Ruthenium Complexes as Promising Candidates against Lung Cancer

Lung cancer is one of the most common malignancies with the highest mortality rate and the second-highest incidence rate after breast cancer, posing a serious threat to human health. The accidental discovery of the antitumor properties of cisplatin in the early 1960s aroused a growing interest in metal-based compounds for cancer treatment. However, the clinical application of cisplatin is limited by serious side effects and drug resistance. Therefore, other transition metal complexes have been developed for the treatment of different malignant cancers. Among them, Ru(II/III)-based complexes have emerged as promising anticancer drug candidates due to their potential anticancer properties and selective cytotoxic activity. In this review, we summarized the latest developments of Ru(II/III) complexes against lung cancer, focusing mainly on the mechanisms of their biological activities, including induction of apoptosis, necroptosis, autophagy, cell cycle arrest, inhibition of cell proliferation, and invasion and metastasis of lung cancer cells.


Introduction
Primary bronchogenic carcinoma, also known as lung cancer, is a malignant tumor that originates from the bronchial mucosa or gland. With a high incidence and mortality rate, lung cancer poses a serious threat to human health, while its cases and deaths rise every year [1]. Although its incidence follows breast cancer, lung cancer remains the leading cause of cancer death, with approximately 1.8 million deaths (18%) worldwide [2]. Moreover, the global cancer burden is expected to increase by 47% in 2040 compared to 2020, reaching a total of 28.4 million cases [2]. However, current advances in novel chemotherapeutic agents, targeted therapies, standardized diagnosis, and staging and multidisciplinary treatment of lung cancer have improved patient survival rates [3]. Nevertheless, the prognosis of lung cancer patients is still poor due to insufficient early diagnosis.
Lung cancer can be classified into central and peripheral lung cancer depending on the anatomical part affected, as well as into two main pathological entities: non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC). NSCLC can be further subdivided into three histological subtypes: lung squamous cell carcinoma, lung adenocarcinoma, and large cell carcinoma. NSCLC, which accounts for about approximately 85% of lung cancer cases, has shown an increased mortality rate in recent years. Radical surgery is the most common treatment applied to early-stage NSCLC patients, while chemotherapy is mainly used for NSCLC patients in advanced or recurrent stages [3]. Moreover, NSCLC (KP1019) [36][37][38][39] and sodium [trans-RuCl 4 (1H-indazole) 2 ] (KP1339) [40] (Figure 1). The pharmacokinetics of KP1019, which was characterized by a small volume of distribution, low clearance, and long half-life, has been researched in a phase I dose-escalation study, in which five out of six patients treated with KP1019 experienced disease stabilization with no severe side effects [36,39]. With these pioneering works, Ru complexes are attracting increasing attention from chemical researchers. The recent years have witnessed the development of Ruthenium complexes as second-generation metal-based anticancer agents, possessing high potency of targeting cancer cells due to their low toxicity, the ability to induce apoptosis, selective anti-invasion, and anti-metastasis activity [41][42][43]. Some of them have also shown anti-angiogenic properties and could therefore be used to inhibit angiogenesis, the basis of tumor growth and metastasis [44]. Moreover, with the purpose of improving their in vivo stability, solubility, cellular uptake, and effectiveness, some research groups with meticulous design have developed special drug delivery systems (nanoparticles, liposomes, etc.), which could encapsulate Ru-based compounds appropriately [30,[45][46][47].
Molecules 2021, 26, x FOR PEER REVIEW 3 of 21 selective activity against lung metastases of some solid metastasizing tumors at a concentration with relatively mild toxicity [32,33,35]. The other two important promising Ru(III) complexes that have entered clinical trials were indazolium [trans-RuCl4(1H-indazole)2] (KP1019) [36][37][38][39] and sodium [trans-RuCl4(1H-indazole)2] (KP1339) [40] (Figure 1). The pharmacokinetics of KP1019, which was characterized by a small volume of distribution, low clearance, and long half-life, has been researched in a phase I dose-escalation study, in which five out of six patients treated with KP1019 experienced disease stabilization with no severe side effects [36,39]. With these pioneering works, Ru complexes are attracting increasing attention from chemical researchers. The recent years have witnessed the development of ruthenium complexes as second-generation metal-based anticancer agents, possessing high potency of targeting cancer cells due to their low toxicity, the ability to induce apoptosis, selective anti-invasion, and anti-metastasis activity [41][42][43]. Some of them have also shown anti-angiogenic properties and could therefore be used to inhibit angiogenesis, the basis of tumor growth and metastasis [44]. Moreover, with the purpose of improving their in vivo stability, solubility, cellular uptake, and effectiveness, some research groups with meticulous design have developed special drug delivery systems (nanoparticles, liposomes, etc.), which could encapsulate Ru-based compounds appropriately [30,[45][46][47]. Based on these promising results, in this review, we summarize the recent findings on the anticancer mechanisms of Ru-based compounds targeting lung cancer, including apoptosis, autophagy, necroptosis, anti-metastasis, and cell cycle arrest (Figure 2 and Table 1).  Based on these promising results, in this review, we summarize the recent findings on the anticancer mechanisms of Ru-based compounds targeting lung cancer, including apoptosis, autophagy, necroptosis, anti-metastasis, and cell cycle arrest ( Figure 2 and Table 1). selective activity against lung metastases of some solid metastasizing tumors at a concentration with relatively mild toxicity [32,33,35]. The other two important promising Ru(III) complexes that have entered clinical trials were indazolium [trans-RuCl4(1H-indazole)2] (KP1019) [36][37][38][39] and sodium [trans-RuCl4(1H-indazole)2] (KP1339) [40] (Figure 1). The pharmacokinetics of KP1019, which was characterized by a small volume of distribution, low clearance, and long half-life, has been researched in a phase I dose-escalation study, in which five out of six patients treated with KP1019 experienced disease stabilization with no severe side effects [36,39]. With these pioneering works, Ru complexes are attracting increasing attention from chemical researchers. The recent years have witnessed the development of ruthenium complexes as second-generation metal-based anticancer agents, possessing high potency of targeting cancer cells due to their low toxicity, the ability to induce apoptosis, selective anti-invasion, and anti-metastasis activity [41][42][43]. Some of them have also shown anti-angiogenic properties and could therefore be used to inhibit angiogenesis, the basis of tumor growth and metastasis [44]. Moreover, with the purpose of improving their in vivo stability, solubility, cellular uptake, and effectiveness, some research groups with meticulous design have developed special drug delivery systems (nanoparticles, liposomes, etc.), which could encapsulate Ru-based compounds appropriately [30,[45][46][47]. Based on these promising results, in this review, we summarize the recent findings on the anticancer mechanisms of Ru-based compounds targeting lung cancer, including apoptosis, autophagy, necroptosis, anti-metastasis, and cell cycle arrest (Figure 2 and Table 1).

Ru(III) Complexes
Ru(III) complexes are rapidly evolving into next-generation anticancer drugs. Ru(III) hydrazone complexes owing uncoordinated phenolic oxygen were offered as better drug candidates than Ru(II) complexes with the same ligands because the phenolic oxygen in Ru(III) complexes could effectively interact with biomolecules by hydrogen bond, while phenolic oxygen of Ru(II) complexes was deprotonated for coordinating with Ru metal [50]. Moreover, several Ru(III) complexes that have entered clinical trials in different phases, such as KP1019 [36][37][38][39], KP-1339 [40], and NAMI-A [31][32][33]65], showed promising anticancer activity with limited side effects and have been used to prepare various derivatives.

Apoptosis
Recent studies have shown that Ru(III) complexes inhibit tumor cell proliferation by inducing apoptosis, which refers to genetically identified programmed cell death and plays a vital role in normal tissue homeostasis [66]. Apoptosis is not only involved in the occurrence and growth of tumors but can also trigger cancer cell death, making it a suitable pathway for the development of anticancer drugs [67]. There are two main types of apoptosis pathways: the intrinsic pathway, which depends on mitochondria, and the extrinsic pathway, which depends on death receptors [67,68].

Anti-Metastasis
Cancer therapies generally fail when genetically unstable cancer cells adapt to the tissue microenvironment, leading to tumor metastasis [70,71]. Tumor metastasis involves several steps, including loss of cell adhesion, increased motility and invasiveness, entry and survival into the circulation, and eventual settlement into new tissues or distant organs [72]. Metastatic tumors are resistant to various cancer therapies, especially drugs and radiotherapy [73], indicating the need for the development of new compounds targeting the tumor metastasis pathway [74]. To date, various analogs have been designed and synthesized to prevent metastasis and/or inhibit the growth of metastatic tumors. Antineoplastic Ru(III) complexes are currently the most effective, such as NAMI-A, which can greatly reduce lung metastasis and the formation of solid metastasizing tumors in mice [75]. Therefore, a series of NAMI-A derivatives have been prepared that could modify important metastasis parameters such as tumor invasion, matrix metalloproteinases (MMP) activity, and cell cycle progression [42]. The new analogs also maintained the potent characteristics of NAMI-A and could selectively interact with solid metastatic tumors. Furthermore, the introduction of different ligands improved their stability in aqueous solutions [76]. Two NAMI-A derivatives bearing a pyridine ligand, Ru9/G26b and Ru10/G94a (Figure 4), displayed little direct cytotoxicity to human (A549) and mouse Lewis lung cancer cells but had a significant suppressive effect on the invasion and migration of cancer cells [51]. Like with NAMI-A, in vivo studies in 4T1 mammary carcinoma-bearing mice showed that the occurrence and development of lung metastasis were suppressed significantly and that no retinal toxicity or hepatotoxicity was found in mice after intraperitoneal injection of Ru9 and Ru10 at a dose of 17.5 mg/kg per day for consecutive 6 days, with three times in a 1-day interval. Ru9 in particular could suppress im-

Anti-Metastasis
Cancer therapies generally fail when genetically unstable cancer cells adapt to the tissue microenvironment, leading to tumor metastasis [70,71]. Tumor metastasis involves several steps, including loss of cell adhesion, increased motility and invasiveness, entry and survival into the circulation, and eventual settlement into new tissues or distant organs [72]. Metastatic tumors are resistant to various cancer therapies, especially drugs and radiotherapy [73], indicating the need for the development of new compounds targeting the tumor metastasis pathway [74]. To date, various analogs have been designed and synthesized to prevent metastasis and/or inhibit the growth of metastatic tumors. Antineoplastic Ru(III) complexes are currently the most effective, such as NAMI-A, which can greatly reduce lung metastasis and the formation of solid metastasizing tumors in mice [75]. Therefore, a series of NAMI-A derivatives have been prepared that could modify important metastasis parameters such as tumor invasion, matrix metalloproteinases (MMP) activity, and cell cycle progression [42]. The new analogs also maintained the potent characteristics of NAMI-A and could selectively interact with solid metastatic tumors. Furthermore, the introduction of different ligands improved their stability in aqueous solutions [76]. Two NAMI-A derivatives bearing a pyridine ligand, Ru9/G26b and Ru10/G94a (Figure 4), displayed little direct cytotoxicity to human (A549) and mouse Lewis lung cancer cells but had a significant suppressive effect on the invasion and migration of cancer cells [51]. Like with NAMI-A, in vivo studies in 4T1 mammary carcinoma-bearing mice showed that the occurrence and development of lung metastasis were suppressed significantly and that no retinal toxicity or hepatotoxicity was found in mice after intraperitoneal injection of Ru9 and Ru10 at a dose of 17.5 mg/kg per day for consecutive 6 days, with three times in a 1-day interval. Ru9 in particular could suppress important molecules involved in metastasis, such as MMP-2 and MMP-9, and the vascular endothelial growth factor [51]. More interestingly, plasma atomic emission spectrometry showed that Ru9 possessed higher metabolic stability due to having a longer Ru-elimination time in the lungs, indicating its better anti-metastatic effect compared to NAMI-A and Ru10 [51]. In an earlier study, the hetero multinuclear complex [Na 2 ] {[RuCl 4 (DMSO-S)(µ-pyz)] 2 PtCl 2 }, AH197 (Ru11/AH197) was also synthesized [77], and its effect on the motility and DNA electrophoretic mobility of NSCLC (A549) and breast cancer (MDA-MB-231) cells was compared to that of [K] [RuCl 4 (DMSO-S)(µ-pyz)Pt(DMSO-S)Cl 2 ] (IT127), NAMI-A, and Na [trans-RuCl 4 (DMSO)(pyz)] (AH403) [78]. The Ru 2 Pt trinuclear species showed higher inhibitory activity in the order Ru11 > IT127 > NAMI-A > AH403 (Figure 4) [78], while it was shown that the inhibition of cell motility might contribute to the anti-metastatic properties of the complexes [79].

Ru(II) Complexes
Ru(II) complexes are also known for their low toxicity, different modes of action, and non-cross resistance to traditional Pt-based drugs, especially cisplatin. Therefore, recent studies have focused on understanding the anticancer mechanism of Ru(II) complexes to develop more effective Ru(II)-based drug candidates.

Ru(II) Complexes
Ru(II) complexes are also known for their low toxicity, different modes of action, and non-cross resistance to traditional Pt-based drugs, especially cisplatin. Therefore, recent studies have focused on understanding the anticancer mechanism of Ru(II) complexes to develop more effective Ru(II)-based drug candidates.

Apoptosis
With air stability, aqueous solubility, and structural diversity, the versatile halfsandwich Ru(II)-η 6 -p-cymene complexes have been reported as potential anticancer drugs, as they show distinct anti-proliferative activity and can effectively induce apoptosis [80]. For instance, [(Ru(η 6 -p-cymene)Cl) 2 (1,3,5-triaza-7-phosphaadamantane)] (RAPTA-C) ( Figure 5) inhibited tumor metastasis and growth by inducing the apoptosis of Ehrlich ascites carcinoma cells through mitochondrial and p53-JNK pathways [81]. Similarly to RAPTA-C, a bimetallic Ru(II) cymene complex, [(Ru(η 6 -p-cymene) Cl) 2 (1,3-bis(2-methyl-6-(pyridin-2-yl) pyrimidin-4-yl) benzene)] (Ru12) (Figure 5), showed strong anticancer activity toward human NSCLC A549 and A427 cancer cells by inhibiting cell proliferation, migration, and invasion, which was stable in solution state in D 2 O/DMSO-d 6 mixture as well as in solid state under air and light [82]. Mitochondria-mediated apoptosis of NSCLC was also observed upon treatment with Ru12, followed by an increase in the apoptosis regulator Bax and caspase-3/-9 activation. Thus, Ru12 induced DNA damage and cell death via caspasedependent apoptosis by activating poly(ADP-ribose) polymerase(PARP) and triggering the p53-dependent pathway [82]. Moreover, Ru12 inhibited cancer cell migration and invasion, which in turn blocked the expression of the c-Myc(myelocytomatosis) oncogene that is important for cell cycle progression, apoptosis, and cellular transformation, and it has been related to cancer metastasis [82,83]. In a current study, another two sets of organometallic arene Ru(II) complexes against cancer cells (NSCLC A549, colon adenocarcinoma LoVo, and hepato cellular carcinoma HuH-7) have been reported by Balaji, S and coworkers. Structurally, the complexes with p-cymene moiety outperformed the anticancer activity of the complexes containing benzene moiety, in that the latter had less hydrophobic interaction with the cell membrane [84].
showed a relatively better anti-proliferative activity compared to the other complexes due to the increased polarization of I − . Further treatment of A549 cells with Ru14 led to concentration-dependent late apoptosis and cell cycle arrest in the G1/G0 phase. In a subsequent study, a hydrazone moiety was rationally incorporated into two tetranuclear arene Ru(II) complexes, Ru19 and Ru20 (Figure 5), to improve their pharmacological activity [86]. With high stability in DMEM containing 10%FBS and PBS with 10%DMSO, both complexes had high anticancer activity in vitro and could induce the apoptosis of various human cancer cell lines, including cisplatin-resistant lung (A549) cancer cells. To certify the systemic toxicity of Ru19, a series of animal experiments were conducted by the group. Impressively, no damage was observed in major organs, including the kidney, in the mice treatment with Ru19 (6 mg/kg), while a mass of vacuolization in the cell cytoplasm of renal tubules were found in the cisplatin-treated mice, suggesting that the compound Ru19 exhibited lower systemic toxicity and was potentially more tolerated by animals than cisplatin [86]. Apart from targeting cisplatin-resistant cancer cells to address chemoresistance issue, Teixeira, R. G et al. proposed a novel approach that organometallic Ru(II) compounds increased cisplatin cytotoxicity up to 1390-fold at nontoxic doses by inhibiting multidrug resistance-associated protein 1 (MRP1) and the P-glycoprotein 1 (Pgp) transporters [87]. It further promoted Ru(II) compounds as more valuable and prospective agents for lung cancer chemotherapy, in particular for those patients with cisplatin resistance. A series of Ru(II) methylimidazole complexes (Ru21-24) ( Figure 6) with strong antiproliferative activity against various human cancer cells were also synthesized [88]. Although Ru12 induced NSCLC apoptosis via the mitochondria-dependent pathway [82], two pathways were involved in the mechanism of action of Ru21. Specifically, Ru21 activated the BID protein and depleted the mitochondrial membrane potential in A549 cells by regulating the expression of pro-survival and pro-apoptotic Bcl-2 family proteins [88]. Recently, six new bimetallic Ru(II) arene complexes [Ru 2 (η 6 -p-cymene) 2 (1,3-bib) 2 Cl 2 ]X 2 (X = Cl − (Ru13), I − (Ru14), NO 3 − (Ru15), BF 4 − (Ru16), PF 6 − (Ru17), and CF 3 SO 3 − (Ru18); 1,3-bib = 1,3-di(1H-imidazol-1-yl) benzene) were designed and synthesized [85]. Ru13-18 ( Figure 5) performed good stability in aqueous solution, and UV-Vis spectra also suggested that bidentate imidazole-based ligand strengthens the stability of the Ru-arene complex, comparing to mononuclear N-heterocyclic ligand. All showed moderate proliferative or anti-proliferative activity due to their interaction with glutathione on lung (A549) cancer cells, but strong intercalative binding ability to ctDNA. Moreover, Ru14 showed a relatively better anti-proliferative activity compared to the other complexes due to the increased polarization of I − . Further treatment of A549 cells with Ru14 led to concentration-dependent late apoptosis and cell cycle arrest in the G1/G0 phase. In a subsequent study, a hydrazone moiety was rationally incorporated into two tetranuclear arene Ru(II) complexes, Ru19 and Ru20 (Figure 5), to improve their pharmacological activity [86]. With high stability in DMEM containing 10%FBS and PBS with 10%DMSO, both complexes had high anticancer activity in vitro and could induce the apoptosis of various human cancer cell lines, including cisplatin-resistant lung (A549) cancer cells. To certify the systemic toxicity of Ru19, a series of animal experiments were conducted by the group. Impressively, no damage was observed in major organs, including the kidney, in the mice treatment with Ru19 (6 mg/kg), while a mass of vacuolization in the cell cytoplasm of renal tubules were found in the cisplatin-treated mice, suggesting that the compound Ru19 exhibited lower systemic toxicity and was potentially more tolerated by animals than cisplatin [86]. Apart from targeting cisplatin-resistant cancer cells to address chemoresistance issue, Teixeira, R. G et al. proposed a novel approach that organometallic Ru(II) compounds increased cisplatin cytotoxicity up to 1390-fold at nontoxic doses by inhibiting multidrug resistance-associated protein 1 (MRP1) and the P-glycoprotein 1 (Pgp) transporters [87]. It further promoted Ru(II) compounds as more valuable and prospective agents for lung cancer chemotherapy, in particular for those patients with cisplatin resistance.
A series of Ru(II) methylimidazole complexes (Ru21-24) ( Figure 6) with strong antiproliferative activity against various human cancer cells were also synthesized [88]. Although Ru12 induced NSCLC apoptosis via the mitochondria-dependent pathway [82], two pathways were involved in the mechanism of action of Ru21. Specifically, Ru21 activated the BID protein and depleted the mitochondrial membrane potential in A549 cells by regulating the expression of pro-survival and pro-apoptotic Bcl-2 family proteins [88]. Moreover, the Ru(II) polypyridyl complexes Ru25 and Ru28 ( Figure 6) showed completely different mechanisms [52]. Ru25 induced marginal oxidative stress and preferably accumulated in lysosomes, triggering apoptosis via an intrinsic mitochondrial pathway, while enlarged mitochondria were detected in Ru25-treated A549 cells. In contrast, Ru28, which was mainly localized in mitochondria and endoplasmic reticulum, did not have the same effect and induced caspase-independent apoptosis [52]. Another study investigated the mechanism of action of Ru complexes, showing that Ru36 can lead to significant ROS production and induce apoptosis, while increasing the BAX/BCL-2 ratio and PERK levels without affecting the expression of caspase-3 [57].  Figure 7) were also prepared in the same study, and their effect on NSCLC (A549) and triple-negative breast (MDA-MB-231) cancer cells were investigated [57]. The increase in lipophilicity as a strategy to enhance the cellular uptake and the antiproliferative activity of the ruthenium complexes on cancer cells is important [96,97]. Among them, Ru36 was the only lipophilic ROS are generated under normal cell activity and are involved in cellular signaling. However, high ROS levels produced mainly by mitochondrial dysfunction may lead to oxidative damage of cellular structures such as DNA damage and apoptosis [89,90]. Therefore, agents modulating ROS generation have been designed for clinical cancer therapy. For instance, Ru30 [54], Ru31-32 [55], Ru33 [56], Ru36 [57], and Ru38 [58] ( Figures 6 and 7 [91,92] and selectively targeted lung target tumor cells [54], while no toxic effect was observed on normal bronchial epithelial BEAS-2B cells. In addition, the increased ROS levels generated by 3.8 µM Ru30 in A549 lung tumor cells caused oxidative stress, which led to proliferation inhibition, changes in the morphology and organization patterns of the actin cytoskeleton, G2/M phase arrest, apoptosis, changes in the mitochondrial membrane potential, and DNA damage [54]. Ru31 [Ru(dip) 2 (SA)] and Ru32 [Ru(dmp) 2 (SA)] (dip = 4, 7-diphenyl-1,10-phenanthroline; dmp = 2,9-dimethyl-1,10-phenanthroline; SA = salicylate), two Ru(II) complexes bearing O,O-chelated ligands with low toxicity to BEAS-2B, could also induce apoptosis in A549 cells via caspase family proteins and PARP activation, ROS accumulation, DNA damage, MMP reduction, and Cytochrome c release from mitochondria [55]. Significant in vitro anticancer activity, combined with proven solution stability and hydrophobic property, prompted the group to further conduct in vivo experiments to test the toxicity [55]. Accordingly, developing zebrafish embryos were used, incubating with various concentrations (0, 12.5, 25, 50, 100, and 200 µM) of Ru31 in water. Although lower cumulative hatch rate and increased lethality rate, even unhealthy features such as pericardial cysts and spine curvature were notably observed with treatment concentrations up to 200 µM, but there were no apparent side effects found in zebrafish embryos after treatment with concentrations from 12.5 to 50 µM of Ru31. Combined with low toxicity towards both normal BEAS-2B cells in vitro and zebrafish embryos in vivo implied that Ru31 has great potential to develop as a promising therapeutic agent against lung cancer with safety profiles [55].

Autophagy
Unlike apoptosis, autophagy is an evolutionarily conserved degradation pathway for the recycling of cytoplasmic components and plays a significant role in response to metabolic and therapeutic stresses [98,99]. Recent studies presented scientific evidence supporting the participation of autophagy in tumor progression, such as the development of multidrug resistance of cancer cells, but autophagy also can help in killing cancer cells that are resistant to anticancer agents as a potential target for cancer therapy [99,100]. Caspase-related family proteins are key components of induction and transduction of apoptotic signaling in cells and are closely related to BCL-2 family proteins that either induce (pro-apoptotic) or inhibit (anti-apoptotic) apoptosis [93,94]. Ru31 downregulated the expression levels of the anti-apoptotic proteins BCL-2 and BCL-xL, whereas it upregulated the levels of the pro-apoptotic proteins BAX and BAD [55]. Since the high expression levels of BCL-2 and BCL-xL have been associated with cisplatin resistance and tumor recurrence in NSCLC patients [95], Ru31 may indirectly inhibit the resistance and relapse of lung cancer. [Ru(dmp) 2 (pddppn)](ClO 4 ) 2 (pddppn = phenantheno [1,2- [3,2-a:2 ,3 -c]phenazine) (Ru33) could also downregulate the expression of BCL-2, BCL-x, BAK, and BIM, while upregulating the expression of BAG-1 and BAD, thus inducing apoptosis of NSCLC A549 cells via an intrinsic ROS-mediated mitochondrial dysfunction pathway [56]. Moreover, Ru33 effectively inhibited the growth of BEL-7402 (human hepatocellular cell line), HeLa (human cervical cancer cell line), MG-63 (human osteosarcoma cell line), and A549 cancer cells with IC 50 values of 1.6 ± 0.4, 9.0 ± 0.8, 1.5 ± 0.2, and 1.5 ± 0.3 µM, respectively [56].

Autophagy
Unlike apoptosis, autophagy is an evolutionarily conserved degradation pathway for the recycling of cytoplasmic components and plays a significant role in response to metabolic and therapeutic stresses [98,99]. Recent studies presented scientific evidence supporting the participation of autophagy in tumor progression, such as the development of multidrug resistance of cancer cells, but autophagy also can help in killing cancer cells that are resistant to anticancer agents as a potential target for cancer therapy [99,100].
To the best of our knowledge, there are only a few studies on the inhibition of cell growth caused by metal complexes through autophagy induction. For instance, a Ptbased complex, [Pt(O,O'-acac)(γ-acac)(DMS)], induced autophagy in Caki-1 renal cancer cells [101] and rat B50 neuroblastoma [102]. Apart from Pt-based agents, Piccolo M et al. [103] and Irace C et al. [104] have certified that the activation of autophagic pathways is a common feature with Ru(III) complexes against breast cancer cells. These findings could pave the way for the development of other autophagy-inducing metal complexes to overcome apoptosis-resistant cancer cells. In 2016, the mechanism of Ru(II) complex targeting lung cancer cells by autophagy induction was reported by Chen, L et al. for the first time [59]. Specifically, the formation of autophagosomes and acidic vesicular organelles along with LC3-II upregulation was observed in A549 and NCI-H460 cancer cells treated with a Ru(II) imidazole complex, [Ru(Im) 4 (dppz)] 2+ (dppz = pyrido [3.2a:2 ,3 -c]phenazine) (Ru39) (Figure 7). Ru39 caused mitochondrial dysfunction and ROS generation in A549 cells, thus partially inducing caspase-3-dependent apoptosis, which causes cell death, as well as autophagy mediated by the extracellular signal-regulated kinase (ERK) signaling pathway. Eventually, the accumulation of Ru39 in mitochondria could induce autophagy and compete with mitochondria-mediated apoptosis [59]. Fur-thermore, the antitumor activity of Ru39 was further evaluated in vivo mice bearing A549 xenografts, which were treated with Ru39 at 10 or 20 mg/kg. After 28 days, it was observed that the weight and volume of tumors were significantly reduced, and the expressions of LC3-II, cleaved caspase-3, CD-31, and Ki-67 were up-regulated via immunohistochemical analysis. Accordingly, Ru39 could inhibit tumor growth very well in vitro and in vivo and develop as a promising anticancer candidate promoting cell death via both apoptosis and autophagy pathway [59]. Resembled Ru-based complexes were also highly active against glioblastoma cell lines, inducing cell death via apoptosis and autophagy in a p53-independent manner [105].

Necroptosis
Promoting apoptosis is one of the main methods to treat tumors. However, the anti-apoptotic properties may lead to resistance to cytotoxic chemotherapeutic drugs or radiotherapy [106]. Necroptosis is another type of regulated cell death closely related to the receptor-interacting protein kinases 1 and 3 (RIPK1 and RIPK3) and mixed-lineage kinase domain-like protein (MLKL) [107]. Necroptosis begins with the activation of RIPK and MLKL, which increase the levels of Ca 2+ , causing lysosomal membrane permeabilization, and release cathepsins into the cytosol [108]. Although relevant studies are scarce, a recent report has shown that inducing necroptosis in cancer cells can overcome chemotherapy failure due to apoptotic resistance [109], while metal complexes have been shown to induce cancer cell death through necroptosis [110,111].
A series of Ru(II) complexes bearing 1,1-(pyrazin-2-yl) pyreno [4,5- (Figure 8) [60]. All analogs showed significant antitumor activity against drug-resistant cancer cells, including A549 lung cancer cells (IC 50 (Ru46) = 3.0 ± 0.1 µM). More importantly, Ru40-46 prevented the DNA binding of topoisomerases (topo) I and II, which are crucial nuclear enzymes that regulate DNA replication, transcription, recombination, and chromosome segregation during mitosis [60,112]. The characteristic indicators of necroptosis, including ROS burst, plasma membrane permeabilization, and cytosolic ATP reduction, were also reported, while the cell signaling pathway from topo I and II inhibition, DNA damage, and PARP1 activation to necroptosis induced by the activation of RIPK1, RIPK3, and MLKL were elucidated [60]. In contrast, the group also reported a collection of Ru(II) complexes containing asymmetric tridentate ligands that could induce DNA damage and cancer cell death through apoptosis, which were also topo I and II inhibitors [113]. Moreover, caspase-8 has been proven to regulate the switch from apoptosis to necroptosis [114].
Molecules 2021, 26, x FOR PEER REVIEW 13 of 21 CD-31, and Ki-67 were up-regulated via immunohistochemical analysis. Accordingly, Ru39 could inhibit tumor growth very well in vitro and in vivo and develop as a promising anticancer candidate promoting cell death via both apoptosis and autophagy pathway [59]. Resembled Ru-based complexes were also highly active against glioblastoma cell lines, inducing cell death via apoptosis and autophagy in a p53-independent manner [105].

Necroptosis
Promoting apoptosis is one of the main methods to treat tumors. However, the antiapoptotic properties may lead to resistance to cytotoxic chemotherapeutic drugs or radiotherapy [106]. Necroptosis is another type of regulated cell death closely related to the receptor-interacting protein kinases 1 and 3 (RIPK1 and RIPK3) and mixed-lineage kinase domain-like protein (MLKL) [107]. Necroptosis begins with the activation of RIPK and MLKL, which increase the levels of Ca 2+ , causing lysosomal membrane permeabilization, and release cathepsins into the cytosol [108]. Although relevant studies are scarce, a recent report has shown that inducing necroptosis in cancer cells can overcome chemotherapy failure due to apoptotic resistance [109], while metal complexes have been shown to induce cancer cell death through necroptosis [110,111].
A series of Ru(II) complexes bearing 1,1-(pyrazin-2-yl) pyreno [4,5- (Figure 8) [60]. All analogs showed significant antitumor activity against drug-resistant cancer cells, including A549 lung cancer cells (IC50 (Ru46) = 3.0 ± 0.1 μM). More importantly, Ru40-46 prevented the DNA binding of topoisomerases (topo) I and II, which are crucial nuclear enzymes that regulate DNA replication, transcription, recombination, and chromosome segregation during mitosis [60,112]. The characteristic indicators of necroptosis, including ROS burst, plasma membrane permeabilization, and cytosolic ATP reduction, were also reported, while the cell signaling pathway from topo I and II inhibition, DNA damage, and PARP1 activation to necroptosis induced by the activation of RIPK1, RIPK3, and MLKL were elucidated [60]. In contrast, the group also reported a collection of Ru(II) complexes containing asymmetric tridentate ligands that could induce DNA damage and cancer cell death through apoptosis, which were also topo I and II inhibitors [113]. Moreover, caspase-8 has been proven to regulate the switch from apoptosis to necroptosis [114].

Anti-Metastasis
Ru(II) compounds can not only suppress the primary tumor but also effectively inhibit malignant tumor metastasis. A series of recently synthesized Ru(II)-containing polypyridyl ligand complexes (Ru25-29 ( Figure 6) [53] and Ru47 (Figure 9) [61]) showed anti-metastatic properties, which was attributed to their targeting ability to MMPs [53]. Ru25-29 bearing 2,2 -bipyridine substituted with a semicarbazone-2-formylopyridine moiety as one of the ligands and 4,4 -di-tert-butyl-2,2 -dipyridyl or 4,7-diphenyl-1,10-phenanthroline as auxiliary ligands have been studied for their effect on the adhesion properties of human A549 and pancreatic cancer cells [53]. All complexes enhanced the cell adherent properties and could directly inhibit the activity of MMP2 and MMP9 enzymes in vitro. Among them, Ru28 led to the most significant enhancement of cell adhesion with increasing concentration [53]. THBS1, and ITB1) and protein-lysine 6-oxidase, while increasing the expression of the extracellular matrix inhibitor [61]. Ru47 was also studied in hypoxia, as hypoxia results from poor vascular organization of the tumor, promoting drug resistance and malignant progression [116,117]. The results suggested that Ru47 was more cytotoxic against human breast (4T1) and lung (A549) cancer cells than cisplatin, while it induced cell apoptosis and affected endothelial cell vasculature by activating oxidative stress and modulating the mRNA expression profile of ICAM-1 and VCAM-1 genes involved in metastasis and angiogenesis [61].
Two other complexes, Ru48/Ru-hq1 [η6-p-cymene) Ru(5-bromo-8-hydroxyquinolinato) Cl] and Ru49/Ru-hq2 [(η6-p-cymene) Ru (k2-O,N-5,7-dibromo-HyQ) Cl] (Figure 9), have also been screened as potential novel agents for bone, lung, and breast cancer chemotherapy [62]. Both complexes are stable in DMSO and DMEM solution within the time-frame of the biological experiments and attenuated cell viability with greater selectivity and specificity than cisplatin, while they inhibited cell proliferation, migration, and invasion on cell monolayers at lower concentrations (2.5-10 µM). The higher inhibitory effect of Ru48/Ru-hq1 and Ru49/Ru-hq2 on cell invasion compared to cisplatin was further confirm using 3D multicellular spheroid models [62]. Similar results were obtained for Ru19, which inhibited cancer cell migration and invasion and had higher safety margins for animals than cisplatin [86]. Therefore, Ru48-49 and Ru19 may be used as cisplatin alternatives to manage lung cancer and inhibit tumor metastasis.

Cell Cycle Arrest
The cell cycle, which involves four main phases (G1, S, G2, and M), contributes significantly to the regulation of cell self-renewal and differentiation, while cyclin-dependent kinases (CDKs) and cycle proteins play a key role in the process [118]. Therefore, clinical trials have been performed recently to explore the synergistic effect of CDK inhibitors and different anticancer drugs on various cancer types [119,120]. Additional studies have shown that metallodrugs can target CDKs and cycle proteins to block the cell cycle in different phases. For instance, Ru(II), Rh(III), Mn(II), and Zn(II) complexes blocked the cell cycle in the S-phase, thus inhibiting cell proliferation by reducing the levels of cyclins A2/B1/D1/E1, CDK-2/6, and PCNA and increasing the levels of p21, p27, p53, and CDC25A [121].
Given that the dysregulation of the cell cycle is associated with the development of cancer, novel Ru(II)-based drugs that inhibited tumor proliferation were synthesized and identified to disrupt the cell cycle in the G0/G1, G1/S, S, and G2/M phases ( Figure 10). Ru12 [82], Ru14 [85], Ru33 [56], Ru28 [52], and Ru38 [58] are typical examples of compounds causing cell death via cell cycle arrest in the G0/G1 phase. Investigation of the mechanism of action revealed that Ru12 could induce cell cycle arrest in the G0/G1 phase; suppress the expression of cell-cycle-regulatory proteins, such as cyclins D1, A1, and B1, upregulate p53, p21, and p15; and cleave PARP [82]. Interestingly, the anti-proliferative complex Ru33 exhibited different mechanisms against different cancer cells: G2/M phase arrest in BEL-7402 and MG-63 cells and G0/G1 phase arrest in A549 cells [56]. In addition, Ru28 promoted cell accumulation in the G0/G1 phase, while the number of Ru25-treated A549 cells increased remarkably in the S-phase but decreased in the G2/M phase [52]. Furthermore, the Ru(II) methylimidazole complex Ru21 inhibited the growth of A549 lung cells by inducing apoptotic cell death, as confirmed by the generation of a significant apoptosis peak in the sub-G1 phase [88].

Cell Cycle Arrest
The cell cycle, which involves four main phases (G1, S, G2, and M), contributes significantly to the regulation of cell self-renewal and differentiation, while cyclin-dependent kinases (CDKs) and cycle proteins play a key role in the process [118]. Therefore, clinical trials have been performed recently to explore the synergistic effect of CDK inhibitors and different anticancer drugs on various cancer types [119,120]. Additional studies have shown that metallodrugs can target CDKs and cycle proteins to block the cell cycle in different phases. For instance, Ru(II), Rh(III), Mn(II), and Zn(II) complexes blocked the cell cycle in the S-phase, thus inhibiting cell proliferation by reducing the levels of cyclins A2/B1/D1/E1, CDK-2/6, and PCNA and increasing the levels of p21, p27, p53, and CDC25A [121].
Given that the dysregulation of the cell cycle is associated with the development of cancer, novel Ru(II)-based drugs that inhibited tumor proliferation were synthesized and identified to disrupt the cell cycle in the G0/G1, G1/S, S, and G2/M phases ( Figure 10). Ru12 [82], Ru14 [85], Ru33 [56], Ru28 [52], and Ru38 [58] are typical examples of compounds causing cell death via cell cycle arrest in the G0/G1 phase. Investigation of the mechanism of action revealed that Ru12 could induce cell cycle arrest in the G0/G1 phase; suppress the expression of cell-cycle-regulatory proteins, such as cyclins D1, A1, and B1, upregulate p53, p21, and p15; and cleave PARP [82]. Interestingly, the anti-proliferative complex Ru33 exhibited different mechanisms against different cancer cells: G2/M phase arrest in BEL-7402 and MG-63 cells and G0/G1 phase arrest in A549 cells [56]. In addition, Ru28 promoted cell accumulation in the G0/G1 phase, while the number of Ru25-treated A549 cells increased remarkably in the S-phase but decreased in the G2/M phase [52]. Furthermore, the Ru(II) methylimidazole complex Ru21 inhibited the growth of A549 lung cells by inducing apoptotic cell death, as confirmed by the generation of a significant apoptosis peak in the sub-G1 phase [88].

Conclusions/Discussions
Lung cancer remains a life-threatening malignancy due to poor prognosis and drug resistance, which is one of the most severe challenges that still need to be addressed to improve patients' prognosis and survival rate. However, specific drugs preventing tumor metastasis and recurrence have not yet been efficiently developed. In order to design and synthesize effective anticancer agents, transition metal-based compounds have gradually evolved as promising drug candidates due to their cytotoxicity and ability to prevent drug resistance in tumor cells. Among them, Ru(II/III)-based compounds proved to be the most effective, as they show high cytotoxicity, which induces apoptosis, necroptosis, or autophagy and cell cycle arrest, thus inhibiting cell proliferation, invasion, and metastasis (Figures 2 and 10). Most studies suggested that Ruthenium complexes were low in toxicity, easily absorbed, and excreted quickly. More importantly, Ruthenium complexes were easily absorbed by tumor tissues.
In this review, we summarized the recent developments of Ru(II) and Ru(III) complexes and discussed their biological activity and mechanism of action against lung cancer. The existing findings clearly support that Ru complexes can be used to develop effective chemotherapeutic agents for human lung cancer, while they may serve as a guide for the design of other metallodrugs with higher efficiency and better clinical application potential. Nevertheless, the anticancer mechanisms of Ru(II/III) complexes require further investigation, and their efficiency against other cancer types should also be explored in future studies.