JONT Journal

Introduction

Artesunate (ART) is a derivative of artemisinin which is isolated from Artemisia annua [1]. ART is an antimalarial agent, and it is widely used for the treatment of Plasmodium falciparum infection [2]. Reactive oxygen species (ROS) generation by ART can cause damage in proteins, lipids, and nucleic acids in parasites, which can lead to the parasites’ death [3]. Due to its ROS generation capacity, the anticancer properties of ART have been studied in various cancer models [4-7].

The anticancer mechanism of ART has been extensively studied in both in vitro and in vivo models [8]. ART-induced cell deaths are executed through apoptosis, autophagy and autophagy. In A375 melanoma cells, ART treatment induces inhibited expression of signal transducer and activator transcription-3 (STAT-3), matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP9), myeloid cell leukemia 1 (Mcl-1) and B-cell lymphomaextra large (Bcl-xL) which leads to apoptosis [4]. ART induces autophagy through reactive oxygen species (ROS) mediated endoplasmic reticulum (ER) stress and up-regulation of binding immunoglobulin protein (BiP), inositol-requiring enzyme 1 alpha (IRE1α), phosphorylated IRE1α (p-IRE1α), C/EBP homologous protein (CHOP), and death receptor 5 (DR5) in HCT116 and SW480 colon cancer cells [9]. In diffuse large B cell lymphoma (DLBCL) cells, ART treatment downregulates STAT-3 signaling and induces apoptosis, autophagy and ferroptosis [10]. In chronic myeloid leukemia (CML) KBM-5 xenograft model, intraperitoneal injection of ART inhibits p38, extracellular signal-regulated kinase (ERK), signal transducer and activator of transcription 5 (STAT5), cAMP response element-binding protein (CREB) activation, and tumor growth [11]. A dose-dependent tumor regression has been reported in ART treated Panc-1 xenograft tumor model [12].

However, a modest clinical efficacy and dose-limiting toxicities were reported in a phase I study of intravenous ART received cancer patients [13]. The reason for the modest clinical efficacy is perhaps due to nonspecific targeting and rapid clearance from the body. To overcome these hurdles, especially for targeting cancer cells, ART was loaded into poly-D, L-lactide-co- glycolide (PLGA), used as nanoparticles for drug delivery purpose [14, 15]. In addition, to locate and kill cancer cells, ART conjugation with dyes has been developed for theranostic purposes [16, 17].

The major purpose of theranostics is to specifically target and kill cancer cells while not affecting normal cells. Heptamethine cyanine dyes (HMCDs) are a class of fluorescent dyes, which are widely used for biomedical imaging due to their near-infrared (NIR) emission, and noninvasive nature of NIR light [18]. HMCDs such as IR-780, IR-783, MHI-148, and DZ-1 have been extensively studied for tumor targeting [19-21]. Aberrant and over expression of organic anion transporting polypeptides (OATPs) in cancer cells mediates cellular uptake of HMCDs [22, 23]. Among these dyes, DZ-1 shows high tumor selectivity, excellent stability and easy chemical nature for conjugation [24-26].

In this study, we conjugated DZ-1 to ART and investigated the cellular cytotoxic effects, uptake, mode of action, and molecular mechanisms underlying the cytotoxicity of the DZ-1-ART conjugate. The results showed mitochondria and lysosomesmediated cellular uptake, and apoptosis involved cell deaths in DZ-1-ART treated cells. Interestingly, Bax (Bcl‐2-associated X protein), Bak (Bcl-2 antagonist/killer 1) and Bid (BH3 interactingdomain death agonist)- independent DZ-1-ART-induced apoptosis, but caspase-3-dependent DZ-1-ART-induced apoptosis was observed.

Methods Cell Culture

Human colon cancer cell line HCT116, human pancreatic cancer cell line BxPC-3, and human breast cancer cell line MCF-7 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Bax knockout (Bax-/-), Bak knockout (Bak-/-), Bid knockout (Bid-/-) and double knockout Bax and Bak (Bax−/−Bak−/−) HCT116 cells were obtained from Dr. B. Vogelstein (Johns Hopkins University, Baltimore, MD, USA). These cell lines were maintained in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco, Grand Island, NY, USA). MCF-7/Vector and MCF-7/Caspase3 were obtained from Dr. C. J. Froelich (Northwestern University, Chicago, IL, USA). These cell lines were maintained in Dulbecco’s Modified Eagle medium (DMEM) (Gibco, Grand Island, NY, USA). These media were maintained with 10% fetal bovine serum (FBS) (R&D Systems, McKinley Place NE, MN, USA) and cultured in a humidified atmosphere of 5% CO2 at 37ºC.

Chemical and Reagents

ART (Cat#A3731), and E-64 (Cat#E3132-1 MG) were purchased from Millipore Sigma (Saint Louis, MO, USA) Pepstatin A (Cat#1190/10) was purchased from Bio-Techne (Minneapolis, MN, USA). Z-VAD-FMK (Cat# S7023) and Z-DEVD-FMK (Cat# S7312) were purchased from Selleckchem (Houston, TX, USA). DZ-1 and DZ-1-ART were synthesized by Dr. Yi Zhang (CedarsSinai Medical Center, Los Angeles, CA, USA).

Cell Survival Assay

For cell survivals, trypan blue exclusion assay was used. To differentiate live and dead cells, after specific time of DZ-1-ART, E-64, pepstatin A, Z-VAD-FMK and Z-DEVD-FMK treatment, cells were stained with 0.4% trypan blue. LUNA™ Automated Cell Counter (L10001, Logos BioSystem, Anyang, Gyeonggi-do, South Korea) was used to count dead cells and live cells.

Immunoblotting and Antibodies

Immunoblotting was performed as previously described [27]. Primary antibodies, such as anti- PARP-1 (Rabbit mAb; Cat #9532S), anti-caspase-8 (Mouse mAb; Cat #9746S), anti-cleaved caspase-9 (Rabbit mAb; Cat #7237S), anti-caspase-3 (Mouse mAb; Cat#9668S), anti-cleaved caspase-3 (Rabbit mAb; Cat# 9664S), anti-Bak (Rabbit mAb; Cat #12105S), anti-Bax (Rabbit pAb; Cat #2772S), anti-Bid (Rabbit pAb; Cat#2002S), anticathepsin B (Rabbit mAb; 31718S) and anti-cathepsin D (Rabbit pAb; 69854S) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-β-actin (Mouse mAb; Cat #A1978) was purchased from Sigma- Aldrich (Saint Louis, MO, USA). For secondary antibodies, anti-rabbit IgG-HRP (Cat#7074P2) and goat anti-mouse IgG-HRP were obtained from Cell Signaling Technology (Danvers, MA, USA) and Santa Cruz Biotechnology (Dallas, TX, USA), respectively.

Annexin V/PI Staining

To distinguish early and late apoptotic cell deaths, annexin V/PI staining was used. For this purpose, annexin V-FITC/PI apoptosis detection kit (Cat#HY-K1073, Med Chem Express, Monmouth Junction, NJ) was used as per the manufacturer’s instructions. Briefly, HCT116 or BxPC-3 cells were seeded in 12-well plates. After specific time of treatment, cells were washed with cold 1X Dulbecco’s phosphate-buffered saline phosphate buffered saline (DPBS) (Gibco, Grand Island, NY, USA), and trypsinized with 0.25 trypsin EDTA (Gibco, Grand Island, NY, USA). Trypsinized cells were resuspended with complete medium and centrifuged at 1,000g for 5 minutes at 4ºC. The media was discarded, and cells were resuspended with ice-cold PBS. Resuspended cells were again subjected to centrifugation and supernatant was discarded. Cells were resuspended in binding buffer followed by staining with annexin V-FITC and PI. The stained cells were incubated at room temperature for 10 minutes. After 10minutes, cells were then washed with PBS and treated with Hoechst for 10 minutes. To capture fluorescence images, an ECHO fluorescence microscope was used.

Mitochondria labeling

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

JC-1 Assay

To analyze mitochondrial membrane potential (ΔΨm), JC-1 dye (mitochondrial membrane potential probe, Cat#T3168, ThermoFisher Scientific, Eugene, OR, USA) was used. 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.

Lysosomes Labeling

To label lysosomes, LysoTracker™ Green DND-26 (Cat# L7526,

ThermoFisher Scientific, ThermoFisher Scientific, Eugene, OR, USA) kit was used. After 24 treatment of ART, DZ-1, DZ-1-ART, cells were treated with LysoTracker probe containing media. To visualize nucleus, Hoechst staining was used. The images were captured using an ECHO fluorescence microscopy.

Statistical Analysis

One-way analysis of variance, followed by Tukey’s post hoc test were used for statistical analysis. For this purpose, GraphPad Prism 9 software was used. 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 ****P<0. 0001.

Results

DZ-1-ART induces apoptosis in time dependent manner.

DZ-1-ART was synthesized by conjunction of DZ-1 and ART (Fig. 1A). To define the time intervals effects of DZ-1-ART, HCT116 and BxPC-3 cells were treated with 20 µM of DZ-1- ART. After the treatments, both cells were harvested at 4 h, 8 h and 24 h and trypan blue exclusive assay was performed for cell survival analysis. The results from cell survival analysis clearly indicated the gradual increase of cellular cytotoxicity of DZ-1-ART in both HCT116 and BxPC-3 cells (Fig. 1B). Western blotting of DZ-1-ART treated cell lysates confirmed cleavage of poly (ADP-ribose) polymerase 1 (PARP-1), which implies the confirmation of apoptosis in HCT116 and BxPC3 cells. Furthermore, cleaved caspase-8 and cleaved caspase-9 expression supported the involvement of DZ-1-ART-induced apoptosis (Fig. 1C). In addition, Annexin V- FITC/PI staining was performed for more confirmation. In both cells, early apoptosis was observed after 4 h of DZ-1-ART treatment while late apoptosis was observed in both 8 h and 24 h of the treatment (Fig. 1D&E).

Figure 1: DZ-1-ART induces apoptosis in time dependent manner. (A) Chemical structure of DZ-1-ART. (B) HCT116 and BxPC-3 cells were treated with 20 µM of DZ-1-ART at various time intervals. Trypan blue exclusion assay was performed for cell survival analysis. For statistical analysis, one-way ANOVA and Tukey’s multiple comparison test were used (****P<0.0001). From triplicate experiments, mean ± SD are shown as error bar. (C) Cells were harvested using 1X lysis buffer, followed by SDS-PAGE for immunoblotting analysis. Immunoblotting was performed using indicated antibodies. (D) and (E) Annexin V assay was performed for apoptosis detection in HCT116 and BxPC-3 cells.

DZ-1-ART localizes through lysosomes and mitochondria

Previous studies reported that DZ-1 conjugated analogs translocate via lysosomes and mitochondria [16,17]. Conjugation of ART with DZ-1 may alter its translocating pathways. To explore this, HCT-116 cells were treated with 10 µM of ART, DZ-1 and DZ-1-ART for 24 h.

After 24 h, treated cells were stained with LysoTracker Green DND-26 dye and Hoechst. DZ-1- ART treated cells showed higher (NIRF) red fluorescence than DZ-1 treated cells, while control and ART treated cells showed no red fluorescence. Green fluorescence (LysoTracker) was observed in control, ART, DZ-1 and DZ-1-ART treated cells, which confirmed lysosomes staining (Fig. 2A). Taken together, these results confirmed the lysosomal translocation of DZ-1- ART. To check the localization of DZ-1-ART via mitochondria, after 24 h of 10 µM treatment of ART, DZ-1 and DZ-1-ART, HCT116 cells were stained with MitoTracker Green FM dye. Fluorescence microscopy analysis indicated higher red (NIRF) fluorescence with green fluorescence (MitoTracker) in DZ-1-ART treated cells (Fig. 2B). These results supported the mitochondria mediated translocation of DZ-1-ART.

Figure 2: DZ-1-ART translocates through lysosomes and mitochondria. HCT116 cells were treated with 10 µM of ART, DZ-1 and DZ-1-ART for 24 h. (A) For lysosomal location, cells were stained with LysoTracker Green DND-26 dye. (B) Cells were stained with MitoTracker Green FM dye for mitochondrial localization assay. A fluorescence microscope was used to capture images (scale bar 100 µm).

Role of cathepsin B and cathepsin D in DZ-1-ART-induced apoptosis

The roles of cathepsin B (CatB) and cathepsin D (CatD) in DZ-1-ART-induced apoptosis were investigated, since LysoTracker staining revealed lysosomal localization of DZ-1-ART. To investigate CatB roles, HCT116 cells were treated with 10 µM of DZ-1-ART and CatB inhibitor, E-64 (25 µM and 50 µM). Trypan blue exclusion assay indicated high toxicity in 50 µM of E-64 alone and DZ-1-ART and 50 µM treated cells, which indicated cytotoxicity of 50 µM of E-64, whereas 25 µM of E-64 and DZ-1-ART treated cells showed no significant inhibition (Fig. 3A). Western blotting analysis also indicated no CatB mediated inhibition in DZ-1-ART-induced apoptosis (Fig. 3B). HCT116 cells were treated with 10 µM of DZ-1-ART and various concentrations of CatD inhibitor, pepstatin A (10 µM, 25 µM and 50 µM). Cell survival analysis indicated no significant difference between DZ-1-ART and DZ-1-ART with pepstatin A treated cells (Fig. 3C). Immunoblotting results confirmed no inhibition of PARP-1 cleavage in DZ-1- ART and pepstatin A treated cells when compared with DZ-1-ART treated cells (Fig. 4D).

Figure 3: Role of cathepsin B and cathepsin D in DZ-1-ART-induced apoptosis. (A, B) HCT116 cells were treated with 25 µM and 50 µM of E-64 (cathepsin B inhibitor, with or without DZ-1-ART). (A) Trypan blue exclusion assay was performed for cell survival assay. (B) Treated cells were harvested using 1X lysis buffer then SDS -PAGE was performed for immunoblotting analysis. Membrane was developed using indicated antibodies. (C, D) For cathepsin D inhibition, HCT116 cells were treated with various concentration (25 µM, 50 µM and 100 µM) of pepstatin A (cathepsin D inhibitor, with or without DZ-1-ART). (C) Cell death per centage was quantified by trypan blue assay. (D) Immunoblotting was performed against indicated antibodies after cells harvesting and SDS-PAGE. For (A) and (C) values are shown as mean ± SD from triplicates. One-way ANOVA and Tukey’s multiple comparison test were used for statistical analysis (****P <0.0001).

DZ-1-ART disrupts mitochondrial membrane potential

Since lysosomal-mediated cathepsins did not participate in DZ-1-ART-induced apoptosis, mitochondria-associated apoptosis was investigated. For this purpose, mitochondrial membrane potential (MMP) (Δψm) was measured in ART, DZ-1 and DZ-1-ART treated HCT116 cells. After 24 h of the treatments, cells were stained with JC-1 dye and followed by Hoechst staining. Fluorescence microscopy analysis showed green fluorescence in DZ-1-ART treated cells, which indicated monomeric form of JC-1 dye. This green fluorescence confirmed the lower Δψm in DZ-1-ART treated cells. Red fluorescence ranges in control, ART and DZ-1 treated cells showed higher, medium and lower levels of intensity, respectively (Fig. 4). These results directly confirmed low Δψm in DZ-1-ART treated cells.

Figure 4: DZ-1-ART disrupts mitochondrial membrane potential. ART, DZ-1 and DZ-1-ART treated cells were stained with JC-1 dye and followed by Hoechst staining. Images were captured using a fluorescence microscope (scale bar:100 µm).

DZ-1-ART induces apoptosis through caspase activation pathways

Since JC-1 assay confirmed MMP disruption in HCT116 cells, caspase activation was investigated. For this purpose, HCT116 cells were treated with 10 µM of DZ-1-ART and various concentrations of pancaspase inhibitor, Z-VAD-FMK. Trypan blue exclusion assay results showed inhibition of cytotoxicity in DZ-1-ART and Z-VAD-FMK treated cells (dose-dependent manner) (Fig. 5A). Immunoblotting results showed inhibition of PARP-1 cleavage, cleaved caspase-8, cleaved caspase-9 and cleaved caspase-3 in DZ-1-ART and Z-VADFMK treated cells (Fig. 5B). Collectively, these results confirmed caspase activation pathways in DZ-1-ART treated cells. To define specific caspase involvement in DZ-1-ART-induced apoptosis, HCT116 cells were treated with 10 µM DZ-1-ART and various concentrations of caspase-3 inhibitor, Z- DEVD-FMK. Trypan blue assay revealed dose-dependent cytotoxicity inhibition in DZ-1-ART and Z-DEVD-FMK treated cells (Fig. 5C). Immunoblotting analysis reported inhibition of PARP-1 cleavage and caspase-3 cleavage in DZ-1-ART and Z-DEVD-FMK treated cells (Fig. 5D).

Figure 5: DZ-1-ART induces apoptosis through caspase activation pathways. (A, B) For pancaspase inhibition, HCT116 cells were pretreated with various concentrations of Z-VAD- FMK (10 µM, 20 µM and 40 µM), followed by 10 µM of DZ-1-ART. (A) Trypan blue exclusive assay was performed for cytotoxicity quantification. (B) Immunoblotting was used to detect indicated antibodies after harvesting treated cells. (C, D) For caspase-3 inhibition, various concentrations of Z-DEVD-FMK (25 µM, 50 µM and 100 µM) pretreated with HCT116 cells and followed by DZ-1-ART treatment. (C) For cytotoxicity quantification, trypan blue exclusive assay was performed. (D) HCT116 cells were harvested after 24 h of treatment, followed by SDS-PAGE. Immunoblotting was done for indicated antibodies. For (A) and (C), values represent mean ± SD from triplicates. For statistical analysis, one-way ANOVA and Tukey’s multiple comparison test were used (****P <0.0001).

DZ-1-ART induces caspase-3-dependent apoptosis

MCF-7/Vector and MCF-7/Caspase 3 cells were treated with 20 µM of DZ-1-ART and harvested at various time points (4 h, 8 h and 12 h) for trypan blue exclusion assay. DZ-1-ART-induced cell death was observed after 4 h of treatment in MCF-7/Caspase 3 cells, while less cell death was seen in MCF-7/Vector cells (Fig. 6A). Immunoblotting results from DZ-1-ART treated MCF-7/Caspase 3 cells indicated time dependent manner of PARP-1 cleavage, whereas DZ-1- ART treated MCF-7/Vector cells showed no PARP-1 cleavage, due to absence of caspase 3 (Fig. 6B). Z-VAD-FMK and DZ-1-ART treatment at various time points (1-4 h) showed no effect in MCF-7/ Vector cells (Fig. 6C), but the same treatment showed Z-VAD-FMK-mediated apoptosis inhibition in MCF-7/Caspase 3 cells (Fig. 6D). Taken together, these results confirmed caspase- 3-dependent apoptosis in DZ-1-ART-induced apoptosis.

Figure 6: DZ-1-ART induces caspase-3-dependent apoptosis. (A, B) MCF-7/Vector and MCF- 7/Caspase 3 cells were treated with 20 µM of DZ-1-ART. (A) DZ-1-ART treated cells were trypsinized at various time intervals (4-12 h) for trypan blue exclusion assay. Oneway ANOVA and Tukey’s multiple comparison test were performed for statistical analysis, (****P <0.0001). (B) DZ-1-ART treated cells were harvested after indicated time intervals for immunoblotting. Harvested samples were subjected to SDS-PAGE and blotting to indicated antibodies. MCF-7/Vector cells (C) or MCF-7/Caspase 3 cells (D) were treated with 20 µM of DZ-1-ART and 40 µM of Z-VAD-FMK. These treated cells were harvested at various time intervals (1-4 h) and immunoblotting was performed using indicated antibodies.

DZ-1-ART induces Bax, Bak, and Bid-independent apoptosis

To confirm Bid involvement in DZ-1-ART-induced apoptosis, HCT116 WT and HCT116 Bid-/- cells were treated with 1 µM, 5 µM and 10 µM of DZ-1-ART. Cytotoxicity was observed in 10 µM DZ-1-ART treated HCT116 WT and HCT116 Bid-/- cells (Fig. 7A). Immunoblotting results indicated PARP-1 cleavage in both cells (Fig. 7B). These results supported Bid-independent apoptosis in DZ-1ART-induced apoptosis. To reveal Bax and Bak roles in DZ-1-ART-induced apoptosis, HCT116 WT, HCT116 Bax-/-, HCT116 Bak-/-, HCT116 Bax-/-Bak-/- cells were treated with 5 µM and 10 µM of DZ-1-ART. Significant cell deaths were observed in 10 µM DZ1- ART treated HCT116 WT, HCT116 Bax-/-, HCT116 Bak-/- and HCT116 Bax-/-Bak-/- cells (Fig. 7C).Western blotting analysis indicated PARP-1 cleavage in 10 µM DZ-1-ART treated cells (Fig. 7D). These results confirmed Bax and Bak-independent apoptosis of DZ-1-ART-induced apoptosis.

Figure 7: DZ-1-ART induces Bax, Bak, and Bid-independent apoptosis. (A, B) HCT116 WT, HCT116 Bid-/- cells were treated with 1 µM, 5 µM and 10 µM of DZ-1-ART. (A) After 24 h of treatment, cells were trypsinized for cell survival analysis. (B) Immunoblotting was performed for indicated antibodies. (C, D) HCT116 WT, HCT116 Bax-/-, HCT116 Bak-/- and HCT116 Bax-/- Bak-/- cells were subjected to 5 µM and 10 µM of DZ-1-ART treatment for 24 h. (C) The treated cells were trypsinized for trypan blue exclusion assay. (D) The treated cells were harvested and immunoblotting was performed for indicated antibodies. For (A) and (C), one-way ANOVA and Tukey’s multiple comparison test were done for statistical analysis, (****P<0.0001).

Discussion

Targeting therapy enables specific targeting and killing of tumor cells while unaffecting normal cells. To achieve these properties, anticancer agents can be conjugated with biopolymers, small molecules and fluorescence dyes. In this study, DZ-1-ART (conjugated form of ART with NIR dye DZ-1) has been used and cytotoxicity and cell death mechanisms have been evaluated in vitro. This study reveals time-dependent manner DZ-1-ART-induced cytotoxicity in HCT116 and BxPC-3 cells (Fig. 1A), and MCF-7 cells (Fig. 5). It also shows apoptosis-mediated cell death through caspases activation (Fig. 1B). ART induces ferroptosis in various cancer cells [28-31]. However, conjugation of ART and DZ-1 may alter the mechanism of cell death.