region can be at-\n It has been reported that the expression levels of cell mem- tributed to ligand absorption (\u03c0 \u2192\u03c0 *), while the relatively weak\nbrane receptors in cancer cells and normal cells vary notably.23 band at 380\u2013430 nm can be ascribed to metal\u2013ligand charge trans-\nBased on this difference, the rational design of cancer-targeted fer absorption (Fig. S7).31 Upon 405 nm excitation, these com-\ndrugs is a promising strategy. Death receptors (DRs), such as asso- plexes showed yellow emission in PBS, CH3 CN, and CH2 Cl2 at 298 K\nciated protein with death domain (FAS) and tumor necrosis factor (Fig. S8). The photophysical properties of Re1\u2013Re3 are summa-\n(TNF) receptors, are membrane proteins of the TNF-R superfamily rized in Table S1.\nthat are capable of inducing apoptosis and, thus, have attracted\nincreasing research interest in the field of cancer research.24\u201325 In In vitro antitumor evaluation\nliterature, numerous works have reported that targeting receptors\n The cytotoxicities and phototoxicity of complexes Re1\u2013Re3 and\nTNF-related apoptosis-inducing ligand (TRAIL), FAS, and TNF can\n cisplatin were tested against several cancer cell lines (HeLa,\nselectively trigger apoptosis of cancer cells.26\u201327 Therefore, explor-\n HepG2, MCF-7, A549) and a non-tumorigenic LO2 cell line by the\ning the mechanism of action between Re(I) complexes and the\n 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide as-\nmembrane receptor is significant for designing reasonably struc-\n say (Table 1 and Fig. S9). Accordingly, Re2 and Re3 had IC50 values\ntured membrane receptor-targeted anti-cancer drugs.\n ranging from 8.7 to 81.7 \u03bcM, thus showing moderate antitumor ac-\n Our previous studies have shown that some Ru(II) and Ir(III)\n tivity to cancer cells in the dark. Compared to cisplatin, all three\nmetal complexes with guanidine exhibit good photodynamic ther-\n Re(I) complexes displayed no obvious anti-tumor advantages but\napeutic potential.28\u201329 Under light irradiation, such compounds\n did demonstrate higher phototoxicity upon visible light irradia-\ncan be positioned in the mitochondria to activate apoptosis. Based\n tion (425 nm, 40 mW\u00b7cm\u20132 ) under the same conditions. Notably,\non this, we synthesized and characterized three Re(I) complexes\n in HeLa cells, Re1 had little cytotoxicity (IC50 = 185.4 \u03bcM) in the\ncontaining guanidinium groups as ligands, investigated their an-\n dark but displayed the highest phototoxicity index (PI) of >142. Re-\nticancer activity in the presence and absence of light irradiation,\n grettably, Re1 did not show higher selectivity for the tested cancer\nand elucidated the respective mechanisms (Fig. 1).\n cells compared to non-tumorigenic LO2 cells upon visible light ir-\n radiation. Due to its superior phototoxicity, Re1 was chosen as the\nResults and discussion compound of interest for further mechanistic investigations.\nSynthesis, characterization, and photophysical\nproperties Assessment of lipophilicity\nLigand L was synthesized by the methods we previously re- The lipophilicities of the three complexes were examined with\nported.30 Complex Re1 was prepared by mixing L and Re(CO)5 Cl the flask-shaking method. The logPo/w values of Re1, Re2, and\nin methanol/toluene (1:1) and refluxed at 80\u00b0C under the pro- Re3 were determined to be \u22120.95, \u22120.75, and \u22120.26, respectively\n\f Paper | 3\n\n\nTable 1. Cytotoxicity (IC50 /\u03bcM) of the compounds in the absence and presence 425 nm light toward different cell lines\n\n IC50 (\u03bcM)\n\n HeLa HepG2 MCF-7 A549 LO2\n\nComplexes Darka (lightb ) PIc Dark (light) PI Dark (light) PI Dark (light) PI Dark (light) PI\n\nRe1 185.4 \u00b1 3.5 142.6 19.5 \u00b1 1.2 4.1 29.8 \u00b1 0.9 5.8 45.0 \u00b1 1.4 11.3 61.3 \u00b1 3.6 22.7\n (1.3 \u00b1 0.7) (4.7 \u00b1 0.9) (5.1 \u00b1 1.6) (4.0 \u00b1 0.9) (2.7 \u00b1 0.7)\nRe2 81.3 \u00b1 2.2 6.8 38.5 \u00b1 2.0 5.3 45.6 \u00b1 2.9 4.6 49.9 \u00b1 2.9 5.3 53.5 \u00b1 2.9 5.9\n (12.0 \u00b1 1.1) (7.3 \u00b1 1.7) (9.9 \u00b1 1.8) (9.5 \u00b1 1.1) (9.0 \u00b1 1.3)\nRe3 34.4 \u00b1 1.9 38.2 8.7 \u00b1 1.6 12.4 36.1 \u00b1 2.4 22.6 27.2 \u00b1 1.9 30.2 39.2 \u00b1 3.3 24.5\n (0.9 \u00b1 0.3) (0.7 \u00b1 0.2) (1.6 \u00b1 0.4) (0.9 \u00b1 0.1) (1.6 \u00b1 0.6)\nCisplatin 13.3 \u00b1 1.6 1.11 58.4 \u00b1 2.4 1.19 15.4 \u00b1 3.2 2.14 13.5 \u00b1 1.7 1.1 42.4 \u00b1 3.0 2.1\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/5/mfac008/6527583 by guest on 12 May 2026\n (12.0 \u00b1 0.7) (49.2 \u00b1 0.8) (7.2 \u00b1 1.4) (12.8 \u00b1 0.4) (20.4 \u00b1 1.0)\n\na\n Cells were incubated with the indicated complexes in the dark for 48 h. b Cells were incubated with the indicated complexes for 12 h in the dark and then irradiated\nwith light at 425 nm. c PI = phototoxicity index, the ratio of the IC50 values in dark to those obtained upon light irradiation. Each value represents the mean \u00b1 SD\nof three independent experiments.\n\n\n\n\nFig. 2 (A) Confocal microscopy images of HeLa cells after 0, 12, and 24 h incubation with Re1 (100 \u03bcM). (B) Confocal microscopy images of HeLa cells\nafter incubation with Re1 (100 \u03bcM) at indicated conditions: (a) HeLa cells were incubated with Re1 at 37\u00b0C for 12 h; (b) HeLa cells were incubated with\nRe1 at 4\u00b0C for 12 h; (c) HeLa cells were incubated with Re1 at 37\u00b0C for 12 h after pre-incubation with 20 \u03bcM carbonyl cyanide\nmetachlorophenylhydrazone at 37\u00b0C for 1 h; and (d) HeLa cells were incubated with Re1 at 37\u00b0C for 12 h after pre-incubation with 50 \u03bcM chloroquine\nat 37\u00b0C for 1 h.\n\n\n\n(Fig. S10). Many studies have indicated that structure and with lower lipophilicity may achieve nuclear absorption easier, as\nlipophilicity affect the cellular transportation, uptake, location, long as they can first penetrate through the plasma membrane\nand action mechanisms of metal complexes, largely contributing of the cell.34 In addition, due to the relatively lower lipophilicity,\nto their anticancer activities.32 , 33 Moreover, it was found that the the complexes can selectively stain the cell plasma membrane,\nlipophilicity of the three complexes follows the order: improving their probability of binding to death receptors on the\nRe3 > Re2 > Re1, which is positively correlated with their cell membrane surface of tumor cells.35\u201336\ntoxicity to HeLa cells in the dark. As the least lipophilicity com-\nplex, Re1 moved into the nucleus with light irradiation faster\nthan the other complexes. This may be due to the increased en- Cellular uptake mechanism\nergy of cells in the presence of light, which induced more Re1 to As Re(I) complexes possess rich photophysical properties, we can\ncross the cell membrane into the cytoplasm. Therefore, Re1 with monitor their transportation process into cancer cells and intra-\nlower lipophilicity may be more conducive to enter the nucleus. cellular distribution conveniently. Figure 2 shows that Re1 started\nLikewise, Chao\u2019s group speculated that cationic metal complexes to accumulate in the cytoplasm after 6 h then penetrated the\n\f4 | Metallomics\n\n\ncytoplasm after 12 h of co-cultivation with HeLa cells in the dark, Membrane-targeted and activation of death\ndemonstrating green fluorescence (Fig. 2A). receptors\n As previously reported, the two main penetrating mech- To investigate whether Re1 could induce apoptosis through the\nanisms related to the transport of small molecules across death receptor pathway, the co-staining of Re1 and cell membrane\nthe cell membrane include energy-dependent endocytosis (e.g. red fluorescent dye (DiD) was used to study localization upon ex-\nendocytosis and active transport) and energy-independent direct posure to irradiation of 425 nm. The results showed that Re1 ac-\npenetration (e.g. facilitated diffusion and passive diffusion).37\u201339 cumulated in a time-dependent manner in HeLa cells. During the\nAs presented in Fig. 2B, incubating HeLa cells with Re1 at a lower initial 4 h, the green fluorescence of Re1 gradually overlapped with\ntemperature (4\u00b0C) resulted in a decrease in cellular uptake effi- the red fluorescence of DiD, indicating that Re1 is mainly con-\nciency. However, the ability of Re1 to cross the plasma membrane centrated on the membrane, and the complex could have a suffi-\nwas not affected by the presence of the metabolic inhibitor car- cient interaction with the membrane receptors. Then, most of Re1\nbonyl cyanide m-chlorophenyl hydrazone (CCCP) and the endocy- crossed the membrane and accumulated in the cytoplasm in 8 h\ntosis regulator chloroquine. These results suggest that the uptake and was finally enriched in the cells at 12 h. Until 24 h later, an in-\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/5/mfac008/6527583 by guest on 12 May 2026\nof Re1 mainly occurs through an energy-dependent mechanism, creasing amount of Re1 was observed in the nucleus accompanied\nwhich means that light may promote the ability of Re1 to enter by a reduction in the cell membrane (Fig. 4A). Furthermore, the ex-\ncells. pression levels of several death receptor-related proteins in HeLa\n cells were examined. As showed in Fig. 4B, cell treatment with Re1\n under light caused an outstanding dose-dependent increase in the\nInduction of apoptosis expressions of TNFR-1, TNFR-2, FAS, and DR5 (Fig. 4C), while only\nRe1-induced cell death and morphological changes were ex- invisible change was observed in the expression of these proteins\namined using Hoechst 33 342 staining and confocal microscopy. in the dark (Fig. 4B). These results indicated that Re1-induced\nWith the increase of concentration, the morphology of the Re1- apoptosis under light irradiation was mediated via the extrinsic\ntreatment cells changed compared with the control cells in the death receptor pathway.\ndark and under light irradiation (Fig. 3A). After 24 h treatment\nwith Re1, the cells exhibited typical apoptotic morphology, such Cytoplasm localization under the light\nas nuclear fragments, condensed chromatin, and apoptotic bod- irradiations\nies.40 Therefore, these findings demonstrate that Re1 could in-\n To further understand the mechanism by which Re1 enters the\nduce apoptosis in HeLa cells. Subsequently, cellular apoptosis\n cytoplasm under light irradiation of 425 nm, its subcellular lo-\nwas assessed by a flow cytometry assay with PI/Annexin-V stain-\n calization was further evaluated in HeLa cells by fluorescence\ning. Early and late apoptosis were progressively increased un-\n microscopy coupled with a fluorescent probe. Considering Re1\nder different light conditions (Fig. 3B). Of these, Re1 (100 \u03bcM)\n mainly aggregated in the cytoplasm after co-incubation with cells\ntreatment increased the percentage of both early and late\n for 8 h, we explored the localization of the complex during this\napoptosis to 3.90% and 29.2%, respectively, compared with the\n period. As shown in Fig. 5A, Re1 demonstrated a high degree of\nvehicle-treated cells (2.66% and 5.02%, respectively) in the dark.\n co-localization with the organelle-specific stain MitoTracker\u00ae Red\nMoreover, Re1 (10 \u03bcM) treatment also enhanced the percentage\n CMXRos (MTR), with a Pearson co-localization coefficient (PCC) of\nof early and late apoptosis by 1.87% + 83.7%, respectively, com-\n 0.89. Under the same conditions, the PCC between Re1 and Lyso-\npared with the vehicle-treated cells (3.83% + 9.25%, respectively)\n tracker Red (LTR) was only 0.48. These results confirm that Re1\nunder light irradiation. These data suggest that the ability of\n spent most of the time in mitochondria after entering the cyto-\nRe1 to induce apoptosis in HeLa cells is stronger with light\n plasm. Further studies are required to confirm whether or not\nillumination.\n Re1 is able to induce apoptosis of HeLa cells via a mitochondrial-\n Apoptosis is an intricate and precise process regulated by sev-\n dependent pathway. Therefore, the expressions of Bcl-2, Bax, Cyto-\neral relevant proteins, which can be classified into extrinsic (death\n c, and PARP, which are typical apoptotic proteins of the mito-\nligand) and intrinsic (mitochondrial) pathways.41 Caspases be-\n chondrial apoptosis pathway, were detected using western blot\nlonging to a family of cysteine proteases play important roles in\n analysis. As presented in Fig. 5B and C, under light conditions,\nthe death of apoptotic cells in diverse biological systems.42 In par-\n the expressions of Bax, Cyto-c and PARP were up-regulated with\nticular, caspase-3 is considered as a central modulator of apop-\n increasing concentrations of Re1, while Re1 down-regulated the\ntosis and is activated in both extrinsic and intrinsic pathways,\n expression levels of Bcl-2. Besides, similar results were observed\nwhich can be mediated by caspase-8 and -9 initiators, respec-\n under dark conditions, except for the PARP protein, whose expres-\ntively.43 To further clarify the latent mechanism of Re1, west-\n sion level had no obvious change. Together, these results suggest\nern blotting was performed to measure the expression levels of\n that Re1-induced apoptosis under light irradiation via endoge-\ncaspase-3, -8 and -9. As presented in Fig. 3C, Re1 treatment with-\n nous pathways involving mitochondria.\nout irradiation caused the dose-dependent activation of caspase-3\nand remarkable elevation of cleaved caspase-3, while changes of\ncaspase-8 and -9 expression levels in HeLa cells were negligible Apoptosis-related mitochondrial events\n(Fig. 3C). However, after Re1 treatment under light irradiation for As a mediator of apoptosis, reactive oxygen species (ROS) can\n24 h, the expression levels of caspase-3, -8, and -9 were reduced trigger a range of mitochondria-related events, such as apoptosis\nsignificantly, whereas levels of cleaved caspase-3 increased re- and the decline of mitochondrial membrane potential (MMP).44\u201346\nmarkably (Fig. 3D). Taken together, these results demonstrate that What is more, induction of apoptosis by PDT is thought to\nRe1 may trigger HeLa cell apoptosis through caspase-dependent occur through the production of intracellular ROS.47 Hence, it\nextrinsic and intrinsic pathways under irradiation, which fol- is significant to investigate the ability of Re1 to stimulate ROS\nlows a different mechanism than that which occurs in dark generation and accumulation for elucidating the potential mech-\nconditions. anism of apoptosis. In this work, ROS levels were measured via\n\f Paper | 5\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/5/mfac008/6527583 by guest on 12 May 2026\n\n\n\n\nFig. 3 (A) Hoechst 33 342 stained HeLa cells after treatment of Re1 at the indicated concentrations for 24 h. (B) Flow cytometric quantification of\nAnnexin V and PI double labeled HeLa cells after treatment with Re1 for 24 h. (C) Western blot analysis of Re1 on the expression of caspase-3, cleaved\ncaspase-3, caspase-8, and caspase-9. HeLa cells were incubated with indicated concentrations of Re1 for 24 h in the dark. (D) The same treatment as\n(B) under light irradiation. Photoirradiation (425 nm, 40 mW\u00b7cm\u20132 , 10 min) was performed after the cells were incubated with Re1 for 30 min. (*P < 0.05,\n**P < 0.01).\n\f6 | Metallomics\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/5/mfac008/6527583 by guest on 12 May 2026\nFig. 4 (A) Confocal microscopy images of HeLa cells incubated with Re1 (5 \u03bcM) and red fluorescent dye (DiD; 5 \u03bcM, 0.5 h) at 37\u00b0C for 0, 4, 8, 12, and 24 h.\nRe1 was excited at 405 nm and DiD was excited at 552 nm. (B) Western blot analysis of Re1 on the expression of TNFR-1, TNFR-2, FAS, and death\nreceptor 5. HeLa cells were incubated with indicated concentrations of Re1 for 24 h, in the dark. (C) The same treatment as (B), under light irradiation.\nPhotoirradiation (425 nm, 40 mW\u00b7cm\u20132 , 10 min) was performed after the cells were incubated with Re1 for 30 min. (*P < 0.05, **P < 0.01).\n\n\n\n2\u0004 ,7\u0004 -dichlorodihydrofluorescein diacetate (DCFH-DA) fluores- green fluorescence (Fig. 6C). Similar results were recorded by\ncence. Results show that the fluorescence intensity in HeLa flow cytometry. As depicted in Fig. 6D, the red/green fluorescence\ncells increased in a concentration-dependent manner after intensity ratio decreased in a concentration-dependent manner\ntreatment with Re1 both in the absence and presence of light in the dark (control: 79.0 \u00b1 1.9; 20 \u03bcM Re1: 72.1 \u00b1 1.7; 50 \u03bcM Re1:\n(Fig. 6A). Moreover, the cellular ROS levels were examined by flow 51.2 \u00b1 2.8; 100 \u03bcM Re1: 37.5 \u00b1 3.4) and light conditions (control:\ncytometry. After 12 h of co-incubation with Re1 (100 \u03bcM in the 81.6 \u00b1 3.2; 2 \u03bcM Re1: 52.3 \u00b1 3.7; 5 \u03bcM Re1: 34.7 \u00b1 2.3; 10 \u03bcM\ndark and 10 \u03bcM under illumination at 425 nm), the mean 2\u0004 ,7\u0004 - Re1: 10.0 \u00b1 1.3). This result indicates that lower concentrations\ndichlorofluorescein (DCF) green fluorescence intensity increased of Re1 under light irradiation could impair the mitochondrial\nto approximately 2.9-fold and 5.6-fold higher than the control integrity.\ngroups (Fig. 6B). Furthermore, HeLa cells were pretreated with Since mitochondria are regarded as the energy factory of cells\nN-acetylcysteine, an ROS scavenger, to further verify the effect of in which ATP is created, mitochondrial dysfunction is accompa-\nROS on cell death. As shown in Fig. S11, after pre-incubating with nied by reduced ATP.51\u201353 After Re1 treatment, ATP levels in HeLa\nN-acetylcysteine, the cell viability of Re1-treated cells increased cells were reduced to 68.1% (2 \u03bcM), 37.3% (5 \u03bcM), and 8.3% (10 \u03bcM)\nfrom 90.9 \u00b1 2.6% to 92.8 \u00b1 5.0% (Re1, 20 \u03bcM), 87.9 \u00b1 5.7% \u00b1 to under light irradiation compared to the control. However, the re-\n91.2 \u00b1 7.3% (Re1, 50 \u03bcM), and 83.5 \u00b1 3.1% to 90.9 \u00b1 2.2% (Re1, sults obtained in the dark were not notably altered versus control,\n100 \u03bcM) in the dark and from 63.1 \u00b1 5.6% to 82.2 \u00b1 7.6% (Re1, confirming that Re1 can affect mitochondrial integrity under light\n2 \u03bcM), 33.7 \u00b1 4.0% to 59.0 \u00b1 5.8% (Re1, 5 \u03bcM), and 15.3 \u00b1 2.9% to conditions.\n34.8 \u00b1 3.3% (Re1, 10 \u03bcM) under the light conditions, respectively.\nAll these data demonstrate that Re1 can induce ROS generation\nunder different light conditions. Nucleus entry and cell cycle arrest\n Disruption of MMP is a landmark event of cellular In the cell membrane DiD staining experiment, after light irra-\napoptosis.48\u201350 When MMP decreases, JC-1 fluorescence is dis- diation, Re1 entered the nucleus at 24 h, which was confirmed\nplayed as green monomers, while higher MMP correlates to red by Hoechst 33 342. As shown in Fig. 7A, the green fluorescence\nJC-1 aggregates, indicating normal mitochondria. After treatment of Re1 closely overlaps with the blue fluorescence represent-\nwith Re1 for 12 h (in the dark and under light irradiation), HeLa ing cell nuclei. It is well known that genetic information and\ncells exhibited a reduction in red fluorescence and increase in encodes proteins are contained in the cell nucleus, which\n\f Paper | 7\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/5/mfac008/6527583 by guest on 12 May 2026\nFig. 5 (A) Confocal microscopic images of HeLa cells incubated with Re1 (5 \u03bcM, 8 h), MitoTracker\u00ae Red CMXRos (MTR; 100 nM, 0.5 h) and Lyso-tracker\nRed (LTR; 100 nM, 0.5 h). Re1 excitation at 405 nm, and MTR and LTR excitation at 552 nm. (B) Western blot analysis of Re1 on the expression of Bcl-2,\nBax, Cyto-c, PARP. HeLa cells were incubated with indicated concentrations of Re1 for 24 h in the dark. (C) The same treatment as (B) under light\nirradiation. Photoirradiation (425 nm, 40 mW\u00b7cm\u20132 , 10 min) was performed after the cells were incubated with Re1 for 30 min. (*P < 0.05, **P < 0.01).\n\n\ngoverns the mechanisms of cell proliferation, differentiation, controlled by the balance established among expressed proteins.\nand function.54\u201355 Numerous studies have revealed that cell To elucidate the effect of Re1 with PDT treatment on the cell cycle\nproliferation is a cell cycle\u2013dependent process in cancer devel- and cell apoptosis, the expressions of p53, p21, cyclin-dependent\nopment, and thus many antitumor drugs are designed based kinases (CDK)1, and Cyclin B1 protein were analyzed by western\non this mechanism to combat cancer.56\u201358 Compared with the blot. As a cell cycle checkpoint, and a vital downstream effector\ncontrol group, Re1 showed a negligible effect on the cell cycle in of p53, p21 mediates the inactivation of various cyclin\u2013CDK\ndark conditions, while the combination of Re1 and light arrested complexes.59 Under the light, Re1 simultaneously activated p53\nthe cell cycle in the G2/M phase in a concentration-dependent and p21 and significantly down-regulated CDK1 and Cyclin B1,\nmanner (Fig. 7B). Following a 24 h treatment with Re1 (10 \u03bcM), the which are two key factors of G2/M phase arrest60\u201362 (Fig. 7D).\npercentages of cells were reduced at the G0/G1 (Control: 49.4 \u00b1 No apparent changes to CDK1 and Cyclin B1 were observed\n1.3%; Re1: 35.2 \u00b1 1.0%) and S phases (control: 43.2 \u00b1 1.8; Re1: in the dark, while the expression levels of p53 and p21 were\n25.7% \u00b1 1.8), while there was an accumulation in the G2/M phase up-regulated (Fig. 7C). These findings prove that Re1 could trigger\n(control: 7.4% \u00b1 2.0; Re1: 32.1% \u00b1 2.3) (Table S2 and Fig. S12). G2/M cycle arrest to further induce apoptosis via regulating the\n In addition, the equilibrium between cell proliferation and expression of related proteins, namely p53, p21, CDK1, and Cyclin\ndifferentiation is reflected in the appearance of the nucleus and is B1, under light irradiation.\n\f8 | Metallomics\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/5/mfac008/6527583 by guest on 12 May 2026\n\n\n\n\nFig. 6 Effects of Re1 on reactive oxygen species (ROS) production. HeLa cells were incubated with Re1 and labeled with DCFH-DA, then analyzed using\nconfocal microscopy (A) and flow cytometry (B). Effects of Re1 on mitochondrial integrity: (C) Fluorescence imaging of JC-1 labeled cells via confocal\nmicroscopy; (D) Effects of Re1 on mitochondrial membrane potential (MMP) analyzed by JC-1 staining and flow cytometry. HeLa cells were treated with\nRe1 at the indicated concentrations for 12 h. JC-1 was excited at 488 nm. (E) Intracellular ATP levels in HeLa cells after incubating with Re1 at different\nconcentrations. The light group of (A)-(E): Photoirradiation (425 nm, 40 mW\u00b7cm\u20132 , 10 min) was performed after the cells were incubated with Re1 for\n30 min. (*P < 0.05, **P < 0.01)\n.\n\f Paper | 9\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/5/mfac008/6527583 by guest on 12 May 2026\n\n\n\nFig. 7 (A) Co-localization of Re1 (5 \u03bcM, 24 h) and Hoechst 33 342 dye in HeLa cells. (B) Effects of Re1 on the distribution of HeLa cells in cell cycle\npopulation at indicated conditions for 24 h treatment. (*P < 0.05) (C) Western blot analysis of Re1 on the expression of P53, P21, CDK1, Cyclin B1. HeLa\ncells were incubated with indicated concentrations of Re1 for 24 h, in the dark. (D) The same treatment as (B), under light irradiation. Photoirradiation\n(425 nm, 40 mW\u00b7cm\u20132 , 10 min) was performed after the cells were incubated with Re1 for 30 min.\n\n\n\nEffect on cell migration dependent inhibition of wound healing integrity compared to the\nThe ability to inhibit cell migration is an important marker for control group (Fig. 8).\nthe antitumor activity of agents.63\u201365 To assess the effect of Re1\non cell migration, a cell scratch experiment was conducted. After\ntreating with Re1 at different concentrations for 24 and 48 h un-\n Conclusions\nder dark conditions, the distance between the scratches was sig- In this study, three new rhenium\u2013guanidine complexes were syn-\nnificantly lower. Comparatively, HeLa cells treated with Re1 under thesized and characterized. The results display that complex Re1\nlight irradiation exhibited a significantly time- and concentration- has great potential as a photosensitizer based on the various\n\f10 | Metallomics\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/5/mfac008/6527583 by guest on 12 May 2026\nFig. 8 Wound healing assay performed on HeLa cells. (A) The cells were incubated with Re1 (20, 50, and 100 \u03bcM) for 0, 24, and 48 h in the dark. (B) The\ncells were incubated with Re1 (2, 5, and 10 \u03bcM) for 0, 24, and 48 h. Photoirradiation (425 nm, 40 mW\u00b7cm\u20132 , 10 min) was performed after the cells were\nincubated with Re1 for 30 min. Statistical data of wound healing assay of (C) dark and (D) light. (Wound closure (%) = [1 \u2212 (distance at indicated\ntime)/(distance at 0 h)] \u00d7 100%).64\n\n\n\nmechanisms by which it induces apoptosis both in the absence and the Project of Scientific Research Development Fund of Dong-\nand presence of light. Specifically, in the dark, Re1 is mainly guan People\u2019s Hospital (K202015).\ndistributed in the cytoplasm and triggers apoptosis by ROS ele-\nvation, reduction of ATP production, and loss of MMP at relatively\nhigh concentrations. Interestingly, Re1 induces apoptosis through Conflicts of interest\nthree pathways under light irradiation of 425 nm, including death The authors declare no competing financial interests.\nreceptor-mediated, mitochondria-mediated, and cell cycle arrest\nin HeLa cells. Further experiments demonstrated that Re1 ac-\ncumulated on the cell membrane during the first 4 h and pro- Data availability\nmoted the activation of death receptors, leading to the extrinsic\n The data underlying this article are available in the article and in\napoptotic pathway through caspase-8 activation. 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