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. Author manuscript; available in PMC: 2025 Dec 17.
Published in final edited form as: J Cell Biochem. 2025 Sep;126(9):e70062. doi: 10.1002/jcb.70062

Current Advances in Anticancer Properties of Heptamethine Carbocyanine DZ‐1 Conjugated to Artesunate: Generation of Reactive Oxygen Species

Badrinath Narayanasamy 1, Sarah Helmueller 1, Yi Zhang 1, Yong J Lee 1
PMCID: PMC12706113  NIHMSID: NIHMS2111515  PMID: 40891511

Abstract

Heptamethine cyanine dyes and anticancer agents based conjugates are being developed for enhanced targeting and killing of cancer cells. DZ-1 dye conjugated agents induced cytotoxicity and mechanism of action have been shown in previous studies. In this study, a conjugated form of DZ-1 and artesunate (DZ-1-ART) was used to evaluate its cytotoxicity and elucidate the mechanism of actions in various cancer cell lines. Cells survival assays indicated dose-dependent cytotoxic activities of DZ-1-ART in HCT116, BxPC-3, and OVCAR-3 cell lines. Immunoblotting and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay confirmed involvement of apoptosis in DZ-1-ART-induced cytotoxicity. To elucidate the anticancer mechanism of the action of DZ-1-ART, MitoTracker and JC-1 assay were used. The results showed that translocation of DZ-1-ART in the mitochondria was followed by disruption of mitochondrial outer membrane potential. Dichlorofluorescin diacetate assay confirmed the generation of reactive oxygen species (ROS) in DZ-1-ART treated cancer cells. An antioxidant, N-acetyl cysteine treatment with DZ-1-ART showed reduction in cell death as well as suppression of ROS generation. When compared to HCT116 wild-type cells, Bak and Bax-deficient HCT116 cells also showed similar levels of cytotoxicity of DZ-1-ART. Taken together, this study’s results reported that DZ-1-ART could induce mitochondria-mediated, ROS-generated, and Bak and Bax-independent apoptosis in cancer cells.

Keywords: artesunate, heptamethine carbocyanine DZ-1, mitochondria, oxidative stress, reactive oxygen species

1 |. Introduction

Cancer chemotherapy has significantly increased the survival rates of cancer patients [1]. However, drug resistance and nonspecific cytotoxicity to normal cells are the major obstacles of conventional therapies [2]. To overcome nonspecific toxicities of chemotherapeutic agents, targeted therapy has been developed [3]. Conjunction of chemotherapeutic agents with various molecules, which target overexpressed receptors of cancer cells could minimize the cytotoxicity to normal cells [4, 5].

Heptamethine cyanine dyes (HMCDs) have been developed for noninvasive imaging of tumors [6]. Due to the physical and chemical nature of HMCDs, such as near-infrared (NIR) light emission and cationic structure, these dyes are being widely developed in cancer imaging and targeting purposes [7]. Overexpression of influx pumps such as organic anion-transporting polypeptides and hypoxia-inducible factor 1-α by cancer cells facilitate tumor selectivity of HMCDs [8, 9].

DZ-1 is a type of HMCD, which targets mitochondria, and its NIR emission was used to develop imaging of hepatocellular carcinoma (HCC) [10, 11]. Owing to imaging property of DZ-1, several studies have shown its tumor-specific targeting and drug delivery efficacy with chemotherapeutic agents [12–16].

Artesunate (ART) is a first-line drug for severe malaria, which induces DNA damage and reactive oxygen species (ROS) in Plasmodium falciparum [17, 18]. Ferroptosis is a form of non-apoptotic cell death characterized by iron-dependent accumulation of lipid peroxides and depletion of polyunsaturated fatty acids in the plasma membrane of cells [19, 20]. In addition, various anticancer mechanisms underlying ART-induced cell death have been reported across multiple cancer models. Lysosome-mediated cell death and degradation of intracellular iron and ferritin have been observed in ART-treated HeLa cervical cancer cells [21]. ART induced G2/M cell cycle arrest, and the activation of ataxia-telangiectasia mutated (ATM) mediated cell death was reported in MCF7 breast cancer cells [22]. ROS-dependent G2/M arrest, ROS-independent G1 arrest, ROS and iron-dependent apoptosis were observed in ART-treated triple-negative MDA-MB-468 and HER2-enriched SK-BR-3 breast cancer cells [23]. ART-induced ferroptosis was reported in U251 glioblastoma cells. This effect was mediated by glutathione depletion, lipid peroxidation, and GPX4 depletion [24].

In our previous work, we have shown that ART treatment can enhance tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis via endoplasmic reticulum (ER) stress in HCT116 cells [25]. In this study, DZ-1-ART, a conjugated form of ART, was used as treatment in various cancer cell lines, and mitochondria-targeted and ROS-induced apoptosis were shown. Conjugation of DZ-1 and ART may increase its targeting efficacy as well as cytotoxicity in cancer cells. In addition, this conjugation can be used for theranostic purposes, such as diagnostics and therapeutic aspects in cancer therapy.

2 |. Materials and Methods

2.1 |. Synthesis of DZ-1-Art

DZ-1 (350 mg, 0.51 mmol) and ART (195 mg, 0.51 mmol) were dissolved in anhydrous dichloromethane (10 mL). At room temperature, this solution was added into EDC·HCl (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; 146 mg, 0.76 mmol) and DMAP (4-dimethylaminopyridine; 6 mg, 0.05 mmol). The reaction mixture was stirred at room temperature for 18 h. The solvent was then removed under reduced pressure, and the crude residue was purified by silica-gel column chromatography (CH2Cl2/MeOH 50:1) to afford compound DZ-1-ART as a dark green solid (364 mg, 68% yield).

2.2 |. Cell Culture

Human colon cancer cell line HCT116, human pancreatic cancer cell line BxPC-3, and human ovarian cancer cell line OVCAR-3 were obtained from American Type Culture Collection (ATCC). Double knockout Bax and Bak (Bax−/−Bak−/−) HCT116 cells were obtained from Dr. B. Vogelstein (Johns Hopkins University, Baltimore, MD). All cell lines were maintained in Roswell Park Memorial Institute (RPMI)−1640 medium. This medium was maintained with 10% fetal bovine serum and cultured in a humidified atmosphere of 5% CO2 at 37°C.

2.3 |. Chemical and Reagents

ART (Cat# A3731), N-acetyl cysteine (NAC) (Cat# A7250), and 2′,7′-Dichlorofluorescin diacetate (DCFDA) (Cat# D6883), ferrostatin-1 (Cat# SML0583), and liproxstatin-1 (Cat# SML1414) were purchased from Millipore Sigma. DZ-1 and DZ-1-ART were synthesized by Dr. Yi Zhang (Cedars-Sinai Medical Center).

2.4 |. Cell Survival Assay

A trypan blue exclusion assay was used for the cell survival assay. To distinguish between live and dead cells, after 24 h of ART, DZ-1 and DZ-1-ART treatment, cells were stained with 0.4% trypan blue. LUNA™ Automated Cell Counter (L10001, Logos BioSystem) was used to count dead cells and live cells.

2.5 |. Western Blot Analysis and Antibodies

Western blot was performed as previously described [26]. The following primary antibodies were used for immunoblotting: anti-PARP-1 (Cat# 9532), anti-caspase-8 (Cat# 9746), anti-cleaved caspase-9 (Cat# 7237), anti-Bak (Cat #12105), anti-Bax (Cat# 2772), and anti-ATF-4 (Cat# 11815) were obtained from Cell Signaling Technology. Anti-β-actin (Cat# A1978) was obtained from Sigma-Aldrich. For secondary antibodies, anti-rabbit IgG-HRP (Cat# 7074P2) and goat anti-mouse IgG-HRP were purchased from Cell Signaling Technology and Santa Cruz Biotechnology, respectively.

2.6 |. Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Assay

To detect apoptosis, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method was used. HCT116 cells were seeded in Nunc glass-bottom dishes. After 24 h of treatment, cells were washed with 1X phosphate-buffered saline (PBS). In Situ Cell Death Detection Kit, Fluorescein (Roche, cat # 11684795910) was used as per the manufacturer’s instructions. Briefly, TUNEL reaction mixture was added with treated cells and incubated at 37°C for 1 h. After 1 h, cells were then washed with PBS and treated with Hoechst for 10 min. Then an ECHO fluorescence microscope was used to capture images.

2.7 |. Mitochondria Labeling

To label mitochondria, MitoTrackerTM Green FM kit (Cat# M7514) was used. After 24 h of ART, DZ-1 and DZ-1-ART treatment, HCT116 cells were incubated with MitoTracker Green FM dye according to manufacturer’s instructions. Hoechst staining was done for visualizing the nucleus. To capture images, an ECHO fluorescence microscopy was used.

2.8 |. JC-1 Assay

JC-1 dye (mitochondrial membrane potential probe, ThermoFisher Scientific, Cat# T3168) was used to analyze mitochondrial membrane potential (ΔΨm). After 24 h of ART, DZ-1 and DZ-1-ART treatment, cells were stained with JC-1 dye as per the manufacturer’s instructions, and an ECHO fluorescence microscopy was used to capture images.

2.9 |. Reactive Oxygen Species (ROS) Assay

To evaluate ROS generation, 2’,7’-dichlorofluorescin diacetate (DCFDA) assay was used. HCT116 cells were treated with ART, DZ-1, DZ-1-ART and DZ-1-ART with/without NAC, and after 24 h of treatment, cells were treated with DCFDA. After 45 min of incubation with DCFDA, Hoechst staining was done. An ECHO fluorescence microscopy was used to capture images.

2.10 |. Treatment With Hyperthermia

Cells plated in Petri dishes were wrapped in Parafilm to prevent leakage during incubation in the water bath. The samples were submerged in a 42°C water bath for 2, 4, or 6 h. After those periods, samples were analyzed accordingly.

2.11 |. Statistical Analysis

For statistical analysis, one-way analysis of variance, followed by Tukey’s post hoc test were used. GraphPad Prism 9 software was used for this purpose. Values are represented as mean ± standard deviation (SD) and p values of less than 0.05 were defined as statistically significant. Significance of p-value is indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0. 0001.

3 |. Results

3.1 |. DZ-1-ART Induces Cytotoxicity

Previous studies reported cytotoxicity of DZ-1 conjugated drugs in various cancer cell lines [12–16]. In this study, we synthesized DZ-1 dye conjugated to ART (DZ-1-ART) (Figure 1A), and investigated cytotoxicity of DZ-1-ART in HCT116 cell line (Figure 1B,C). When compared with ART and DZ-1 treated cells, the morphology of DZ-1-ART treated cells showed blebbed and more detached cells (Figure 1B). To quantify cytotoxicity, we performed trypan blue exclusion assay. The results showed a significantly higher cytotoxicity in DZ-1-ART-treated cells than ART and DZ-1-treated cells (Figure 1C). The significance of p-value is 0.001 or 0.0001 at 10 μM or 20 μM, respectively.

FIGURE 1 |.

FIGURE 1 |

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DZ-1-ART induces cytotoxicity in HCT116 cells. Cells were treated with various concentrations (1–20 μM) of artesunate (ART), DZ-1, or DZ-1 conjugated to ART (DZ-1-ART) for 24 h. (A) Schematic diagram of the synthesis of DZ-1-ART. (B) For morphology assay, phase-contrast images were captured under a light microscope. Representative images are shown here (scale bar 200 μm). (C) For cell survival assay, Trypan blue exclusion assay was used to detect cell death percentage. DZ-1-ART-treated cells (10 μM and 20 μM) were compared with control. One-way ANOVA and Tukey’s multiple comparison test were used for statistical analysis (****p: < 0.0001). Error bars show the mean ± SD from triplicates.

3.2 |. DZ-1-ART Induced Cytotoxicity Through Apoptosis

Next, to detect the type of cell death, we performed Western blot and TUNEL assay. Western blot analysis showed clear cleaved PARP-1, cleaved caspase-8, and cleaved caspase-9 in DZ-1-ART-treated HCT116 cells, which confirmed the apoptosis involvement in DZ-1-ART-induced cytotoxicity (Figure 2A–D). For further confirmation, we used TUNEL assay for apoptosis detection in HCT116 cells. DZ-1-ART cells showed TUNEL-positive cells (green fluorescence), while ART and DZ-1-treated cells showed no TUNEL-positive cells (Figure 2E). The cytotoxicity and apoptosis effects of DZ-1-ART were also evaluated in BxPC-3 and OVCAR-3 cells (Figure 3). Cell death assay confirmed higher levels of DZ-1-ART induced cytotoxicity in both BxPC-3 and OVCAR-3 cells (Figure 3A). To check the type of cell death, DZ-1-ART-treated BxPC-3 and OVCAR-3 cells were harvested and subjected to Western blot analysis. The results confirmed cleaved PARP-1, which is a hallmark of apoptosis, as well as cleavage of caspase-8 and caspase-9 (Figure 3B).

FIGURE 2 |.

FIGURE 2 |

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DZ-1-ART induces apoptosis in HCT116 cells. Cells were treated with various concentrations (1–20 μM) of ART, DZ-1, or DZ-1-ART for 24 h. (A) For western blot analysis, cells were harvested using a 1X lysis buffer and denaturing SDS-PAGE was performed, and then immunoblot analysis was performed using indicated antibodies. (B) Densitometric analysis of PARP-1 cleavage. (C, D) Densitometric analysis of cleaved caspase-8 and cleaved caspase-9. (E) HCT116 cells were subjected to 10 μM of ART, DZ-1, and DZ-1-ART treatment for 24 h, and TUNEL assay was performed. Images were captured under a fluorescence microscope. Representative images are shown here (scale bar 200 μm).

FIGURE 3 |.

FIGURE 3 |

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DZ-1-ART induces cytotoxicity through apoptosis in various cell lines. HCT116, BxPC-3, and OVCAR-3 cells were treated with various concentrations (1–30 μM) of DZ-1-ART for 24 h (A) Trypan blue exclusion assay was used to detect cytotoxicity. One-way ANOVA and Tukey’s multiple comparison test were used for statistical analysis (****p: < 0.0001). Error bars show the mean ± SD from triplicates. (B) Whole-cell extracts were analyzed with an immunoblotting assay using indicated antibodies.

3.3 |. DZ-1-ART Targets Mitochondria and Disrupts Mitochondrial Membrane Potential

To explore the translocation of DZ-1 and DZ-1-ART, we used MitoTracker green dye. DZ-1 dye can be detected at NIR. MitoTracker green dye can bind with mitochondria. Control, ART, DZ-1, and DZ-1-ART-treated HCT116 cells showed green fluorescence that demonstrates mitochondria binding, but NIR range showed red fluorescence that confirmed translocation of DZ-1 and DZ-1-ART. However, the results showed more red fluorescence in DZ-1-ART-treated cells than in DZ-1-treated cells, which confirmed mitochondrial translocation of DZ-1-ART (Figure 4A). Next, to explore the effect of mitochondrial translocation DZ-1-ART, we used JC-1 assay. This assay can be used to measure mitochondrial membrane potential. After 24 h of treatment, control, ART, and DZ-1-treated HCT116 cells showed higher, medium, and lower red fluorescence, respectively, while DZ-1-ART-treated HCT116 cells showed diminished red fluorescence. DZ-1-ART-treated HCT116 cells showed higher green fluorescence than any other treated cells (Figure 4B). The formation of red fluorescence showed aggregation of JC-1 dye, which indicated healthy mitochondria, whereas green fluorescence indicated JC-1 monomer, which indicated mitochondrial depolarization. Our results confirmed DZ-1-ART induced mitochondrial depolarization in HCT116 cells.

FIGURE 4 |.

FIGURE 4 |

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DZ-1-ART translocates into mitochondria and disrupts mitochondrial membrane potential. HCT116 cells were treated with 10 μM of ART, DZ-1-, or DZ-1-ART for 24 h. (A) For mitochondrial localization assay, cells were stained with MitoTracker dye. (B) For mitochondrial integrity assay, cells were stained with JC-1 dye. Images were captured under a fluorescence microscope. Representative images are shown here (scale bar 200 μm).

3.4 |. DZ-1-ART Generates ROS Through Mitochondria

We postulate that DZ-1-ART mitochondrial translocation and its induced mitochondrial depolarization can generate ROS from mitochondria. To examine this possibility, we employed DCFDA assay. DCFDA is a nonfluorescent probe, which cleaved into a fluorescent molecule, dichlorofluorescein (DCF) by intracellular esterase. This DCF is converted into green fluorescence by ROS [27]. DZ-1-ART-treated HCT116 cells showed higher levels of green fluorescence than ART and DZ-1-treated cells, which indicated high levels of ROS generation by DZ-1-ART. Antioxidant NAC treatment with DZ-1-ART-treated cells showed gradually diminished green fluorescence (Figure 5). Taken together, these results confirmed DZ-1-ART enhanced and facilitated ROS generation in HCT116 cells.

FIGURE 5 |.

FIGURE 5 |

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DZ-1-ART induces reactive oxygen species (ROS), and N-acetyl cysteine (NAC) reduces ROS level. HCT116 cells were treated with 10 μM of DZ-1, ART, or DZ-1-ART for 24 h. The level of ROS was detected using DCFDA probe, and the inhibitory effect of NAC was analyzed. A fluorescence microscope used to capture images (scale bar 200 μm). DCFDA, 2',7'-dichlorofluorescin diacetate.

3.5 |. NAC Inhibits DZ-1-ART Induced Apoptosis

NAC is an antioxidant, which scavenges ROS. To examine the effects of NAC on DZ-1-ART treatment, HCT116 cells were treated with both DZ-1-ART and NAC. Morphological analysis showed less cells death in 1.0 mM of NAC and 10 μM of DZ-1-ART treated cells (Figure 6A). Cell death analysis also showed a significant reduction of cytotoxicity in 1.0 mM of NAC and 10 μM of DZ-1-ART treated cells (Figure 6B). Furthermore, NAC and DZ-1-ART-treated cells were harvested after 24 h of treatment for Western blot analysis. The results showed inhibition of cleaved PARP-1, caspase-8, and caspase-9 in NAC and DZ-1-ART-treated cells (Figure 6C). The inhibition of PARP-1, caspase-8, and caspase-8 by NAC confirmed that NAC may suppress the DZ-1-ART-induced apoptosis in HCT116 cells.

FIGURE 6 |.

FIGURE 6 |

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N-acetyl cysteine (NAC) inhibits DZ-1-ART-induced cytotoxicity. HCT116 cells were treated with 10 μM of DZ-1-ART in the presence or absence of various concentrations (0.1–1 mM) of NAC for 24 h. (A) Morphology was observed under a light microscope. Representative images are shown here (scale bar 200 μm). (B) Cytotoxicity was detected using Trypan blue exclusion assay. One-way ANOVA and Tukey’s multiple comparison test were used for statistical analysis (****p: < 0.0001). Error bars show the mean ± SD from triplicates. (C) Whole-cell extracts were analyzed with an immunoblotting assay using indicated antibodies.

3.6 |. DZ-1-ART Induces Bak and Bax-Independent Apoptosis

Bcl-2 homologous antagonist killer (Bak) and Bcl-2–associated X (Bax) are proapoptotic proteins, which form Bak/Bax pore formation on mitochondrial membranes. To examine Bak and Bax roles in DZ-1-ART-induced apoptosis, we used HCT116 wild-type (WT) and HCT116 Bak−/−Bax−/− cells. Both cells were treated with DZ-1-ART (1–20 μM). After 24 h of treatment, cells survival assay and Western blotting analysis were performed. Cells survival assay showed similar levels of cells death in both HCT116 WT and HCT116 Bak−/− Bax−/− cells (Figure 7A). Cleaved PARP-1 protein was observed in both cells. Bak and Bax expressions were only seen in HCT116 WT cells and not in HCT116 Bak−/− Bax−/− cells (Figure 7B). These results supported the occurrence of Bak and Bax independent apoptosis in HCT116 cells.

FIGURE 7 |.

FIGURE 7 |

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DZ-1-ART induces Bak and Bax-independent apoptosis. (A) HCT116 WT and HCT116 Bak−/−Bax−/− cells were treated with various concentrations (1–20 μM) DZ-1-ART for 24 h. Trypan blue exclusion assay was used to detect cell death percentage. One-way ANOVA and Tukey’s multiple comparison test were used for statistical analysis. Error bars show the mean ± SD from triplicates. ns, no statistical significance. (B) Whole-cell extracts were analyzed with an immunoblotting assay using indicated antibodies.

3.7 |. Ferroptosis Has No Effect on DZ-1-ART-Induced Apoptosis

To rule out a potential role of ferroptosis in DZ-1-ART–induced cytotoxicity, HCT116 cells were treated with DZ-1-ART in combination with ferroptosis inhibitors, including ferrostatin-1 and liproxstatin-1. Immunoblotting analysis revealed that PARP-1 cleavage was still evident in cells treated with DZ-1-ART and ferrostatin-1, as well as in those treated with DZ-1-ART and liproxstatin-1 (Figure 8A). These results indicate that DZ-1-ART-induced apoptosis is not affected by ferroptosis.

FIGURE 8 |.

FIGURE 8 |

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Effect of ferroptosis inhibitors or hyperthermia on DZ-1-ART-induced apoptosis. (A) HCT116 cells were treated with 10 μM of DZ-1-ART in the presence or absence of 10 μM ferrostatin-1 or 2 μM liproxstatin-1. After 24 h, cells were harvested and subjected to immunoblotting using the indicated antibodies. (B) HCT116 cells were treated with DZ-1-ART for 2, 4, and 6 h, with or without exposure to heat at 42°C. Cells were then harvested and analyzed by immunoblotting for the indicated proteins.

3.8 |. Effect of Hyperthermia on DZ-1-ART-Induced Apoptosis

Since the DZ-1 dye is activated under NIR irradiation, it may generate heat upon exposure. To assess the potential effect of heat on DZ-1-ART-induced apoptosis, HCT116 cells were treated with 10 μM DZ-1-ART at 42°C for varying periods (2–6 h). Immunoblotting analysis revealed PARP-1 cleavage after 6 h of treatment with DZ-1-ART alone, as well as with the combination of DZ-1-ART and heat (Figure 8B). These results indicate comparable levels of apoptosis in both conditions, confirming that mild hyperthermia does not enhance DZ-1-ART-induced apoptosis.

4 |. Discussion

Previous studies showed ART-induced ferroptosis as well as apoptosis in various cancer cell lines [24, 28–31]. However, each study reported different mechanisms of actions. In this study, we reported DZ-1-ART induced mitochondrial-targeted, ROS-generated, and Bax/Bak-independent apoptosis in HCT116 cells. DZ-1-ART treatment showed significant toxicity (Figure 1) and cleavages of PARP-1, caspase-8, and cleavage-9 in colon cancer cell line HCT116, human pancreatic cancer cell line BxPC-3, and human ovarian cancer cell line OVCAR-3 (Figures 2B and 3B). Moreover, DZ-1-ART-treated HCT-116 cells showed TUNEL-positive cells, which confirmed DNA degradation stage of apoptosis (Figure 2B). NIR emission of red fluorescence and MitoTracker-based green fluorescence of DZ-1-ART-treated HCT116 cells confirmed mitochondrial targeting of DZ-1-ART (Figure 4A). DZ-1-ART-treated HCT116 cells showed green fluorescence, directly proportional to monomeric formation of JC-1 dye that clearly indicated disruption of mitochondrial integrity (Figure 4B).

Mitochondrial translocation and mitochondrial outer membrane (MOM) disruptions by DZ-1-ART can induce ROS generation from mitochondria. The green fluorescence of DCFDA assay of DZ-1-ART-treated HCT116 cells confirmed DZ-1-ART-induced ROS generation (Figure 5). NAC and DZ-1-ART-treated HCT116 cells showed reduction in cytotoxicity and diminished activation of PARP-1, caspase-8, and caspase-9 (Figure 6). These results may confirm the targeting and apoptosis efficacy of DZ-1 conjugation with ART. In our previous work, we reported ER stress-induced p53-independent, p53 upregulated modulator of apoptosis mediates enhanced apoptosis of ART or erastin in combination with TRAIL treatment in PANC-1, BxPC-3, and HCT116 cells [25]. However, DZ-1 conjugated with ART may alter kinetics and targeting mechanism of DZ-1-ART, which may directly translocate to mitochondria and induce ROS-mediated apoptosis.

Bak and Bax oligomerization is an essential process for pore formation in the MOM. The translocation of Cytochrome C from mitochondria to cytosol has occurred through Bak and Bax-formed pores [32]. We used HCT116 wild-type and HCT116 Bak−/− Bax−/− cells to confirm Bak and Bax involvement in DZ-1-ART-induced apoptosis (Figure 7). Surprisingly, DZ-1-ART-treated HCT116 Bak−/− Bax−/− cells also showed almost the same level PARP-1 cleavage as compared to HCT116 wild-type cells (Figure 7). These results confirmed DZ-1-ART-induced Bak and Bax-independent apoptosis in HCT116 cells.

It is also well known that ROS is generated through several intracellular sources, including the mitochondrial electron transport chain and peroxisomal cytochrome P-450 oxidases, as well as endogenous enzyme systems [33]. Previous studies suggest that disruption of the mitochondrial electron transport chain is the main source of ROS generation [34]. Translocated DZ-1-ART may directly disrupt the mitochondrial electron transport chain and cause ROS generation. This possibility needs to be clarified in the near future. Moreover, we previously observed that ROS can be sensed through thioredoxin (TRX) and glutaredoxin (GRX), resulting in activation of the ASK1 (apoptosis signal-regulating kinase 1)-MEK (mitogen-activated protein/extracellular signal-regulated kinase)-MAPK (mitogen-activated protein kinase) signal transduction pathway [35]. These sensor molecules may be converted to the intramolecular disulfide form of TRX-(S-S) and GRX-(S-S) during treatment with DZ-1-ART. This oxidized form of TRX and GRX may dissociate from ASK1 and consequently activate the MEK-JNK1 apoptotic signal transduction pathway [36]. Obviously, further studies are necessary to understand the involvement of the ASK1-MEK-JNK signal pathways in DZ-1-ART-induced apoptosis. Mitochondria are central hubs for oxidative phosphorylation, the tricarboxylic acid cycle, fatty acid oxidation, the urea cycle, and heme synthesis [37]. Due to their role in oxidative metabolism, mitochondria are also susceptible to oxidative damage and may be involved in DZ-1-ART-induced ROS generation. As shown in Figure 1A, DZ-1-ART is a conjugate of the DZ-1 dye and ART linked via an ester bond. This design enables selective tumor targeting by the DZ-1 dye, while ART may contribute to mitochondrial ROS production. Once inside the mitochondria, DZ-1-ART may be cleaved by mitochondrial esterases, leading to the release of ART. Within the mitochondria, ART’s endoperoxide bridge could be activated by Fe2+/Fe3+ through Fenton-like reactions, resulting in ROS generation.

A previous study reported that DZ-1 alone does not induce cytotoxicity in cancer models under NIR irradiation [10]. However, its tumor-targeting capability makes DZ-1 a promising scaffold for engineering with photosensitizing agents to generate ROS under NIR light for photodynamic therapy (PDT). Additionally, the ability of DZ-1 to absorb and emit light under NIR irradiation may lead to localized heat generation, suggesting its potential utility in photothermal therapy (PTT). In the present study, we focused on DZ-1-ART-induced ROS production and apoptosis in cancer cells. We also demonstrated that mild hyperthermia did not enhance DZ-1-ART–induced apoptosis. Collectively, these findings may support the future development of DZ-1 and DZ-1-ART as multifunctional agents for both PDT and PTT applications.

In conclusion, DZ-1-ART, a conjugated form of ART with DZ-1, directly targets mitochondria, where it disrupts MOM potential and generates ROS. Mitochondria-generated ROS induces caspase-9 and caspase-8 activation and Bak and Bax-independent apoptosis.

Acknowledgments

This study was supported by the following grants: National Institutes of Health R21CA259243 (Yong J. Lee), R01CA265827 (Yong J. Lee), R21CA256419 (Yi Zhang), and Department of Defense W81XWH-22-1-1095 RA210084 (Yong J. Lee).

Funding:

This study was supported by the following grants: National Institutes of Health R21CA259243 (Yong J. Lee), R01CA265827 (Yong J. Lee), R21CA256419 (Yi Zhang), and Department of Defense W81XWH-22-1-1095 RA210084 (Yong J. Lee).

Abbreviations:

ART

artesunate

ASK1

apoptosis signal-regulating kinase 1

ATM

ataxia-telangiectasia mutated

Bak

Bcl-2 homologous antagonist killer

Bax

Bcl-2–associated X protein

Bcl-2

B-cell lymphoma 2

Bcl-xL

B-cell lymphoma-extra large

DCF

dichlorofluorescein

DCFDA

dichlorofluorescin diacetate

ER

endoplasmic reticulum

GPX4

glutathione peroxidase 4

GRX

glutaredoxin

GSH

glutathione

HCC

hepatocellular carcinoma

HIF1α

hypoxia-inducible factor 1-α

HMCDs

heptamethine cyanine dyes

HRP

horseradish peroxidase

MAPK

mitogen-activated protein kinase

MEK

mitogen-activated protein/extracellular signal-regulated kinase

MOM

mitochondrial outer membrane

NAC

N-acetyl cysteine

NIR

near-infrared

OATPs

organic anion-transporting polypeptides

PARP-1

poly [ADP-ribose] polymerase 1

PDT

photodynamic therapy

PTT

photothermal therapy

PUMA

p53 upregulated modulator of apoptosis

RPMI

Roswell park memorial institute

ROS

reactive oxygen species

TRAIL

tumor necrosis factor-related apoptosis-inducing ligand

TRX

thioredoxin

TUNEL

terminal deoxynucleotidyl transferase dUTP nick-end labeling

Footnotes

Conflicts of Interest

DZ-1-ART is licensed to DaZen Theranostics Inc., of which Yi Zhang is a shareholder position. A joint patent for DZ-1-ART (US20230226198A1) has been filed. Other authors declare no conflicts of interest.

Data Availability Statement

Research materials that were generated in the studies will be made freely available to the scientific research community as soon as this manuscript has been documented in a publication. Raw data were generated at the Cedars-Sinai Medical Center. Derived data supporting the findings of this study are available from the corresponding author, Dr. Yong J. Lee, upon request.

References

  • 1.Galmarini D, Galmarini CM, and Galmarini FC, “Cancer Chemotherapy: A Critical Analysis of Its 60 Years of History,” Critical Reviews in Oncology/Hematology 84, no. 2 (2012): 181–199. [DOI] [PubMed] [Google Scholar]
  • 2.Palumbo MO, Kavan P, Miller WH Jr., et al. , “Systemic Cancer Therapy: Achievements and Challenges That Lie Ahead,” Frontiers in Pharmacology 4 (2013): 57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Zhong L, Li Y, Xiong L, et al. , “Small Molecules in Targeted Cancer Therapy: Advances, Challenges, and Future Perspectives,” Signal Transduction and Targeted Therapy 6, no. 1 (2021): 201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Bhattacharya S, Anjum MM, and Patel KK, “Gemcitabine Cationic Polymeric Nanoparticles Against Ovarian Cancer: Formulation, Characterization, and Targeted Drug Delivery,” Drug Delivery 29, no. 1 (2022): 1060–1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Cooper E, Choi PJ, Denny WA, Jose J, Dragunow M, and Park TIH, “The Use of Heptamethine Cyanine Dyes as Drug-Conjugate Systems in the Treatment of Primary and Metastatic Brain Tumors,” Frontiers in Oncology 11 (2021): 654921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Yang X, Shi C, Tong R, et al. , “Near IR Heptamethine Cyanine Dye-Mediated Cancer Imaging,” Clinical Cancer Research 16, no. 10 (2010): 2833–2844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Choi PJ, Park TH, Cooper E, Dragunow M, Denny WA, and Jose J, “Heptamethine Cyanine Dye Mediated Drug Delivery: Hype or Hope,” Bioconjugate Chemistry 31, no. 7 (2020): 1724–1739. [DOI] [PubMed] [Google Scholar]
  • 8.Buxhofer-Ausch V, Secky L, Wlcek K, et al. , “Tumor-Specific Expression of Organic Anion-Transporting Polypeptides: Transporters as Novel Targets for Cancer Therapy,” Journal of Drug Delivery 2013, no. 1 (2013): 863539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Shi C, Wu JB, Chu GCY, et al. , “Heptamethine Carbocyanine Dye-Mediated Near-Infrared Imaging of Canine and Human Cancers Through the HIF-1α/OATPs Signaling Axis,” Oncotarget 5, no. 20 (2014): 10114–10126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.An J, Zhao N, Zhang C, et al. , “Heptamethine Carbocyanine DZ-1 Dye for Near-Infrared Fluorescence Imaging of Hepatocellular Carcinoma,” Oncotarget 8, no. 34 (2017): 56880–56892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Zhang C, Zhao Y, Zhang H, et al. , “The Application of Heptamethine Cyanine Dye DZ-1 and Indocyanine Green for Imaging and Targeting in Xenograft Models of Hepatocellular Carcinoma,” International Journal of Molecular Sciences 18, no. 6 (2017): 1332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Mrdenovic S, Zhang Y, Wang R, et al. , “Targeting Burkitt Lymphoma With a Tumor Cell-Specific Heptamethine Carbocyanine-Cisplatin Conjugate,” Cancer 125, no. 13 (2019): 2222–2232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Mrdenovic S, Wang Y, Yin L, et al. , “A Cisplatin Conjugate With Tumor Cell Specificity Exhibits Antitumor Effects in Renal Cancer Models,” BMC Cancer 23, no. 1 (2023): 499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Ou Y, Wang R, Chu GCY, et al. , “Novel DZ-SIM Conjugate Targets Cancer Mitochondria and Prolongs Survival in Pancreatic Ductal Adenocarcinoma,” Advanced Therapeutics 5, no. 10 (2022): 2200021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Ou Y, Chu GCY, Lyu J, et al. , “Overcoming Resistance in Prostate Cancer Therapy Using a DZ-Simvastatin Conjugate,” Molecular Pharmaceutics 21, no. 2 (2024): 873–882. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Qin J, Zhang C, Zhao Y, et al. , “Small Mitochondria-Targeting Fluorophore With Multifunctional Therapeutic Activities Against Prostate Cancer via the HIF1α/OATPs Pathway,” Molecular Pharmaceutics 20, no. 12 (2023): 6226–6236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Gopalakrishnan AM and Kumar N, “Antimalarial Action of Artesunate Involves DNA Damage Mediated by Reactive Oxygen Species,” Antimicrobial Agents and Chemotherapy 59, no. 1 (2015): 317–325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Ruwizhi N, Maseko RB, and Aderibigbe BA, “Recent Advances in the Therapeutic Efficacy of Artesunate,” Pharmaceutics 14, no. 3 (2022): 504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Dixon SJ, Lemberg KM, Lamprecht MR, et al. , “Ferroptosis: An Iron-Dependent Form of Nonapoptotic Cell Death,” Cell 149, no. 5 (2012): 1060–1072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Magtanong L, Ko PJ, and Dixon SJ, “Emerging Roles for Lipids in Non-Apoptotic Cell Death,” Cell Death & Differentiation 23, no. 7 (2016): 1099–1109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Yang ND, Tan SH, Ng S, et al. , “Artesunate Induces Cell Death in Human Cancer Cells via Enhancing Lysosomal Function and Lysosomal Degradation of Ferritin,” Journal of Biological Chemistry 289, no. 48 (2014): 33425–33441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Wen L, Liu L, Wen L, Yu T, and Wei F, “Artesunate Promotes G2/M Cell Cycle Arrest in MCF7 Breast Cancer Cells Through ATM Activation,” Breast Cancer: Basic and Clinical Research 25 (2018): 681–686. [DOI] [PubMed] [Google Scholar]
  • 23.Greenshields AL, Fernando W, and Hoskin DW, “The Anti-Malarial Drug Artesunate Causes Cell Cycle Arrest and Apoptosis of Triple-Negative MDA-MB-468 and HER2-Enriched SK-BR-3 Breast Cancer Cells,” Experimental and Molecular Pathology 107 (2019): 10–22. [DOI] [PubMed] [Google Scholar]
  • 24.Song Q, Peng S, Che F, and Zhu X, “Artesunate Induces Ferroptosis via Modulation of p38 and ERK Signaling Pathway in Glioblastoma Cells,” Journal of Pharmacological Sciences 148, no. 3 (2022): 300–306. [DOI] [PubMed] [Google Scholar]
  • 25.Hong SH, Lee DH, Lee YS, et al. , “Molecular Crosstalk Between Ferroptosis and Apoptosis: Emerging Role of ER Stress-Induced p53-Independent PUMA Expression,” Oncotarget 8, no. 70 (2017): 115164–115178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Nam SY, Amoscato AA, and Lee YJ, “Low Glucose-Enhanced TRAIL Cytotoxicity Is Mediated Through the Ceramide–Akt–FLIP Pathway,” Oncogene 21, no. 3 (2002): 337–346. [DOI] [PubMed] [Google Scholar]
  • 27.Figueroa D, Asaduzzaman M, and Young F, “Real Time Monitoring and Quantification of Reactive Oxygen Species in Breast Cancer Cell Line MCF-7 by 2′, 7′–dichlorofluorescin Diacetate (DCFDA) Assay,” Journal of Pharmacological and Toxicological Methods 94 (2018): 26–33. [DOI] [PubMed] [Google Scholar]
  • 28.Chen Y, Wang F, Wu P, et al. , “Artesunate Induces Apoptosis, Autophagy and Ferroptosis in Diffuse Large B Cell Lymphoma Cells by Impairing STAT3 Signaling,” Cellular Signalling 88 (2021): 110167. [DOI] [PubMed] [Google Scholar]
  • 29.Jiang Z, Wang Z, Chen L, et al. , “Artesunate Induces ER-Derived-ROS-Mediated Cell Death by Disrupting Labile Iron Pool and Iron Redistribution in Hepatocellular Carcinoma Cells,” American Journal of Cancer Research 11, no. 3 (2021): 691–711. [PMC free article] [PubMed] [Google Scholar]
  • 30.Liu W, Zhou H, Lai W, et al. , “Artesunate Induces Melanoma Cell Ferroptosis and Augments Antitumor Immunity Through Targeting Ido1,” Cell Communication and Signaling 22, no. 1 (2024): 378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Roh JL, Kim EH, Jang H, and Shin D, “Nrf2 Inhibition Reverses the Resistance of Cisplatin-Resistant Head and Neck Cancer Cells to Artesunate-Induced Ferroptosis,” Redox Biology 11 (2017): 254–262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Westphal D, Kluck RM, and Dewson G, “Building Blocks of the Apoptotic Pore: How Bax and Bak Are Activated and Oligomerize During Apoptosis,” Cell Death & Differentiation 21, no. 2 (2014): 196–205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Gamaley IA and Klyubin IV, “Roles of Reactive Oxygen Species: Signaling and Regulation of Cellular Functions,” International Review of Cytology 188 (1999): 203–255. [DOI] [PubMed] [Google Scholar]
  • 34.Venkataraman S, Wagner BA, Jiang X, et al. , “Overexpression of Manganese Superoxide Dismutase Promotes the Survival of Prostate Cancer Cells Exposed to Hyperthermia,” Free Radical Research 38, no. 10 (2004): 1119–1132. [DOI] [PubMed] [Google Scholar]
  • 35.Song JJ and Lee YJ, “Catalase, but Not MnSOD, Inhibits Glucose Deprivation-Activated ASK1-MEK-MAPK Signal Transduction Pathway and Prevents Relocalization of Daxx: Hydrogen Peroxide as a Major Second Messenger of Metabolic Oxidative Stress,” Journal of Cellular Biochemistry 90, no. 2 (2003): 304–314. [DOI] [PubMed] [Google Scholar]
  • 36.Song JJ and Lee YJ, “Differential Role of Glutaredoxin and Thioredoxin in Metabolic Oxidative Stress-Induced Activation of Apoptosis Signal-Regulating Kinase 1,” Biochemical Journal 373, no. 3 (2003): 845–853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Murphy MP, “How Mitochondria Produce Reactive Oxygen Species,” Biochemical Journal 417, no. 1 (2009): 1–13. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Research materials that were generated in the studies will be made freely available to the scientific research community as soon as this manuscript has been documented in a publication. Raw data were generated at the Cedars-Sinai Medical Center. Derived data supporting the findings of this study are available from the corresponding author, Dr. Yong J. Lee, upon request.

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