Rhodium(III) Complex Noncanonically Potentiates Antitumor Immune Responses by Inhibiting Wnt/β-Catenin Signaling.

{"full_text": " pubs.acs.org/jmc Article\n\n\n\n Rhodium(III) Complex Noncanonically Potentiates Antitumor\n Immune Responses by Inhibiting Wnt/\u03b2-Catenin Signaling\n Feng-Yang Wang,\u00a7 Liang-Mei Yang,\u00a7 Xiao-Lin Xiong,\u00a7 Jing Yang, Yan Yang, Jiu-Qin Tang, Lei Gao,\n Yuan Lu, Yuan Wang, Taotao Zou,* Hong Liang,* and Ke-Bin Huang*\n Cite This: J. Med. Chem. 2024, 67, 13778\u221213787 Read Online\n\n\n ACCESS Metrics & More Article Recommendations *\n s\u0131 Supporting Information\nSee https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.\n\n\n\n\n ABSTRACT: Metal-based chemoimmunotherapy has recently garnered\n significant attention for its capacity to stimulate tumor-specific immunity\n Downloaded via MOSCOW STATE UNIV on May 12, 2026 at 13:47:15 (UTC).\n\n\n\n\n beyond direct cytotoxic effects. Such effects are usually caused by ICD via the\n activation of DAMP signals. However, metal complexes that can elicit antitumor\n immune responses other than ICD have not yet been described. Herein, we\n report that a rhodium complex (Rh-1) triggers potent antitumor immune\n responses by downregulating Wnt/\u03b2-catenin signaling with subsequent\n activation of T lymphocyte infiltration to the tumor site. The results of\n mechanistic experiments suggest that ROS accumulation following Rh-1\n treatment is a critical trigger of a decrease in \u03b2-catenin and enhanced secretion\n of CCL4, a key mediator of T cell infiltration. Through these properties, Rh-1\n exerts a synergistic effect in combination with PD-1 inhibitors against tumor\n growth in vivo. Taken together, our work describes a promising metal-based\n antitumor agent with a noncanonical mode of action to sensitize tumor tissues to ICB therapy.\n\n\n 1. INTRODUCTION pathways associated with immune evasion.40\u221243 For example,\n Several specific, metal-based agents have been reported to \u03b2-catenin inhibits the transcription of CCL4-mediated\n exhibit cytotoxicity, provoking anticancer immunity.1\u22124 A infiltration and activation of CD103+ DCs and CD8+ T cells,\n typical mechanism of these complexes is known as leading to a subsequent lack of response to immune\n immunogenic cell death (ICD), in which the drug-treated checkpoint blockade.44 A couple of studies have confirmed\n tumor tissue promotes T cell proliferation, which is dependent the ability of metal compounds to inhibit the Wnt/\u03b2-catenin\n on the antigen-presenting process from dendritic cells (DCs) pathway;45,46 however, whether the antitumor immunity could\n activated by the immunostimulatory signal, including cell be achieved by these metal-based agents remained unknown.\n In view of their high structural diversity and tight relationship\n surface exposure of calreticulin (CRT), secretion of high\n with antitumor immunity as reported, metal complexes would\n mobility group box 1 (HMGB1), and release of ATP.5\u22129 In\n offer a suitable pool for mining new Wnt/\u03b2-catenin pathway\n recent years, an increasing number of metal complexes, based\n inhibitors with potent immunostimulatory activity.\n on platinum, iridium, gold, ruthenium, and copper, that exhibit\n Rhodium complexes have shown promising anticancer\n potent ICD activity, have been developed.10\u221226 Importantly,\n activity, but few studies have reported their biological\n synergistic antitumor effects have also been confirmed when\n mode(s) of action.47\u221251 Here, we screened a series of rhodium\n combining metal-based ICD agents with immune checkpoint\n compounds and identified a potent Wnt/\u03b2-catenin pathway\n blockade (ICB) therapeutics, as exemplified by the use of\n inhibitor, Rh-1. We found that ROS accumulation was a key\n oxaliplatin with PD-1 inhibitors.27,28 Beyond their role in ICD,\n event in \u03b2-catenin blockage and CCL4 secretion. Moreover,\n metal complexes have demonstrated the ability to modulate\n Rh-1 potentiated the PD-1 blockade in inhibiting tumor\n inflammation or immune responses through diverse pathways,\n growth in animal models. Similarly, we observed the\n including regulating zinc homeostasis, targeting specific\n manifestation of this synergistic effect as enhanced T cell\n proteins, etc.29\u221233\n In addition to the activation of T cells through the antigen-\n presenting process, the success of tumor cell eradication is also Received: March 10, 2024\n dependent on a sufficient number of tumor-infiltrating Revised: July 2, 2024\n lymphocytes (TILs).34\u221236 However, a variety of evasion Accepted: August 1, 2024\n strategies by which tumor cells reduce T cell infiltration have Published: August 12, 2024\n been described.37\u221239 Of these, the Wnt/\u03b2-catenin pathway has\n been identified as one of the most crucial oncogenic signaling\n\n \u00a9 2024 American Chemical Society https://doi.org/10.1021/acs.jmedchem.4c00583\n 13778 J. Med. Chem. 2024, 67, 13778\u221213787\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\n\n\n\nFigure 1. Chemical structures of the Rh(III) complexes.\n\ninfiltration by Rh-1-stimulated CCL4 secretion in the tumor quinoline and pyridine, and they differ in the substituent\nmicroenvironment. Based on our findings, Rh-1 or similar groups of the benzene rings, which can affect the lipophilicity\nmetal-based Wnt/\u03b2-catenin inhibitors may emerge as chemo- and electronic effects in a series of structures with the same N\ntherapeutic agents to complement current or future ICB heterocycle. These substitution groups are also mainly divided\ntherapeutic strategies. into two categories: electron-withdrawing groups containing\n Cl, F and NO2, and electron-supplying groups containing H,\n2. RESULTS AND DISCUSSION Me and OMe. These rhodium(III) complexes dissolved readily\n 2.1. Synthesis and Characterization. Reedijk and co- in common organic solvents, such as DMF and DMSO, and\nworkers have reported that 4-methyl-2-N-(2-pyridylmethyl)- are soluble in PBS after dilution from DMF stock solution (1%\naminophenol (Hpyrimol) coordinated with copper, iron, zinc, DMF unless otherwise stated).\net al. could result in multiple biological synergistic effects.52\u221254 2.2. Stability of Rh Complexes. The stability of Rh\nBased on the theory of structure\u2212activity relationship, we complexes was investigated using HPLC and UV absorption\nsynthesized 12 new ligands based on the Hpyrimol structure, spectroscopy under physiological conditions (Tris-KCl-HCl\nand then 12 novel rhodium(III) complexes have also been buffer, pH 7.35). As illustrated in Figures S52 and S53\nsynthesized (Figure 1) and fully characterized by 1H NMR, (Supporting Information), the time-dependent HPLC chro-\n13 matograms (12, 24, and 48 h) demonstrate that there are no\n C NMR and ESI-MS (see details in the synthetic section,\nSupporting Information). We also performed X-ray crystallog- observable changes in the incubation solution. This indicates\nraphy on Rh-1 and Rh-5. According to the X-ray that the Rh complexes remained stable in the TBS buffer for up\ncrystallography data (Table S1), the Rh(III) metal center is to 48 h at room temperature. To further validate the HPLC\ncoordinated by two Cl\u2212 ions, one H2O molecule, and two N results, the time-dependent UV absorption spectra of the Rh\natoms and one O atom from the tridentate ligand. Two five- complex were also monitored, as shown in Figure S54. The UV\nmembered chelating rings are formed and share the common absorption spectra showed no significant red or blue shifts after\nRh\u2212N edge in the two complexes (Figures 2 and S51). The incubation for different durations under physiological con-\nrhodium(III) metal center shows a twisted octahedral ditions. This further corroborates the reliability of the HPLC\nconfiguration with the six coordination mode, in which the test results.\nmetal center and tridentate ligand form a planar structure. 2.3. In Vitro Cytotoxic Activity of Rhodium(III)\nThese metal complexes are mainly divided into two categories Complexes. We investigated the in vitro cytotoxicity of the\nbased on the different core nitrogen heterocycles including rhodium complexes and their ligands in triple-negative breast\n cancer (TNBC, 4T1 and MDA-MB-231), urinary bladder\n cancer (T-24), liver cancer (BEL-7404 and HepG2) and\n gastric cancer (MGC-803) cell lines. None of the ligands or\n Rh(III)Cl3 salt elicited cytotoxicity against the tested cell lines\n (IC50 > 40 \u03bcM), but almost all rhodium complexes showed\n some degree of cytotoxicity against all tested cancer cell lines\n (IC50 values ranging from 2.48 to 25.61 \u03bcM), indicating that\n the coordination between the ligands and metal salt exhibited a\n cytotoxic synergism. As shown in Table S2, the IC50 values of\n the rhodium complexes with electron-withdrawing groups were\n significantly lower than those of the rhodium complexes with\n electron-donating groups in all tested cell lines, among which\n two complexes with chlorine atoms are the most toxic against\n TNBC cell lines (IC50 values of 2.48\u22124.56 \u03bcM for Rh-1 and\n 6.34\u22126.78 \u03bcM for Rh-7). In addition, quinine-based rhodium\nFigure 2. X-ray crystallography structure of Rh-1 at a 50% probability complexes (Rh-1-6) tended to exhibit higher activities against a\nlevel. All hydrogen atoms were removed for clarity. given cell line than the pyridine-based rhodium complexes\n 13779 https://doi.org/10.1021/acs.jmedchem.4c00583\n J. Med. Chem. 2024, 67, 13778\u221213787\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\n\n\n\nFigure 3. (A) Western blot analysis of \u03b2-catenin protein after treatment with 12 different Rh(III) complexes; (B\u2212C) Western blot analysis of\nproteins related to Wnt/\u03b2-catenin signaling and chemokine CCL4 after treatment with Rh-1.\n\n(Rh-7-12) with the same R group. The difference in treatment with Rh-1. We also observed downregulation of\ncytotoxicity among rhodium complexes may be attributed to the transcription of two Wnt-dependent genes, dickkopf 1\nthe varying lipophilicity or electronic effects of their ligands. (DKK1) and cyclin D1, and the protein levels of the related\nRh-1 showed the strongest activity (IC50 value of 2.48 \u03bcM) transcription factor, T cell factor 7 (TCF7), and CD44 upon\nout of all the complexes against TNBC 4T1 cell line, which treatment with Rh-1 (Figure 3B,C).40\u221243 These data indicate\nwas also more potent than that of the positive control, cisplatin that the Wnt/\u03b2-catenin signaling pathway is substantially\n(IC50 value of 4.21 \u03bcM). In addition, the IC50 values of Rh-1 inhibited by Rh-1.\nagainst different cancer cell lines exhibited apparent differences Previous studies have shown that Wnt signaling down-\n(IC50 values ranging from 2.48 to 13.02 \u03bcM), suggesting a regulation can improve T cell recruitment or infiltration, since\ncertain extent of selectivity to cancer cell types. Considering downregulated Wnt/\u03b2-catenin signaling inhibits the expression\nthe excellent cytotoxicity of Rh-1 against 4T1 cell line and the of the transcriptional repressor, activating transcription factor 3\nfact that TNBC is a type of highly aggressive cancer that is (ATF3), and that ATF3 inhibition can promote CCL4\nhard to be cured, 4T1 cell line was chosen for further study. production, which is a chemokine-associated with T-cell\n 2.4. Inhibition of Wnt/\u03b2-Catenin Signaling: Candidate infiltration or recruitment in tumors.44 Therefore, we next\nScreening. In addition to cytotoxicity, we also sought to pursued whether Wnt/\u03b2-catenin signaling regulated by Rh-1\nidentify a candidate inhibitor of Wnt/\u03b2-catenin signaling to promotes CCL4 production. Consistent with the previous\npotentially stimulate anticancer immunity against TNBC. To study, we observed decreased ATF3 and increased CCL4\nthis end, we treated 4T1 cells with the 12 compounds (at their expression in tumor cells treated with Rh-1 (Figure 3C),\nIC50 concentrations) for 48 h, and then examined \u03b2-catenin indicating that Rh-1 has the potential to promote T cell\nexpression, a key component of the Wnt signaling pathway, by infiltration of tumors.\nWestern blot analysis. As shown in Figure 3A, rhodium 2.5. Mechanism of Rh-1 Regulating Wnt/\u03b2-Catenin\ncomplexes with an electron-withdrawing group (R) showed Signaling. Some metal complexes with anticancer activity\ndiffering downregulation of the \u03b2-catenin protein. In particular, stimulate ROS production and induce ER-stress.55\u221258 Previous\ntwo rhodium complexes with chlorine groups, Rh-1 and Rh-7, studies have also indicated that ER stress can result in\nexhibited the most significant inhibition of Wnt/\u03b2-catenin downregulation of Wnt/\u03b2-catenin signaling, since activating\nsignaling. Due to the apparent downregulation of \u03b2-catenin transcription factor 4 (ATF4), an ER stress-inducible tran-\nand excellent cytotoxicity induced by Rh-1 against the 4T1 cell scription factor, can promote the expression of C/EBP\nline, we chose this complex as a representative rhodium homologous protein (CHOP).59,60 CHOP acts as a specific\ncompound for subsequent experiments aimed at elucidating inhibitor of Wnt-TCF signaling, since this inhibition blocks the\nthe mechanism(s) of cytotoxicity and immune-regulation in binding of TCF to its DNA recognition site.61,62 Therefore, we\nthe 4T1 cancer cell line. examined whether the downregulation of \u03b2-catenin protein\n Further confirming the downregulation of Wnt/\u03b2-catenin induced by Rh-1 is related to ER-stress. After incubation of\nsignaling induced by Rh-1, we observed a time-dependent 4T1 cells with 3 \u03bcM Rh-1 for 0\u221248 h, the expression levels of\ndownregulation of the protein levels of two target genes in the phosphorylated RNA-dependent protein kinase-like endoplas-\nWnt/\u03b2-catenin pathway, the receptor tyrosine kinase (MET) mic reticulum kinase (PERK), phosphorylated eukaryotic\nand lymphoid enhancer-binding factor 1 (LEF1), after initiation factor 2a (eIF2a), and CHOP all showed time-\n 13780 https://doi.org/10.1021/acs.jmedchem.4c00583\n J. Med. Chem. 2024, 67, 13778\u221213787\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\n\n\n\nFigure 4. (A) WB analysis of ER-stress-related proteins in 4T1 cells treated with Rh-1 (at the IC50 concentration) for the indicated durations; (B)\nThe ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) significantly decreased after Rh-1 treatment; (C) Representative\nhistogram plot of ROS stained by DCF-DA. Pretreatment with the antioxidant (NAC, 5 mM) resulted in a reduction of the ROS levels induced by\nRh-1; (D) Proliferative inhibition by Rh-1 was significantly lower in the presence of 5 mM NAC compared with Rh-1 alone; (E) The ROS induced\nby 3 \u03bcM Rh-1 was inhibited by the NADPH oxidase inhibitor, DPI and MT. The data are shown as mean \u00b1 std, n \u2265 3, DPI, diphenylene iodonium\nchloride; MT, mito-tempo; (F) GSH peroxidase and reductase activities were examined in the presence of Rh-1; (G) TrxR activity inhibition\noccurred approximately in parallel with the proliferative inhibition of 4T1 cells upon Rh-1 treatment; (H) Western blot analysis of proteins related\nto ROS and the downregulation of \u03b2-catenin and CCL4.\n\ndependent increase (Figure 4A), indicating the induction of with Rh-1 for 24 h, GPx activity was slightly decreased and GR\nER-stress. The ER-stress response plays a critical role in activity remained nearly unchanged. In contrast, TrxR activity\ntriggering various cell death pathways. Particularly, apoptosis is was dose-dependently inhibited after the 24 h treatment, along\nconsidered to be one of the significant death pathways with inhibition of cell proliferation (Figure 4F,G), indicating\nresulting from the ER-stress response.55\u221258 Thus, we examined that TrxR may be the target of Rh-1. In addition, the\nthe activation of caspase family proteins associated with expression of TrxR was found to be significantly down-\napoptosis. We observed elevated caspase-3 and caspase-9 regulated by Rh-1 treatment, in a time-dependent manner\nproteolytic activities following treatment with 3 \u03bcM Rh-1 (Figure S56), indicating that Rh-1 not only inhibited TrxR\n(Figure S55), indicating that Rh-1 induces apoptosis which activity but also decreased TrxR expression, disrupting\nmay be mediated by the ER-stress response. To understand the intracellular redox balance. Cisplatin and oxaliplatin are clinical\nmechanism of Rh-1-induced ER-stress, we assessed ROS metal-based drugs that can induce ROS production. However,\nproduction. The ratio of intracellular GSH to GSSG, which is oxaliplatin could not inhibit \u03b2-catenin expression (Figure S57),\ninversely related to ROS production,63,64 was significantly and the downregulation of \u03b2-catenin expression by cisplatin\ndecreased after treatment with Rh-1 (Figure 4B). Consistent was much lower than that by Rh-1 (Figures 3B and S58). To\nwith this finding, the ROS scavenger, N-acetylcysteine (NAC),\n further understand the role of TrxR inhibition in \u03b2-catenin\ndecreased ROS production and growth inhibition by Rh-1 in\n suppression, we next compared \u03b2-catenin expression in 4T1\n4T1 cells (Figure 4C,D). To further identify the source of\nROS, we used two more ROS scavengers, DPI and MT, which cells treated with auranofin and the gold(III) porphyrin\nare cognate inhibitors of NADPH oxidase and mitochondria, [Au(TPP)]Cl. Auranofin, a classical metal-based inhibitor of\nrespectively.65 Surprisingly, MT did not affect ROS level, while TrxRs,71 showed notable efficacy in suppressing \u03b2-catenin. In\nDPI significantly decreased the ROS (Figure 4E). Together, contrast, [Au(TPP)]Cl, another metal-based agent with\nthese results indicate that ROS production induced by Rh-1 weaker activity than that of auranofin against TrxRs,71 failed\nmay be dependent on the SOD system, rather than to decrease \u03b2-catenin (Figure S58). Taken together, these data\nmitochondrial damage. Antioxidant protein, glutathione indicate that targeting TrxR may be a general trigger for \u03b2-\nperoxidase (GPx), can decrease ROS, oxidizing GSH to catenin downregulation. Since ROS production is a common\nGSSG, which is then replenished by glutathione reductase outcome of TrxR inhibition, we next sought to figure out the\n(GR) that reduces GSSG back to GSH. Peroxiredoxin, one of role of ROS in Wnt/\u03b2-catenin signaling. ER-stress-related\nthe substrates of Trx, which can be reduced by TrxR, may also proteins, apoptosis-related proteins, \u03b2-catenin, and chemokine\ndecrease ROS. Thus, the combined intracellular activities of CCL4 were inhibited after pretreating cells with NAC (Figures\nTrxR, GPx, and GR make up the main antioxidant systems that 4H, S55 and S59). Together, these results indicate that Rh-1\nimpact ROS accumulation in cells.66\u221271 After treating cells may cause the inhibition of Wnt/\u03b2-catenin signaling, CCL4\n 13781 https://doi.org/10.1021/acs.jmedchem.4c00583\n J. Med. Chem. 2024, 67, 13778\u221213787\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\n\n\n\nFigure 5. Induction of antitumor immune cell infiltration in vivo. (A) Tumor volumes of BALB/c mice bearing 4T1 cells (n = 7) after treatment\nwith the indicated formulations; (B) Survival of BALB/c mice bearing 4T1 cells (n = 7) after treatment with the indicated formulations; (C) Flow\ncytometry analysis of T cell types in 4T1 tumors of BALB/c mice after treatment with Rh-1; (D) IHC analysis of CD3+ T cells in tumors from\nmice treated with Rh-1 or \u03b1PD-1 therapy alone or combined therapy; (E) IFC analysis of CD8+ T cells and CD4+ FoxP3+ T cells in tumors from\nmice treated with Rh-1 alone or combined therapy with Rh-1 and \u03b1PD-1; (F) WB analysis of proteins involved in Wnt signaling, CCL4 and\nimmune checkpoint PD-L1 and PD-1; (G) Flow cytometry analysis of Ki-67+ in CD8+ T cells and CD4+FoxP3+ T cells in 4T1 tumors from BALB/\nc mice after treatment with Rh-1; (H) Flow cytometry analysis of Granzyme B+ and IFN-\u03b3+ TNF-\u03b1+ in 4T1 tumors from BALB/c mice after\ntreatment with Rh-1 or combined Rh-1 and \u03b1PD-1.\n\nproduction, and ER-stress-dependent apoptosis as the ultimate CD4+FoxP3+ Treg cells (Figures 5G and S62) as well as PD-\nconsequences of ROS production. L1 and PD-L2 expression in the tumor microenvironment\n 2.6. In Vivo Chemoimmunotherapy Activity. To study (Figure 5F). Together, these data indicate that CD8+ T cells\nthe in vivo response of 4T1 tumors to Rh-1, we subcutaneously infiltrate into breast tumors under Rh-1 treatment, but the\nimplanted mice with 8 \u00d7 104 4T1 cells and allowed the tumors tumor microenvironment is still immunosuppressive. In\nto grow to approximately 60 mm3 before treatment. The addition, the key proteins of the Wnt signaling pathway, \u03b2-\nchemotherapy agent, cisplatin, which is effective in breast catenin, and LEF1, are significantly downregulated, while the\ncancer patients, was used as a control. Cisplatin (2 mg/kg) or CCL4 chemokine that promotes T-cell infiltration or recruit-\nRh-1 (6 mg/kg) was administered (i.p.) once every 2 days. ment in tumors is increased (Figure 5F), all of which is\nRh-1 treatment led to significantly slower tumor growth and consistent with the results of the in vitro experiments (Figure\nincreased survival compared with that of both control mice and 3C). For the role of CCL4 in T cell recruitment, we\ncisplatin-treated mice (Figure 5A,B), meanwhile, no obvious administered an anti-CCL4 antibody to shield its function\norgan toxicity was observed (Figure S60). \u03b2-catenin on days 3, 7, and 11 after the start of Rh-1 treatment. The T\nsuppression is known to be associated with immune cells in tumors treated with Rh-1 plus the anti-CCL4 antibody\nresponses.40\u221243 We assessed the effects of Rh-1 on T cells in were lower than under treatment with Rh-1 alone (Figure 5C).\nmurine 4T1 tumors and found a significant increase in the These data indicate that CCL4 is an important chemokine\nnumber of CD3+ T cells that were predominantly composed of promoting T cell infiltration. We next examined the possibility\nCD3+ CD8+ cells (Figures 5C and S61) after 7 days of that the antitumor immunity following Rh-1 administration is\ntreatment. Next, we tested whether CD8+ T cells were critical from the classical immunogenic cell death (CICD) pathway by\nfor the therapeutic benefits of Rh-1. To this end, we detecting HMGB-1, an important hallmark of CICD.59,60 The\nadministered an anti-CD8 antibody to shield the function of secretion of HMGB-1 into the culture media by 4T1 cells\nCD8+ T cells on days 3, 7, and 11 after the start of Rh-1 treated with Rh-1 (3.0 \u03bcM) for 24 or 48 h was not significantly\ntreatment. The depletion of CD8+ T cells abrogated the Rh-1- altered, as determined by ELISA. Taken together, the above\ninduced increase in survival and inhibition of tumor growth, results suggest that Rh-1 stimulates anticancer immunity\nwith most mice dying within 20 days after tumor implantation, primarily by affecting the Wnt signaling pathway.\nwhereas most mice survived past 30 days with Rh-1 treatment Given the recent clinical success of PD-1 blockade and the\nin the absence of the antibody (Figure 5B). Thus, CD8+ T cells PD-L1 promoting effect of Rh-1, we hypothesized that\nare critical for the efficacy of Rh-1 therapy in this mouse model immune checkpoint blockade would synergize with Rh-1\nof breast cancer, while CD4+ T cells are less important therapy to enhance antitumor immunity by boosting CD8+ T-\n(Figures 5C and S61). When assessing the proliferation cell response. Treatment with anti-PD-1 alone did not\npotential of CD8+ TILs, we did not find obvious incensement obviously affect tumor growth or survival (Figure 5A,B).\nin the expression of the nuclear proliferation marker, Ki-67, in Notably, combining PD-1 pathway blockade (utilizing an anti-\nCD8+ TILs in mice administered Rh-1 (Figures 5G and S62), PD1 antibody) with Rh-1 resulted in a substantial delay in\nbut treatment with the formulation led to an increase in tumor growth and enhanced survival compared to either\n 13782 https://doi.org/10.1021/acs.jmedchem.4c00583\n J. Med. Chem. 2024, 67, 13778\u221213787\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\ntherapy alone. Approximately 30% of the mice survived beyond 4. EXPERIMENTAL SECTION\n60 days and achieved complete remission (Figure S64), The relative purity of all target compounds used in the biophysical\nwhereas none of the mice in the Rh-1 group survived past 50 and biological studies was \u226595%, which were routinely confirmed by\ndays (Figure 5A,B). We observed minimal to no increase in HPLC (waters e2695).\nCD3+ T cells in tumors treated with anti-PD-1 treatment 4.1. Materials and Reagents. The cancer cell lines including T-\nalone, but combining Rh-1 with anti-PD-1 led to a significant 24, BEL-7404, MGC-803, HepG2, MDA-MB-231, 4T1 in this study\nincrease in CD3+ T cells, compared with any of the were obtained from the Laboratory Cell Service Center at Chinese\nmonotherapy formulations (Figure 5D). Combination therapy Academy of Sciences (Shang Hai, China). BALB/c mice at the age of\nalso led to a substantial increase in CD8+ T cells in the tumor 4 weeks were purchased from Changsha\u2019s animal research center (Hu\ncompared with Rh-1 alone (Figure 5E) with no difference in Nan, China). Dulbecco\u2019s Modified Eagle Medium (DMEM), Roswell\n Park Memorial Institute 1640 Medium (RPMI 1640), and fetal\nCD4+ FoxP3+ Treg cells in the tumor. We also found an bovine serum (FBS) were procured from Gibco Company. Penicillin,\nincreased fraction of granzyme B-producing CD8+ T cells, as streptomycin, and trypsin were obtained from HyClone Company.\nwell as polyfunctional CD8+ T cells producing both IFN-\u03b3 and Penicillin, streptomycin and trypsin were purchased from HyClone\nTNF-\u03b1, in the mice treated with anti-PD-1 and Rh-1 (Figures Company. Ethidium Homodimer-1 (EthD-1) and Calcein-AM were\n5H and S63). Together, these results indicate that the from Abbkine (USA) and Aladdin (Shanghai, China). Bovine albumin\nintratumoral CD8+ T-cell response is synergistically enhanced (BSA) and methylthiazolyldiphenyl-tetrazolium bromide (MTT)\nby combination therapy with Rh-1 with anti-PD-1. were obtained from Sigma. Human serum albumin (HSA) was\n purchased from Solarbio (Beijing, China). The following antibodies\n3. CONCLUSIONS were used for Western Blot: p-eIF2\u03b1 (#3398), p-PERK (#675505),\n CHOP (#5554), \u03b2-catenin (#862601), LEF1 (#653101), LEF1\nIn summary, we identified a rhodium complex, Rh-1, as a new (#653101), MET (#689902), Cyclin D1 (#681902), CD44\nWnt/\u03b2-catenin pathway inhibitor. This inhibition was achieved (#ab243894), \u03b2-actin (#664801), \u03b2-catenin (#862601), ATF3\nby cellular ROS production triggered by Rh-1 (Scheme 1). As (#ab254268), TCF7 (#615701), DKK1 (#PA5-23187), CCL4\n (#710391), PD-L1 (#60475), PD-L2 (#49189). All chemicals, unless\nScheme 1. Graphic Summary of Rh-1-Induced Antitumor otherwise noted, were from commercial sources.\nImmunity 4.2. Synthesis and Characterization. General procedures for\n the synthesis of metal Rh complexes are illustrated below. Equimolar\n amounts (0.1 mol) of quinoline-2-carbaldehyde or pyridine-2-\n carbaldehyde and 2-amino-phenol derivatives in methanol were\n reacted for 4 h at 60 \u00b0C. After cooling, the solution was filtered\n and evaporated under reduced pressure. The crude product was\n recrystallized from CH3OH/n-hexane. Next, the ligands (1 mmol)\n and RhCl3\u00b73H2O (2 mmol) were dissolved in 15 mL CH3OH/CHCl3\n solution (v/v = 3:2), and the mixture was stirred at 60 \u00b0C for 8 h. The\n resulting yellow solution was filtered and crystals or solid powders\n were obtained by slow evaporation of the solvent. For detailed data\n and spectra of all compounds, please refer to the corresponding part\n of Supporting Information.\n 4.3. Cytotoxicity Assay. The tested cancer cell lines included\n TNBC (4T1 and MDA-MB-231), urinary bladder cancer (T-24), liver\n cancer (BEL-7404 and HepG2) and gastric cancer (MGC-803) cell\n lines. Our previously published papers can be referred for the detailed\n cell culture conditions and screening methods cytotoxicity.12\n 4.4. In Vivo Mouse Studies. To test the acute toxicity of Rh-1,\n six-week-old male and female BALB/c mice, weighing 20\u221222 g, were\n randomly divided into 7 groups (n = 6). The Rh-1 was administered\n via intraperitoneal injection at the specified doses once a day for 12\n days. The animals were closely monitored for any signs of toxicity,\nan outcome of Wnt/\u03b2-catenin inhibition, CCL4 is up-regulated and their body weights were recorded daily.\nto promote T cell infiltration into the tumor tissue, along with Subcutaneous xenograft models were established by injecting 8 \u00d7\nmarkers of activated CD8+ T cells, such as granzyme B, IFN-\u03b3, 104 4T1 cells into immunocompetent female BALB/c mice (7 mice\n per group). When the tumor volume grown to about 60 mm3,\nand TNF-\u03b1. Experiments using Rh-1 with a PD-1 inhibitor\n cisplatin (2 mg/kg) and Rh-1 (6 mg/kg) were injected intra-\ndemonstrated their synergism in combating tumor progression. peritoneally every 2 days. Tumor volumes were monitored by digital\nAlthough the molecular basis behind this ROS-dependent calipers every 3 days, and calculated volume with the formula: tumor\ninhibition of Wnt/\u03b2-catenin signaling warrants additional volume = (shortest diameter)2 \u00d7 (longest diameter) \u00d7 0.5. The\n growth curves of tumor were drawn with the average tumor volume to\nstudy, this work is instructive for the future design of metal- the number of days. All mice were euthanized when the tumor volume\nbased agents with better performance. grown to approximately 2 \u00d7 103 mm3. The inhibition rates of tumor\n Despite the significant success of combining ICD agents growth (IRT) were figured out with the formula: IRT = 100% \u00d7\n (mean tumor weight of the control group\u2212mean tumor weight of the\nwith cancer immunotherapy strategies, emerging mechanisms\n experimental group)/mean tumor weight of the control group.\nthat reduce the ICD effect have been identified in certain The rat monoclonal antibody (mAb; BioLegend, San Diego, CA),\ncancer types.72,73 Therefore, metal complexes with novel antimouse PD-1 (RMP1-14) or the rat immunoglobulin G2a (IgG2a;\nproinflammatory functions, such as Rh-1, will be important to RTK2758) mAb (BioLegend) was injected intraperitoneally with\n dose of 200 \u03bcg/mouse on days 7, 10, and 13. For the experiment of\novercoming the ongoing resistance to existing cancer chemo- CD8+ T cell blocking, the dose 100 \u03bcg/mouse of anti-CD8 (Lyt 3.2)\nimmunotherapies. mAb (BioXCell) was injected intraperitoneally on days \u22122 and 0, and\n\n 13783 https://doi.org/10.1021/acs.jmedchem.4c00583\n J. Med. Chem. 2024, 67, 13778\u221213787\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\nthen every 7 days. To block CCL4, 50 \u03bcg anti-CCL4 (#46907) mAb orcid.org/0000-0001-9129-4398; Email: zoutt3@\n(R&D Systems) or control rat IgG2a (#54447) mAb (R&D Systems) mail.sysu.edu.cn\nwas administered intraperitoneally 7 days after tumor inoculation and Hong Liang \u2212 State Key Laboratory for Chemistry and\nthen every 3 days. Tumors were harvested on day 14 for the Molecular Engineering of Medicinal Resources, Key\npreparation of TILs, which were analyzed with flow cytometry. All\nmouse experiments were approved by the Committee for Animal\n Laboratory for Chemistry and Molecular Engineering of\nExperimentation of Guangxi Normal University (approval number: Medicinal Resources (Ministry of Education of China),\n201903-012). Collaborative Innovation Center for Guangxi Ethnic\n 4.5. Flow Cytometric Analysis of Tumor-Infiltrating Lym- Medicine, School of Chemistry and Pharmaceutical Sciences,\nphocytes. A tumor-infiltrating lymphocyte (TIL) enrichment Guangxi Normal University, Guilin 541004, China;\nprotocol was used for mouse tumors. Tumors were weighed, cut, Email: hliang@gxnu.edu.cn\nand placed in collagenase type I and incubated on a shaker at 37 \u00b0C\nfor 30 min. The dissociated tumor was then filtered (70 mm) to get a Authors\nsingle-cell suspension. TILs were enriched from the single-cell\nsuspension using a sucrose gradient (40%/70% Percoll; GE Feng-Yang Wang \u2212 State Key Laboratory for Chemistry and\nHealthcare). Mouse TILs were stained with the following directly Molecular Engineering of Medicinal Resources, Key\nlabeled antibodies (all from BioLegend): anti-CD3e (145-2c11), anti- Laboratory for Chemistry and Molecular Engineering of\nCD4 (RM4-5), anti-CD8a (53\u22126.7), anti-Ki67 (16A8), anti-PDL1 Medicinal Resources (Ministry of Education of China),\n(10F.9G2), anti-FoxP3 (MF-14), and antigranzyme B (GB11). Collaborative Innovation Center for Guangxi Ethnic\n 4.6. Statistics. The data processing included the Student\u2019s t-test, Medicine, School of Chemistry and Pharmaceutical Sciences,\nwith p \u2264 0.05 considered significant, using SPSS 13.0.\n Guangxi Normal University, Guilin 541004, China\n\n\u25a0\n*\n ASSOCIATED CONTENT\ns\u0131 Supporting Information\n Liang-Mei Yang \u2212 State Key Laboratory for Chemistry and\n Molecular Engineering of Medicinal Resources, Key\n Laboratory for Chemistry and Molecular Engineering of\nThe Supporting Information is available free of charge at Medicinal Resources (Ministry of Education of China),\nhttps://pubs.acs.org/doi/10.1021/acs.jmedchem.4c00583. Collaborative Innovation Center for Guangxi Ethnic\n Molecular formula strings and biological data (CSV) Medicine, School of Chemistry and Pharmaceutical Sciences,\n Crystallographic data for complex Rh-1 (CIF) Guangxi Normal University, Guilin 541004, China\n Crystallographic data for complex Rh-5 (CIF) Xiao-Lin Xiong \u2212 Guangdong Key Laboratory of Chiral\n The methods of some experiments; lH and 13C NMR Molecule and Drug Discovery, School of Pharmaceutical\n spectra of all complexes synthesized in this work; ESI- Science, Sun Yat-Sen University, Guangzhou 510006, China\n MS spectra of complexes; X-ray crystallographic data of Jing Yang \u2212 State Key Laboratory for Chemistry and\n complexes Rh-1 and Rh-5; stability and UV/vis Molecular Engineering of Medicinal Resources, Key\n absorption spectrograms of complexes Rh-1 and Rh-2 Laboratory for Chemistry and Molecular Engineering of\n in aqueous solution; IC50 values in several cell lines; Medicinal Resources (Ministry of Education of China),\n expression levels of proteins related to ER-stress, TrxR, Collaborative Innovation Center for Guangxi Ethnic\n GR, GPx and \u03b2-catenin; results of in vivo mouse studies Medicine, School of Chemistry and Pharmaceutical Sciences,\n and immunohistochemistry analyses; flow cytometry Guangxi Normal University, Guilin 541004, China\n analysis of apoptosis-related proteins; Western blot Yan Yang \u2212 Guangdong Key Laboratory of Chiral Molecule\n analysis of the relationship between ROS and apoptosis and Drug Discovery, School of Pharmaceutical Science, Sun\n (PDF) Yat-Sen University, Guangzhou 510006, China\n Jiu-Qin Tang \u2212 State Key Laboratory for Chemistry and\n Crystallographic data for the structural analysis have\n Molecular Engineering of Medicinal Resources, Key\n been deposited in the Cambridge Crystallographic Data\n Laboratory for Chemistry and Molecular Engineering of\n Centre, CCDC No. 2166861 and 2172152 for Rh-1 and\n Medicinal Resources (Ministry of Education of China),\n Rh-5. The data can be obtained free of charge at http://\n Collaborative Innovation Center for Guangxi Ethnic\n www.ccdc.cam.ac.uk, or from the Cambridge Crystallo-\n Medicine, School of Chemistry and Pharmaceutical Sciences,\n graphic Data Centre, 12 Union Road, Cambridge\n Guangxi Normal University, Guilin 541004, China\n CB21EZ, UK; fax: (+44) 1223-336-033; E-mail:\n Lei Gao \u2212 State Key Laboratory for Chemistry and Molecular\n deposit@ccdc.cam.ac.uk.\n Engineering of Medicinal Resources, Key Laboratory for\n\n\u25a0 AUTHOR INFORMATION\nCorresponding Authors\n Chemistry and Molecular Engineering of Medicinal Resources\n (Ministry of Education of China), Collaborative Innovation\n Center for Guangxi Ethnic Medicine, School of Chemistry and\n Ke-Bin Huang \u2212 State Key Laboratory for Chemistry and Pharmaceutical Sciences, Guangxi Normal University, Guilin\n Molecular Engineering of Medicinal Resources, Key 541004, China\n Laboratory for Chemistry and Molecular Engineering of Yuan Lu \u2212 State Key Laboratory for Chemistry and Molecular\n Medicinal Resources (Ministry of Education of China), Engineering of Medicinal Resources, Key Laboratory for\n Collaborative Innovation Center for Guangxi Ethnic Chemistry and Molecular Engineering of Medicinal Resources\n Medicine, School of Chemistry and Pharmaceutical Sciences, (Ministry of Education of China), Collaborative Innovation\n Guangxi Normal University, Guilin 541004, China; Center for Guangxi Ethnic Medicine, School of Chemistry and\n orcid.org/0000-0003-4773-4442; Email: kbhuang@ Pharmaceutical Sciences, Guangxi Normal University, Guilin\n mailbox.gxnu.edu.cn 541004, China\n Taotao Zou \u2212 Guangdong Key Laboratory of Chiral Molecule Yuan Wang \u2212 Guangdong Key Laboratory of Chiral Molecule\n and Drug Discovery, School of Pharmaceutical Science, Sun and Drug Discovery, School of Pharmaceutical Science, Sun\n Yat-Sen University, Guangzhou 510006, China; Yat-Sen University, Guangzhou 510006, China\n 13784 https://doi.org/10.1021/acs.jmedchem.4c00583\n J. Med. Chem. 2024, 67, 13778\u221213787\n\fJournal of Medicinal Chemistry pubs.acs.org/jmc Article\n\nComplete contact information is available at: (10) Wong, D. Y. Q.; Ong, W. W.; Ang, W. H. Induction of\nhttps://pubs.acs.org/10.1021/acs.jmedchem.4c00583 immunogenic cell death by chemotherapeutic platinum complexes.\n Angew. Chem., Int. Ed. 2015, 54, 6483\u22126487.\nAuthor Contributions (11) Tham, M. J. R.; Babak, M. V.; Ang, W. H. PlatinER: A Highly\n\u00a7 Potent Anticancer Platinum(II) Complex that Induces Endoplasmic\n F.-Y.W., L.-M.Y., and X.-L.X. contributed equally to this work. Reticulum Stress Driven Immunogenic Cell Death. Angew. Chem., Int.\nNotes Ed. 2020, 59, 19070\u221219078.\nThe authors declare no competing financial interest. (12) Huang, K. B.; Wang, F. Y.; Feng, H. W.; Luo, H.; Long, Y.;\n Zou, T.; Chan, A. S. C.; Liu, R.; Zou, H.; Chen, Z. F.; Liu, Y. C.; Liu,\n\n\u25a0 ACKNOWLEDGMENTS\nThis work was supported by the National Nature Science\n Y. N.; Liang, H. An aminophosphonate ester ligand-containing\n platinum(ii) complex induces potent immunogenic cell death in vitro\n and elicits effective anti-tumour immune responses in vivo. Chem.\nFoundation of China (Grant No. 22177022) and the Natural Commun. 2019, 55, 13066\u221213069.\nScience Foundation of Guangxi Province of China (Grant: (13) Sen, S.; Hufnagel, S.; Maier, E. Y.; Aguilar, I.; Selvakumar, J.;\n DeVore, J. E.; Lynch, V. M.; Arumugam, K.; Cui, Z.; Sessler, J. L.;\nAD17129007).\n Arambula, J. F. Rationally Designed Redox-Active Au(I) N-\n\n\u25a0 ABBREVIATIONS\nATF, activating transcription factor; ATP, adenosine triphos-\n Heterocyclic Carbene: An Immunogenic Cell Death Inducer. J. Am.\n Chem. Soc. 2020, 142, 20536\u221220541.\n (14) Wernitznig, D.; Meier-Menches, S. M.; Cseh, K.; Theiner, S.;\n Wenisch, D.; Schweikert, A.; Jakupec, M. A.; Koellensperger, G.;\nphate; CCL4, cc chemokine ligand 4; CHOP, CCAAT/\n Wernitznig, A.; Sommergruber, W.; Keppler, B. K. Plecstatin-1\nenhancer-binding protein homologous protein; CRT, calreti- induces an immunogenic cell death signature in colorectal tumour\nculin; DAMP, damage-associated molecular pattern; DKK1, spheroids. Metallomics 2020, 12, 2121\u22122133.\ndickkopf 1; eIF2a, eukaryotic initiation factor 2a; ER, (15) Kaur, P.; Johnson, A.; Northcote-Smith, J.; Lu, C.;\nendoplasmic reticulum; GPx, glutathione peroxidase; GR, Suntharalingam, K. Immunogenic Cell Death of Breast Cancer Stem\nglutathione reductase; HMGB1, high mobility group box 1; Cells Induced by an Endoplasmic Reticulum-Targeting Copper(II)\nICB, immune checkpoint blockade; ICD, immunogenic cell Complex. 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