Dithiocarbazate–Zn(II) complexes for photodynamic therapy and chemotherapy against lung cancer

Abstract

Combining photodynamic therapy with chemotherapy is a promising strategy for treating tumors. Herein, a series of new dithiocarbazate–Zn(II) complexes, including [Zn^II(L1)₂] Zn1, [Zn^II(L2)₂] Zn2, [Zn^II(L3)₂] Zn3, and [Zn^II(L4)₂] Zn4, have been synthesized for chemotherapy and photodynamic therapy of lung cancer. These complexes can be used for live-cell fluorescence-based imaging, are photostable, and exhibit hemolysis comparable to cisplatin. Among them, complex Zn3 shows good chemotherapeutic ability, and its chemotherapeutic mechanism relies on multiple pathways. At the same time, complex Zn3 generates superoxide anion radicals (˙O₂⁻) under light irradiation, leading to cancer cell death through photodynamic therapy. Its remarkable anticancer effect is achieved through multimodal photodynamic therapy/chemotherapy. This study paves the way for the use of novel Zn(II) complexes as anticancer agents, providing new strategies for multimodal cancer therapy.

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Introduction

Lung cancer is a highly prevalent type of cancer and a significant barrier to increasing life expectancy in every country in the world.^1,2 According to estimates from the GLOBOCAN in 2020, lung cancer is the leading cause of cancer death (18.0%) and the second most frequently diagnosed cancer (11.4%) in the world, with nearly 1.8 million deaths and 2.2 million new cancer cases annually.³ Globally, the cancer burden is projected to double by 2050, led by lung cancer.⁴ Moreover, the 5-year relative survival rate for lung cancer patients after diagnosis is only 10%–20% in most countries.^3,5 Various therapies against lung cancer including surgery, radiotherapy, chemotherapy, photodynamic therapy, and immunotherapy have been developed. However, because of the high heterogeneity of lung cancer, the efficacy of monotherapy is often limited.^6,7 Therefore, multimodal therapies that combine different therapeutic approaches have attracted considerable interest; they utilize different therapeutic mechanisms that can act synergistically, resulting in higher therapeutic efficiency.^8,9 In particular, the combination of photodynamic therapy and chemotherapy is promising for the treatment of lung cancer.^10–14

Photodynamic therapy is a minimally invasive treatment modality with few side effects.¹⁵ In photodynamic therapy, photosensitizers are accumulated in tumors and generate reactive oxygen species (ROS) upon irradiation with light, thereby inducing tumor regression.¹⁶ After photodynamic therapy, a series of therapeutic effects occur, such as damage or permeabilization of the tumor vasculature, tumor cell apoptosis, tumor cell necrosis, and immune system response activation to induce tumor regression.¹⁷ Moreover, photodynamic therapy can be repeated on the same site without a decrease in efficacy.¹⁸ However, photodynamic therapy as a monotherapy often results in incomplete tumor killing because of limited light penetration in tissues, disease recurrence caused by cells that survived photodynamic therapy, tumor hypoxia, and tumor migration.^14,19,20 Drug chemotherapy can effectively eradicate tumors, but often causes side effects such as drug resistance and toxicity, thereby severely reducing the quality of life of patients.^21,22 Several studies demonstrated that the combination of photodynamic therapy with chemotherapy shows multifold advantages: (1) photodynamic therapy can be used to treat the tumor tissue locally with minimal damage to adjacent tissues, whereas chemotherapy can eliminate surviving tumor cells, especially in deep or large tumors that are difficult to penetrate with lasers; (2) photodynamic therapy reduces tumor solid pressure, reduces extravascular barriers, and permeabilizes tumor vessels, which may enhance the accumulation of chemotherapeutic drugs in the tumor, thereby improving the therapeutic effect; (3) the ROS generated during photodynamic therapy can directly damage lysosomes and endosomes to promote the delivery of intracellular drugs to targets such as mitochondria, nucleus, and microtubules; (4) DNA damage from combined photodynamic therapy and chemotherapy cannot be easily repaired by drug-resistant DNA-repairing enzymes.^22–28 To date, many studies have attempted to realize the synergistic chemo-photodynamic therapy through the chemical linking or nanoloading of photosensitizers and anticancer drugs together to obtain higher therapeutic effects.^{14,21,29–40} However, chemical linking often relies on the use of additional toxic chemicals. Nanoloading generally requires complex operations and often causes toxicity problems because of the accumulation of nanomaterials in vivo. Therefore, designing and developing a molecule with both chemotherapeutic and photodynamic effects seems a promising strategy for tumor therapy.

Recently, Zn(II)-based complexes have attracted attention owing to their photophysical properties and chemotherapeutic effects.^41–46 Dithiocarbazinates and their derivatives, as a class of Schiff base ligands with multiple pharmaceutical and biological activities, can coordinate with a variety of metal ions to form versatile metal complexes.⁴⁷ Herein, we propose a series of dithiocarbazate–Zn(II) complexes (Scheme 1) as potential photodynamic therapy and chemotherapy agents for lung cancer treatment. Porphyrins, phthalocyanines, and chlorins constitute the basic structures of many photodynamic therapeutic agents, which are not only difficult to synthesize and purify but also suffer from other limitations such as aggregation and poor stability in biological fluids.⁴⁶ In this work, dithiocarbazate–Zn(II) complexes were easily synthesized and purified, showing good water stability and low hemolysis. Among them, complex Zn3 exhibited good chemotherapeutic and photodynamic activities. The mechanisms of the photodynamic and chemotherapeutic effects of complex Zn3 were investigated using various methods. The mechanism of photodynamic action can be mainly divided into two types: type I and type II.⁴⁸ In the type II process, O₂ obtains energy directly from the excited photosensitizer, resulting in the formation of ROS. For the type I process, ROS are obtained by direct transfer of hydrogen atoms or electrons when the photosensitizer interacts with the substrate, thus avoiding the large consumption of oxygen.⁴⁹ Thus, type I photoreactivity may lead to strong cytotoxic effects under low-oxygen conditions, which is beneficial for overcoming tumor hypoxia.^49–51 However, hypoxic-active type I photosensitizers remain an underexplored research area and are still rarely reported.⁵² Interestingly, complex Zn3 can cause cellular damage by exploiting type I processes. In addition, studies on the chemotherapeutic mechanism of action of complex Zn3 revealed that Zn3 suppresses cancer cells by triggering multiple mechanisms. Our results provide new insights into the development and utilization of Zn(II)-based complexes and the treatment of lung cancer.

Scheme 1 Synthesis of complexes Zn1–Zn4.

Results and discussion

Characterization of the Zn(II) complexes

The dithiocarbazate Schiff-base ligands HL1–HL4 and complex Zn1 were synthesized using the previously reported methods.^53–56 Complexes Zn2–Zn4 were prepared by reacting 1 equivalent of Zn(II) with 2 equivalents of ligands in CH₃OH. All Zn(II) complexes were characterized using X-ray crystallography. The obtained crystallographic information is summarized in Tables S1 and S2.†

X-ray crystallography shows that these Zn(II) complexes have similar coordination geometries but exhibit two distinct crystal systems, triclinic (Zn1) and monoclinic (Zn2–Zn4). As shown in Fig. 1, all Zn(II) complexes are in mononuclear six-coordination form, showing distorted octahedral coordination geometry. One S atom, one imine N atom, and one pyridine N atom from one ligand and one imine N atom from another ligand occupy the basal coordination sites. The coordination sites at the axial positions are occupied by one S atom and one pyridine N atom from the second ligand. Similar Zn(II)-based complexes have been previously reported; in these complexes, both ligands can both be deprotonated (thiol form) at the thioamide N atom or both in the neutral thione form.^43,57–60 Herein, two Schiff-base ligands were coordinated in their deprotonated form via the S/N/N donor atoms, forming four 5-membered bichelate rings around the Zn(II) center. The Zn–S, Zn–N_imine, and Zn–N_pyridine bond lengths and the angles with Zn atom as the vertex are within the range reported for other similar Zn(II) complexes.^61–65 In general, average Zn–N_imine bond lengths (2.113(2)–2.128(2) Å) are lower than the Zn–N_pyridine bond lengths (2.212(2)–2.376(2) Å).

Fig. 1 ORTEP views of the complexes Zn1–Zn4 with displacement ellipsoids drawn at the 30% probability level. All H atoms are omitted for clarity.

Although these Zn(II) complexes have similar structures, only Zn1, Zn3, and Zn4 complexes show significant π⋯π interactions in the solid state. As presented in Fig. S1,† complex Zn1 is linked into a dimeric structure by π⋯π interactions between pyridine rings, with an angle of 0°, shift distance of 1.672 Å, and centroid–centroid distance of 3.854 Å. Adjacent monomer units in complex Zn3 are linked into a 1D zigzag chain by π⋯π interactions between the pyridine rings, with an angle of 171.201°, shift distance of 1.741 Å, and centroid–centroid distance of 3.793 Å (Fig. S2†). In complex Zn4, the adjacent monomer units are linked into a dimeric structure by π⋯π stacking between the aromatic rings, with the angle of 0°, shift distance of 1.498 Å, and centroid–centroid distance of 3.737 Å (Fig. S3†).

To determine the nature of the aforementioned π⋯π stacking interactions in these complexes, energy decomposition analysis based on the classical molecular force field (EDA-FF) was performed. The EDA-FF method can decompose the weak interactions of these system into repulsion interactions, electrostatic interactions and dispersion interactions. As depicted in Table S3,† the EDA-FF results show the same trend, that is, the dispersion interaction accounted for approximately 60%, and the electrostatic interaction accounted for the least (about 15%). This indicates that the attractive dispersion interactions dominate in whole π⋯π stacking interactions.

Photophysical properties

The UV-vis absorption spectra of complexes Zn1–Zn4 in methanol were obtained (Fig. 2A). The bands at 300–500 nm were primarily assigned to spin-allowed ligand-centered n → π* and/or π → π* transitions. To gain insight into the properties of charge transfer between Zn and the ligands in the UV-vis absorption spectra, the time-dependent density functional theory (TD-DFT) calculations were also performed for these Zn(II) complexes. Based on the calculation results (Table S4†), the relatively bands at 300–500 nm were primarily attributed to ligand-to-ligand charge-transfer transitions, followed by a minor contribution from Zn-to-ligand charge-transfer transitions.

Fig. 2 (A) Absorption spectra of complexes Zn1–Zn4 (30 μM) measured in methanol. (B) The photostability of complexes Zn1–Zn4 (30 μM) under continuous LED irradiation at 470 nm. (C) Fluorescence spectra of complexes Zn1–Zn4 (10 μM) measured in methanol followed by excitation at 470 nm.

The photostability of complexes Zn1–Zn4 was examined by monitoring the UV-vis absorption spectra of their methanol solutions under continuous LED light irradiation at 470 nm for 30 min. No significant changes are observed in the absorption properties of all complexes throughout the experiment (Fig. 2B), demonstrating their photostability.

Upon excitation at 470 nm, complexes Zn1–Zn4 exhibit green emission (Fig. 2C). The emission lifetimes of complexes Zn1–Zn4 fall in the range of 2–5 ns, indicating the fluorescent nature of the emission. These complexes exhibit fluorescence in the green region of the visible spectrum, suggesting that they may be applied for cellular fluorescence imaging. Next, the fluorescence imaging properties of complexes Zn1–Zn4 in living lung cancer A549 cells were investigated using confocal microscopy. The cells were incubated with the complexes (10 μM) for various times. As shown in Fig. 3, these Zn(II) complexes are efficiently taken up by the cells and can be used for cellular imaging because fluorescent signals are clearly observed in living cells. It was previously reported that the position of an Ir complex luminescence does not change over time in the cell (luminescence is always observed throughout the cell).⁶⁶ Interestingly, our results show that these Zn(II) complexes primarily accumulated in the cytoplasm within the first 60 min and partly migrated to the nucleus during further incubation (120 min).

Fig. 3 Confocal fluorescence images of living A549 cells incubated with complexes Zn1–Zn4 (10 μM) in real time (15, 30, 60, 120 min); λ_ex = 488 nm, λ_em = 520–570 nm. Scale bar: 100 μm.

Hemocompatibility assay

The interaction between human blood components, especially human erythrocytes, and drugs is an important phenomenon.⁶¹ The effects of these Zn(II) complexes on blood were hence assessed through a hemolysis assay using human red blood cells. The hemolytic behavior of these Zn(II) complexes was studied at different concentrations up to 40 μM using Triton X-100 as a positive control.⁶⁷ According to ISO/Technical Report 7406, the allowable level of hemolysis of biological materials needs to be less than 5%. All Zn(II) complexes exhibited negligible red hemoglobin release (% hemolysis <5%) in a concentration range of 0–40 μM (Fig. S4†), indicating excellent blood compatibility. The hemolytic activity of cisplatin, a commonly used clinical metal-based anticancer drug, was also used as a reference. All Zn(II) complexes showed hemolysis comparable to cisplatin.

In vitro cytotoxicity

To study the efficacy of the Zn(II) Zn1–Zn4 complexes as anticancer agents, their cytotoxicity toward human lung carcinoma cells (A549, A549/DDP, and Calu-1) in the dark and upon irradiation (photocytotoxicity) at 470 nm (15 min, 20 mW cm⁻²) was investigated. Complexes Zn1, Zn3, and Zn4 cause cell death in all investigated lung cancer cell lines with IC₅₀ values in the range of 4.55–16.78 μM in the dark. Interestingly, upon exposure to light, all complexes, especially complexes Zn2 and Zn3, show higher anticancer activity (Table 1), indicating that these complexes are highly phototoxic.

Table 1 IC₅₀ values (μM) of the Zn(II) complexes for different lung cancer cell lines at 48 h

Complex		Cell growth inhibition, IC50 ± SD (μM)
		A549	A549/DDP	Calu-1
Zn1	Dark	13.36 ± 1.47	13.98 ± 0.74	16.42 ± 0.81
	Light	5.12 ± 0.31	5.87 ± 0.57	9.07 ± 1.79
Zn2	Dark	>20	>20	>20
	Light	0.36 ± 0.11	0.29 ± 0.04	0.24 ± 0.05
Zn3	Dark	4.55 ± 0.75	5.57 ± 0.41	8.63 ± 0.71
	Light	0.26 ± 0.05	0.25 ± 0.01	0.22 ± 0.03
Zn4	Dark	14.66 ± 1.23	15.06 ± 1.18	16.78 ± 1.48
	Light	7.63 ± 0.56	7.44 ± 0.38	9.48 ± 0.87

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Having confirmed a close relationship between ROS and phototoxicity, we investigated ROS generation inside the A549 cells after irradiation. Cells treated with complexes Zn1–Zn4 and a ROS probe in the dark showed little fluorescence. In contrast, a remarkable increase in ROS production (strong red fluorescence) occurred when light irradiation followed the treatment with these Zn(II) complexes (Fig. S5†), which confirms that these Zn(II) complexes are promising photodynamic therapy agents because ROS generation is the main mechanism of photodynamic therapy-induced cell death.⁶⁸ Overall, these results show that complexes Zn1, Zn3, and Zn4 can be used for both chemotherapy and photodynamic therapy of tumors. Tumor recurrence from surviving cells after photodynamic therapy is a significant concern, which is partly due to the limited diffusion distance and short half-life of ROS in cells.^14,18,69 Chemotherapy can eliminate surviving tumor cells after photodynamic therapy, thereby reducing the possibility of cancer recurrence.²² At the same time, photodynamic therapy has shown the potential to enhance the efficacy of chemotherapy.²⁴ Thus, agents with both photodynamic and chemotherapeutic properties can ablate cancer cells more effectively.

Zn3 in 3D multicellular tumor spheroids

After evaluating the cytotoxicity on cell monolayers, the cytotoxic effect of complex Zn3 with good chemotherapeutic and photodynamic therapy capabilities was investigated in 3D multicellular tumor spheroids. Notably, 3D tumor spheroids are closer than monolayer cell models to tumor models for clinical treatment; thus, they are a widely used tissue model for assessing drug delivery.^70,71 3D tumor spheroids can also replicate certain pathological elements of solid tumors such as spatial architecture and hypoxia at the tumor center.^72,73 Consequently, 3D A549 multicellular tumor spheroids were incubated with complex Zn3. One of the reasons why many of the investigated anticancer drugs fail during translation from cancer monolayers to in vivo models is impaired drug delivery due to drug penetration through the extracellular barrier. The penetration of the complex was first analyzed via Z-stack imaging microscopy. After 12 h of incubation, fluorescence signals are observed at each slice depth (Fig. S6†), indicating complete penetration of the complex in the 3D tumor spheroids.

We further investigated the potential antisolid tumor activity of complex Zn3 in 3D A549 multicellular tumor spheroids. The 3D tumor spheres in the control group were very dense and had a smooth surface. The 3D spheres treated with complex Zn3 became loose, with cell leakage in the dark (Fig. S7†), suggesting that complex Zn3 also exhibits a chemotherapeutic effect in 3D tumor spheroids. The 3D tumor spheres treated with complex Zn3 followed by irradiation with 470 nm light exhibit significant surface disintegration, with more cells lost from the spheroid than in the dark, and loss of 3D structure at day 3 (Fig. 4). In addition, these results indicate that the photodynamic therapy capabilities of complex Zn3 are superior to its chemotherapeutic capabilities because photodynamic therapy did more damage to 3D spheres at lower doses and shorter times than chemotherapy.

Fig. 4 Representative pictures of 3D A549 tumor spheroids at different times before (0 h) and after they were exposed once to 470 nm-LEDs. The spheroids were incubated without or with complex Zn3 at the indicated concentrations for 24 h. They were then illuminated in PBS with LEDs (470 nm, 20 mW cm⁻²) for 15 min. After this exposure, tumor spheroids were suspended in fresh medium and imaged. After 24 h, 48 h and 72 h, the second, third and fourth irradiation steps were performed as described above.

Photodynamic mechanism of Zn3

The photodynamic mechanisms are currently generally divided into type I and type II pathways, both inducing the production of ROS.⁷⁴ Type II photodynamic pathway is manifested as the generation of singlet oxygen ¹O₂ through direct energy transfer. The photochemical type I pathway is more complex than the type II pathway and involves the transfer of photo-induced electrons of biological substrates, which after several steps generate free radical species such as hydrogen peroxide (H₂O₂), peroxynitrite anion (ONOO⁻), superoxide anion radical (˙O₂⁻), and hydroxyl radical (˙OH). To identify the specific ROS generated after the treatment with complex Zn3, A549 cells were incubated with several selective ROS scavengers, including NaN₃ as a ¹O₂ scavenger, D-mannitol as a ˙OH scavenger, sodium pyruvate as an H₂O₂ scavenger, tiron as an ˙O₂⁻ scavenger, and ebselen as an ONOO⁻ scavenger.⁶⁸ Upon light excitation, the cells treated with complex Zn3 in the presence of NaN₃/D-mannitol/sodium pyruvate/ebselen exhibit cell morphologies similar to that of cells treated with complex Zn3 alone, which ruled out the possibility that ¹O₂, ˙OH, H₂O₂, and ONOO⁻ are involved in the photodynamic effect (Fig. 5). Interestingly, in the presence of tiron, the intracellular production of ROS was significantly inhibited, which suggested the generation of ˙O₂⁻ in the cells after light irradiation in the presence of complex Zn3. Low O₂ concentrations in solid tumors remain an obstacle for the use of classical photosensitizers because photodynamic therapy requires molecular oxygen to initiate cell death.^50,75 In contrast to type II photoreactivity, type I photoreactivity can still lead to strong cytotoxicity under hypoxic conditions.⁵² Therefore, complex Zn3 is promising for the photodynamic therapy of tumors because it utilizes the type I pathway, which is beneficial for overcoming the problem of tumor hypoxia.

Fig. 5 Representative image of ROS assay with A549 cells exposed to light (470 nm, 20 mW cm⁻²). Cells were pre-incubated with specific ROS scavengers (NaN₃/D-mannitol/sodium pyruvate/ebselen/tiron) and then treated with complex Zn3 (2 h in the dark followed by 15 min under the irradiation).

Chemotherapeutic mechanisms of Zn3

In view of the good chemotherapeutic capabilities (in the dark) of complex Zn3, its chemotherapeutic mechanism has also been carefully studied. Four-dimensional data-independent acquisition (4D-DIA) proteomic analysis is a fast, efficient, and sensitive method that can help elucidate potential anticancer mechanisms of drugs at the protein level.^76,77 Therefore, we performed 4D-DIA proteomic analysis in the diaPASEF mode to understand the details of the chemotherapeutic mechanism of complex Zn3.

The total number of identified peptides is 86 379, and the corresponding total number of quantified proteins is 7897. Among these quantified proteins, 1065 proteins were found to be significantly differentially expressed based on the following criteria: FC > 1.5 or FC < 0.6667 and P < 0.05. Among these differentially expressed proteins (DEPs), 520 were upregulated and 545 were downregulated (Fig. S8†). Subcellular localization studies (Fig. S9†) show that different numbers of DEPs are mainly concentrated in the nucleus (358), cytoplasm (213), plasma membrane (156), and mitochondria (120).

The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis can help systematically understand cell biological processes, disease mechanisms or characteristics, and drug action mechanisms. We thus performed KEGG pathway enrichment analysis based on DEPs to elucidate the pathways of action significantly affected by complex Zn3. As shown in Fig. 6, the 20 most important pathways cover organismal systems (complement and coagulation cascades and protein digestion and absorption), environmental information processing (cytokine–cytokine receptor interaction, ECM–receptor interaction, PI3K-Akt signaling pathway, and HIF-1 signaling pathway), genetic information processing (mismatch repair, fanconi anemia pathway, base excision repair, DNA replication, and homologous recombination), human diseases (microRNAs in cancer, small cell lung cancer, malaria, pathways in cancer, and toxoplasmosis), cellular processes (p53 signaling pathway and mitophagy-animal), and metabolism (glycosaminoglycan biosynthesis and sphingolipid metabolism) were considered. Among these KEGG pathways, the greatest number of differentially expressed proteins (43) is involved in pathways in cancer, of which 20 are upregulated and 23 are downregulated. Moreover, among these KEGG pathways, those closely related to cell proliferation, death, and migration, including the p53 signaling pathway, ECM–receptor interaction, cytokine–cytokine receptor interaction, PI3K-Akt signaling pathway, and HIF-1 signaling pathway, are jointly involved in the composition of pathways in cancer (Fig. S10†). Details on the differential proteins involved in these KEGG pathways are listed in Table S5.† Therefore, we further validated these pathways through western blotting and flow cytometry.

Fig. 6 KEGG pathway enrichment analysis (top 20) of complex Zn3 treated A549 cells. The abscissa represents the enrichment factor, and the ordinate indicates a significantly enriched KEGG pathway. The color of the point changes from blue to red to represent the change of P-value from large to small. The size of the point represents the number of DEPs annotated for the corresponding function.

Dysregulation of the p53 signaling pathway was detected in almost all tumors, thus the development of therapeutic drugs targeting the p53 signaling pathway has attracted considerable attention.⁷⁸ Activation of the p53 signaling pathway primarily leads to cell cycle arrest, or apoptosis.⁷⁹ We, therefore, investigated the effect of complex Zn3 on apoptosis and cell cycle of A549 cells. As shown in Fig. 7A, complex Zn3 induced dose-dependent apoptosis of A549 cells. To further confirm that complex Zn3 can induce apoptosis, we conducted western blotting analyses to monitor the expression levels of cleaved caspase-3 and cleaved PARP-1. As shown in Fig. 7B, the levels of cleaved caspase-3 and cleaved PARP-1 were upregulated, which further indicated that the apoptosis program was executed. In addition, the effect of complex Zn3 on the cell cycle was monitored through flow cytometry. Flow cytometric analysis reveals that complex Zn3 arrested the cell cycle at the G1 phase (Fig. 7C).

Fig. 7 (A) Apoptotic effects of complex Zn3 on A549 cells. (B) Western blot analysis of the protein expression levels of cleaved caspase-3, PARP-1, cleaved PARP-1, ITGA5, MMP-9, CDKN1A, HIF1A, LTBR, and GAPDH in A549 cells. (C) The cell cycle distribution histograms of A549 cells treated with complex Zn3.

ECM–receptor interactions enable direct or indirect control of cellular activities such as adhesion, migration, and proliferation.^80,81 The KEGG study showed that the ECM–receptor interaction pathway is disturbed by the upregulation of ITGA5, COL4A1, COL4A2, ITGA2, and LAMB3 as well as the downregulation of AGRN, LAMA5, LAMB1, LAMC1, SDC1, HSPG2, DAG1, and LAMA3. ITGA5, as an important protein in the ECM–receptor interaction pathway, plays a crucial role in different tumor cell subpopulations and cell-to-cell interactions.^82,83 Western blotting revealed that the treatment of A549 cells causes dose-dependent upregulation of the ITGA5 level (Fig. 7B), which agrees with the proteomics results. In addition, given that the ECM–receptor interaction plays a key role in cell migration,⁸⁴ the effect of complex Zn3 on the migration of A549 cells was investigated through wound-healing assays and western blotting. As shown in Fig. 8, the cells rapidly migrated, and the gap significantly narrowed in the control group. In contrast, fewer migrated cells and larger gaps are observed after the addition of complex Zn3. We also used western blotting to investigate the expression of extracellular matrix protein MMP-9 that can promote tumor cell migration.⁸⁵ Complex Zn3 inhibited the expression of MMP-9 (Fig. 7B), which matches the results of the wound-healing assays.

Fig. 8 Antimigratory effect of on A549 cells by wound healing assay.

In addition, LTBR protein in cytokine–cytokine receptor interaction, CDKN1A protein in PI3K-Akt signaling pathway and LTBR, CDKN1A and HIF1A proteins in HIF-1 signaling pathway were selected to further verify their expression by western blotting. Western blotting revealed that the treatment of A549 cells causes a dose-dependent increase in the expression of CDKN1A and HIF1A as well as a dose-dependent downregulation in the expression of LTBR (Fig. 7B). The western blotting data are consistent with the proteomics results: complex Zn3 activates the cytokine–cytokine receptor interaction, PI3K-Akt signaling pathway, and HIF-1 signaling pathway.

Collectively, these results suggest that complex Zn3 exerts chemotherapeutic effects by regulating multiple pathways, such as pathways in cancer, p53 signaling pathway, and ECM–receptor interactions.

Conclusions

A series of fluorescent N-heterocyclic dithiocarbazate–zinc(II) complexes were prepared, and their anti-lung cancer properties were assessed. These Zn(II) complexes exhibit excellent blood compatibility and can be used for live-cell imaging. Among them, complex Zn3 exhibited good chemotherapeutic and photodynamic therapy capabilities in both monolayer cancer cell models and 3D A549 multicellular tumor spheroids. Very importantly, under visible light irradiation, treatment with complex Zn3 generates type I ROS in living cells, superoxide anion radicals (˙O₂⁻), which can overcome the O₂-dependence of conventional photosensitizers, thus suggesting promising applications in photodynamic therapy against hypoxic tumors. Moreover, the studies of chemotherapeutic mechanisms demonstrated that complex Zn3 kills lung cancer cells through multiple pathways. Overall, these findings indicate that complex Zn3 can simultaneously express photodynamic and chemotherapeutic effects, which provides a simple method for the multimodal treatment of cancer and extends the horizon of anticancer research on zinc complexes.

Data availability

The data supporting the study is available within the ESI.†

Author contributions

JunGang Deng, YouRu Wu and AiLi Li: investigation, data curation, writing – original draft. WeiPing Pan: investigation, data curation. LiXia Hou: data curation. DaQi Wu: data curation. ZhenLei Zhang: software, methodology. Feng Yang: supervision, writing – review & editing. Yi Gou: supervision, resources, conceptualization, writing – review & editing.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

We thank these projects 22007023, 22267004, 2020GXNSFBA238003 and 2022GXNSFGA035003. We also sincerely thank Peng-Fei Yao (Baise University) and Jin-Long Li (Nantong University) for their help in the work.

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Footnotes

† Electronic supplementary information (ESI) available. CCDC 2265560–2265562. For ESI and crystallographic data in CIF or other electronic format see DOI: https://doi.org/10.1039/d3qi01777j

‡ These authors contributed equally to this work.

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