The Antimalarial Drug Pyronaridine Inhibits Topoisomerase II in Breast Cancer Cells and Hinders Tumor Progression In Vivo

Abstract

Background:

Breast cancer is the most frequently diagnosed cancer in women worldwide. Pyronaridine (PND), an antimalarial drug, was shown to exert anticancer activity on seventeen different human cancer cells, seven from female breast tissue. Additionally, PND induced apoptosis via mitochondrial depolarization, alteration of cell cycle progression, and DNA intercalation. However, the molecular target of PND in cells was not elucidated.

Objective:

Here, we have further investigated PND’s mode of action by using transcriptome analysis. Preclinical studies were also performed to determine whether PND could affect tumor progression in a human breast cancer xenograft in mice. Moreover, we assessed the combined efficacy of PND with well-known anticancer drugs.

Methods:

Transcriptome analyses of PND-treated cancer cells were performed. Topoisomerase II activity was evaluated by an in vitro assay. In addition, daily oral administration of PND was given to mice with human breast cancer xenografts. The differential nuclear staining assay measured in-vitro cell toxicity.

Results:

The transcriptome signatures suggested that PND might act as a topoisomerase II inhibitor. Thus, topoisomerase inhibition assays were performed, providing evidence that PND is a bona fide topoisomerase II inhibitor. Also, in-vivo studies suggest that PND hinders tumor progression. Besides, combination studies of PND with anticancer drugs cisplatin and gemcitabine revealed higher cytotoxicity against cancer cells than individual drug administration.

Conclusion:

The findings provide evidence that PND is a topoisomerase II inhibitor and can hinder cancer progression in an animal model, further demonstrating PND’s favorable characteristics as a repurposed anticancer drug.

1. INTRODUCTION

The search for an effective treatment for many diseases has led researchers to look into well-known drugs that may have additional curative properties, also known as drug repurposing. There are many benefits of using an existing drug over a newly developed drug. When there is an opportunity to utilize this strategy, the risk of failure validating the discovered medication is lower. The time frame from drug development can be reduced considerably, and there is less financial investment needed [1]. Currently, a severe clinical-problem in the field of cancer treatment is the emergence of multidrug resistance (MDR). While different approaches have been utilized to combat MDR, they have not been successful against a variety of tumor types, and there is an urgent need for new treatment tactics [2]. Thus, drug repurposing has gained attention as a productive approach for anticancer drug discovery [1, 3, 4]

Previously, we reported that the antimalarial drug Pyronaridine Tetraphospate (PND) shows potential as a well-tolerated anticancer drug [5]. In this study, we elucidate one of the mechanisms by which PND induces cell death and propose PND as a repurposed drug for the treatment of cancer. Pyronaridine has been shown to exert cytotoxic activity on 17 different cancer cell lines, as well as induce cell death via apoptosis [5]. To further investigate the anticancer potential of PND, we sought to determine its mode of action in cancer cells, more specifically on breast cancer cells. Our previous study demonstrated PND to be more cytotoxic and selective in most of the breast cancer cell lines [5]. Breast cancer is the most common cancer in women worldwide, and approximately 1 in 8 women will be diagnosed with invasive breast cancer in their lifetime [6, 7]. Currently, local and systemic treatment options are available to treat this type of cancer. These include surgery, radiation, neoadjuvant therapy, endocrine therapy, anti-HER2 targeting and chemotherapy [8]. While there are multiple therapies available to treat a variety of breast cancer subtypes, there is a possibility of reoccurrence and drug resistance [4, 6, 9, 10]. Thus investigating PND’s mode of action in breast cancer cells was deemed of high importance.

Moreover, prior studies had revealed that PND acts as a topoisomerase II inhibitor in Plasmodium falciparum [11]. We previously demonstrated that PND intercalates with DNA [6], and in this study, we sought to confirm that PND is a bona fide topoisomerase II inhibitor in mammalian cells. Through transcriptome analysis performed on the MDA-MB-231 breast cancer cell line, we were able to determine that PND induces gene signatures that correlate with those of known topoisomerase II inhibitors. Furthermore, we demonstrate that PND inhibits topoisomerase II in a dose-dependent manner by using the decatenation-supercoiling assay. In addition, we report the first pilot study in which PND is used to reduce tumor size in mice with human breast cancer xenografts. Additionally, we show combinational studies of PND with well-known anticancer drugs to elucidate their cytotoxic potential. Overall, our data strongly suggest that PND is a strong candidate for anticancer therapy.

2. MATERIAL AND METHODS

2.1. Cell lines and culture conditions

The human triple-negative breast cancer MDA-MB-231 cell line (ATCC, Manassas, VA, USA), and its lung metastatic (LM) derivative MDA-MB-231 LM2–4 [12] were cultured in DMEM (Hyclone, Logan UT) supplemented with 10% heat-inactivated fetal bovine serum (FBS: Hyclone), 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific Inc. Rockford, IL). Consistently, all cell lines were incubated at 37°C in a 5% CO₂ humidified atmosphere.

2.2. Preparation of Drugs

Pyronaridine tetraphosphate (PND; 2-methoxy-7-chloro-10[3,5-bis(pyrrolidinyl-1-methyl)-4hydroxyphenyl] aminobenzyl-(b)-1,5-naphthyridine; APExBIO, Houston, TX, USA) was prepared, as previously described [5]. PND was dissolved in Dulbecco’s Phosphate Buffered Saline (PBS; Sigma-Aldrich, St Louis, MO, USA). Cis-diammine-platinum(ll) dichloride (cisplatin) and etoposide, both purchased from Sigma-Aldrich (St Louis, MO USA) were dissolved in PBS and dimethyl sulfoxide (DMSO; Sigma-Aldrich, St Louis, MO, USA), respectively. Gemcitabine was purchased from Selleck Chemicals (Houston, TX, USA), and a stock solution of 1 mM was dissolved in PBS. Thus, aliquots of specific drug dilutions were added directly to wells containing cells in culture media as necessary. Furthermore, PND was dissolved in sterile Milli-Q water that was administered orally during all the in-vivo series of experiments.

2.3. Transcriptome Analyses

Transcriptomic analyses capture a snapshot at a given time of the total number of transcript copies existing in cells [13]. For this purpose, the MDA-MB-231 cells were seeded in 6 well plates at 1,000,000 cells/1 mL. After seeding, cells were exposed to 11 μM PND and incubated for 6 h. Additionally, transcripts from cells incubated with PBS, as solvent control, were also examined and used as a reference for the analysis. After incubation, the cells were harvested, washed, centrifuged, and the supernatant was discarded. The cell pellets were stored at −80°C. Three independent biological replicates were executed for each treatment. The total RNA extraction was performed the next day by using the RNeasy Mini Kit (Qiagen;74,104). After RNA extraction, samples were incubated with 10 μL of DNase I for 15 min at room temperature. The RNA was quantified using Qubit Fluorometer (Thermo Fisher; Q33216), and its integrity was examined using TapeStation 2200 (Agilent Technologies, Santa Clara, CA). The Truseq Stranded mRNA kit was implemented to produce pair-ended libraries, and the RINe from the TapeStation was used to ensure high-quality samples. The transcriptome samples were loaded into the NextSeq 500 system for sequencing (Illumina, San Diego, CA).

2.4. Connectivity Map (CMAP) Analysis

The CMap database contains a large collection of genome-wide transcriptional expression data of drug-treated cancer cells and allows for comparisons of data across experimental platforms of common gene-expression changes [14–16]. The transcriptomics perturbation molecular signatures induced by PND on MDA-MB-231 cells were used as inputs to query CMap using itsclue.io tool (https://clue.io) [14], in order to obtain similarity scores for the signatures associated to the small molecule perturbagens available at CMap.

2.5. Topoisomerase II Enzyme Inhibitory Assay

The PND topoisomerase inhibitory effect was determined by the decatenation of a kinetoplast (k) DNA [17]. The assay was performed according to the manufacturer’s instructions (TopoGEN, Buena Vista, CA, USA). A typical reaction mixture of 20 μl volume includes the following reactants: 0.5 M Tris- HCl (pH8), 1.50 M NaCl, 100 mM MgCl₂, 5 mM dithiothreitol, 300 μg BSA/ml, and 20 mM ATP in water. Samples were pre-incubated at 37°C for 30 minutes prior to the addition of the topoisomerase II enzyme. Subsequently, the reaction was initiated by the addition of the topoisomerase II (diluted 1:2) and incubated for 30 min at 37°C. The reactions were stopped using 5x stop buffer (5% Sarkosyl, 0.125% bromophenol blue, 25% glycerol). Next, samples were analyzed by using 1% (w/v)-agarose-gel electrophoresis dissolved in TAE buffer (0.04 M Tris base, 0.04 M acetate and 0.001 M EDTA) pH 8.0. Afterward, were stained with ethidium bromide (EtBr; 0.5 μg/ml) and imaged through the Gel Doc XR+ imaging system (Bio-Rad, Hercules, CA, USA). The digital images were used to analyze the migration patterns.

2.6. In-vivo Tumor Growth Assessment

C.B-17/IcrHan®Hsd-Prkdc severe combined immunodeficiency (SCID) female mice were purchased from Envigo (Indianapolis, IN). After a two-week acclimation period, we performed subcutaneous implantation of tumor cells to the right flank of the mice. Prior to injection, MDA-MB-231 LM2–4 cells were harvested with 0.25% trypsin, washed with PBS, and resuspended in DMEM. A total of 2.5 million cells /200 μl per mouse were implanted. Mice were monitored daily, observing for variations in body weight and tumor growth. Vernier calipers measured tumor volume and calculated using the formula (length × width²)/2. Experimental endpoints were determined based on institutional guidelines. Results were plotted as event-free survival (Kaplan-Meier analysis) over time. Mice with the primary tumor size progression beyond 1200 mm³ or ≥15% weight loss were euthanized as previously described [18].

2.7. Differential Nuclear Staining Assay

To examine the PND cytotoxic effect in combination with well-known anticancer drugs, the Differential Nuclear Staining (DNS) assay was utilized [19]. Cells were seeded at a density of 10,000 cells/well in 96-well plates and incubated overnight. Cells were then treated with a concentration gradient of cisplatin (95.4 μM, 47.7 μM, 23.8 μM, 11.92 μM, 5.96 μM) or gemcitabine (0.44 μM, 0.22 μM, 0.11 μM, 0.05 μM, 0.02 μM) and incubated for 24 h. Next, 5.5 μM of PND, its CC₅₀ at 24 h, was administered to drug-treated wells, and an additional incubation of 24 h was implemented. The CC₅₀ stands for the cytotoxic concentration of compound that results in 50% cell death. Approximately 2 hours before imaging, a mix of Hoechst 33342 and propidium iodide (PI; Invitrogen) was added to each well at a final concentration of 5 μg/ml each fluorophore. Image acquisition and data analyses, including image segmentation, were performed using the IN Cell Investigator software, specifically designed to assist the IN Cell 2000 bioimager system (GE Healthcare Life Sciences, Pittsburgh, PA). In the study, the following controls were utilized: PBS as solvent/vehicle control, 5.5 μM PND to assess the individual PND cytotoxicity on these cell lines, and hydrogen peroxide (H₂O₂) as a control for cytotoxicity. For each treatment, three biological replicates were performed.

2.8. Statistical Analysis

Each experimental point denotes at least three independent measurements. The statistical significance was determined through the two-tailed paired Student’s t-test (http://studentsttest.com/). A P-value of ≤ 0.05 between the two treatments was deemed significant. For the in-vivo experiments, statistical analysis was performed using GraphPad Prism 7.0 (GraphPad Software, San Diego, CA USA). Tumor growth results are reported as mean and standard deviations to denote the level of variability; the statistical difference was determined through Welch’s t-test. The survival data were depicted by using the Kaplan-Meier analysis and were tested for survival differences using the Gehan – Breslow- Wilcoxon test. The level of significance was set at P<0.05.

3. RESULTS

3.1. Transcriptome Analysis of the Effects of PND Reveals Similarities with known Topoisomerase Inhibitors

Expression signatures of PND-treated cells obtained through a transcriptome analysis were compared to the expression signatures of drug-treated cells. The connectivity map (CMap) is a collection of genome-wide transcriptional expression data and allows for comparisons of data across experimental platforms of common gene-expression changes [13, 14]. The data from the CMap comparison reveals that PND gene expression pattern in MDA-MB-231 cells is very similar to the gene expression profile of drugs that inhibit topoisomerase II, such as: pidorubicine, doxorubicin, and pirarubicin on other cancer cells (Fig. 1).

Fig. (1).

CMap comparison analyses revealed that the PND-induced gene expression profile is similar to that of known topoisomerase inhibitors.

Connections were viewed as a heat map ranked by the summary connectivity score across nine cancer cell lines. The heat-map shows the strongest hits in dark grey while lower or no effects are shown as lighter grey to white boxes. The type of perturbagens are shown in light grey (drug/small molecule) or dark grey (RNAi/other screen). Only the top 15 hits over 99% median score are shown.

3.2. PND Inhibits Topoisomerase II

To test the ability of PND to inhibit topoisomerase II, the decatenation of kinetoplast DNA (kDNA) was analyzed by using an agarose gel electrophoresis approach [17]. The preincubation of increasing concentrations of PND (5, 50, 500 μM) with the topoisomerase II enzyme, followed by the addition of kDNA, showed a dose-dependent inhibition of decatenation with complete inhibition of topoisomerase activity resulting in no decatenation at 500 μM of PND (Fig. 2; lanes 5–7). Etoposide, a well-known topoisomerase II inhibitor, included as a positive control, showed a partial enzyme inhibition with an incomplete decatenation at 1mM (Fig. 2; lane 8) as opposed to the negative control which included (enzyme + kDNA; fig. 2, lane 4), showing complete kDNA decatenation. Our results demonstrate that PND acts as a bona-fide topoisomerase II inhibitor.

Fig. (2). PND caused inhibition of topoisomerase II activity in a dose-dependent manner.

The gel loading lanes are numbered on top of the gel and indicate the following: (1) catenated kinetoplast DNA (kDNA); (2) decatenated kDNA; (3) linear kDNA; (4) kDNA + topoisomerase II; (5) kDNA + topoisomerase + 5 μM of PND; (6) kDNA + topoisomerase + 50 μM of PND; (7) kDNA + topoisomerase + 500 μM of PND; (8) kDNA + topoisomerase II + 1 mM etoposide; (9) kDNA + topoisomerase II + PBS; (10) kDNA + topoisomerase II + DMSO.

3.3. PND Inhibits Tumor Growth in Mice Carrying Human Breast Cancer Tumors

To evaluate whether PND’s activity translates to a reduction of tumor growth in-vivo, we tested PND on mice subcutaneously implanted with MDA-MB-231 LM2–4 tumor cells. Oral administration of PND at 160 mg/kg/day (n=3) inhibited tumor growth (P<0.0001 PND vs. control), as shown in Fig. 3A. Control mice with xenografted tumors and not treated with PND (n=5) did not prevent tumor growth, which proliferated rapidly as compared to PND-treated mice. To assess whether PND exerts any adverse and/or non-specific toxic side effects in-vivo, the bodyweight of PND-treated and untreated control mice was monitored concomitantly throughout the course of the experiment, as previously described [18]. Even though there was a significant difference between weights of control and treated mice P=0.0167, the weight remained constant within each group since the beginning of the study (data not shown). In addition, we used the Kaplan-Meier plot to show tumor responses, as shown in Fig. 3B. The time to 50% event-free survival of the PND treatment was 29 days compared to the control group, which was 23 days. Moreover, there was a significant impact on survival with a P-value of 0.041.

Fig. (3). Effect of PND treatment on tumor growth.

(A) Human MDA-MB-231 LM2–4 cells were implanted subcutaneously in Severe Combined Immunodeficient (SCID) mice. Therapy began when tumors reached the size of approximately 260 mm³; the control mice (n=5) received daily doses of Milli-Q water, and PND treated mice (n=3) received 160 mg/kg daily doses. P<0.0001 PND vs. control (mean values ± s.d.); B) In event-free survival analysis (Kaplan-Meier analysis), significant event-free survival was observed with PND P<0.05.

3.4. Cytotoxic Effect of PND in Combination with known Anticancer Drugs

The combinational effect of PND and well-known anticancer drugs was observed through the Differential Nuclear Staining (DNS) assay [19]. This DNS assay has been shown to be a simple, trustworthy, consistent, and suitable strategy for both primary and secondary screening strategies when searching for potential cytotoxic experimental compounds in vitro [19, 20]. Two clinical drugs, in particular, gemcitabine and cisplatin, were shown to have a higher cytotoxic effect when PND was added concomitantly (Fig. 4). A single concentration of PND was tested in combination with a concentration gradient of gemcitabine or cisplatin. As shown in Fig. 4A, the PND and gemcitabine combination showed a significant increase in cytotoxicity, when compared with gemcitabine (P= 5.92 E −07), or PND (P= 2.7 E −05) alone. A similar effect was observed when cisplatin was tested in combination with PND (Fig. 4B). Thus, it can be concluded from these results that the combination of PND with these well-known drugs produces higher cytotoxicity as compared to single-drug treatment. Therefore, our results demonstrate a synergistic effect of PND with each drug, resulting in more significant cell toxicity as compared to single-drug treatment.

Fig. (4). Testing PND cytotoxicity in combination with gemcitabine (A) or cisplatin (B) on MDA-MB-231 cells.

(A) MDA-MB-231 cells were pre-incubated with a concentration gradient of gemcitabine (CC₅₀, ½ CC₅₀, ¼ CC₅₀, ⅛ CC₅₀, $\raisebox{1ex}{$1$}\!\left/ \!\raisebox{-1ex}{$16$}\right.{\text{CC}}_{50}$) for 24 h, followed by a dose of 5.5 μM of PND and incubated for an additional 24 h. (B) MDA-MB-231 cells were pre-incubated with a concentration gradient of cisplatin (CC₅₀, ½ CC₅₀, ¼ CC₅₀, ⅛ CC₅₀, $\raisebox{1ex}{$1$}\!\left/ \!\raisebox{-1ex}{$16$}\right.{\text{CC}}_{50}$) for 24 h, followed by a dose of 5.5 μM of PND and incubated for an additional 24 h. The following controls were included: PBS as solvent control; PND CC₅₀ 24 h (5.5 μM); and 1 mM of H₂O₂ as a positive control for cytotoxicity incubated for 48 h. A P-value of ≤ 0.05 was considered significant between all comparisons. Two-tailed Student’s paired t-test of combination-treated cells with gemcitabine or cisplatin versus individual treatment (*) and PND only-treated (‡) cells provided consistent significant P (≤ 0.05) values, respectively. Representative results from three independent experiments are shown. Image acquisition, image segmentation, and data analyses were achieved by using the IN Cell Investigator software [19].

4. DISCUSSION

We previously reported that PND exhibited selective cytotoxicity towards cancer cells, as well as demonstrated its ability to intercalate with DNA [5]. In the present study, we aimed to investigate PND’s mode of action on cancer cells by conducting a whole transcriptome analysis. These analyses were used to compare the PND-induced expression signatures to those of other known drugs available on the CMap database. The CMap database encompasses gene profiles of human cancer cell lines exposed to chemicals and other perturbagens [13]. More specifically, the CMap database contains the gene expression profiles of nine cancer-derived human cell lines exposed to 27,927 perturbagens [14, 15]. These comparisons reveal that PND affects the expression of genes that are similar to the gene signatures of topoisomerase II inhibitors. To further confirm that PND is a topoisomerase II inhibitor, a biochemical assay was performed. The results demonstrate that PND inhibits topoisomerase II in a dose-dependent manner. Previous reports have shown that PND may act as a catalytic inhibitor and a topoisomerase II poison in mammalian cells, depending upon exposure concentration [11]. This demonstrates that PND’s activity as a topoisomerase inhibitor is not solely seen in P. falciparum but also in mammalian cells, and our data support that the mechanism of action by which PND causes cytotoxicity in cancer cells is through topoisomerase II inhibition and activation of apoptosis.

To investigate if PND can exert its activity on cancer cells in-vivo, we utilized a severely compromised immune-deficient mouse xenograft human cancer model to observe PND’s effects on experimentally implanted human breast cancer tumors. PND was administered orally on a daily basis, and this treatment resulted in significant inhibition of tumor growth over the course of 20 days. As expected from other PND-treatment studies [11, 21], no noticeable side effects were observed, except for the skin’s yellow coloration, most prominently seen in ears and legs. Previous in-vivo studies using PND also reported increased yellow coloration as a side effect and indicated the total or partial recovery of tissue coloration after a two-week recovery period [11].

In addition, we performed combinational drug studies in vitro, where we observed the cytotoxic effects of PND with other currently used, well-known FDA-approved anticancer drugs, gemcitabine and cisplatin. It has been previously discussed that the combination of two or more drugs that have different mechanisms of action are an alternative approach to increase the success of a drug to be repurposed [22]. In the present study, we used PND in combination with gemcitabine, a nucleoside analog, and cisplatin, a DNA intercalator. We were able to observe that while keeping a constant concentration of PND and reducing the doses of the other drugs, there was a higher cytotoxic effect in breast cancer cells.

CONCLUSION

Through the findings presented in this study, we demonstrate that PND is a strong candidate to become a repurposed anticancer drug, as a consequence of demonstrating both its good clinical safety profile and its potential therapeutic effect. Although there are limitations to the use of in vitro studies to demonstrate effectiveness in humans, PND has already been shown to be safe for both animals and humans [11]. In vitro, PND causes cytotoxicity in cancer cells through topoisomerase II inhibition and induction of apoptosis. In addition, our in-vivo study was the first to demonstrate that PND slows the progression of tumor growth in mice. Further long-term studies must be conducted to assess PND’s effect on tumor growth and eradication. Moreover, the results of our combinational drug studies indicate that PND can be considered along with well-known drugs for new anticancer therapies.