Cytotoxicity of juglone and thymoquinone against pancreatic cancer cells

Abstract

Juglone and thymoquinone are cytotoxic to pancreatic cancer cells. The aim of this study was to investigate, using an analysis of isobolograms, the type and degree of interactions between juglone and thymoquinone on MIA PaCa-2 pancreatic cancer cells. Cell viability was evaluated using the MTT assay. Cell death was determined by flow cytometry. The IC₅₀ value for juglone and TQ in combination was found to be 24.75 μM, which was higher than juglone or TQ alone. Juglone alone killed Mia Paca-2 cells by ferroptosis. At concentrations where 10, 20 or 50% of cells were affected, there existed a moderate antagonistic relationship between juglone and TQ as indicated by the combination index (CI) value determined by the Compusyn software. At concentrations that affected 75% and 90% of cells, there were nearly an additive effect with CI value of 1.09249 and 0.92391, respectively. Moderate synergism was only seen at concentration where 95% of cells were affected, and the corresponding concentration of juglone and TQ at that combination was 40.90 μM and 511.19 μM, respectively.

1. Introduction

The mortality rate due to pancreatic ductal adenocarcinoma, commonly referred to as pancreatic cancer, in both men and women is very high [1]. In the United States, in 2019, the estimated new cases of pancreatic cancer are 56,770 for males and females of which 29,940 are projected for males and 26,830 for females Mortality rate is projected to be 45,750 for both males and females of which 23,800 deaths will occurs among male patients and 21,950 among female patients [1]. Pancreatic ductal adenocarcinoma has a 5-year survival rate of only 8% [2]. The disease is usually diagnosed at an advanced stage. Gemcitabine is the first-line chemotherapeutic agent that is used for the treatment [3]. The tumor response rate to gemcitabine is often less than 10% with a survival benefit of 5.8 months only because of the development of chemoresistance [3,4]. Gemcitabine in combination with drug such as paclitaxel and other drug combination such as 5-fluorouracil/leucovorin with irinotecan also lead to chemoresistance [5,6].

Ras proteins play a key role in the regulation of cell survival and proliferation. Mutated Ras proteins are common in pancreatic, lung, and colorectal cancers and often associated with disease development [7]. KRAS is one of the RAS gene isoforms that is frequently mutated in human cancers [8]. KRAS is one of the four major driver genes for pancreatic cancer. Other genes included the CDKN2A, TP53, and SMAD4. Mutated KRAS oncogene which encodes a small GTPase that mediates downstream signaling from growth factor receptors such as EGFR, is present in 57% of pancreatic cancer, 33% of colon cancer, and 26% of lung cancer [9]. Drugs targeting aberrant Ras function have been the focus of intensive research over the past three decades; however, effective drugs need to be identified. In this study, we investigated a dietary compound in a common food that can target K-Ras signaling pathway in pancreatic cancer cell in vitro.

Juglone (5-hdroxy-1, 4-napthoquinone) is a naphthoquinone found in leaves, roots, husks/shells and barks of several species of walnut trees including Juglans nigra (black walnut), Juglans regia (English or Persian walnut), Juglans sieboldiana (Japanese walnut), and Juglans cinerea (butternut or white walnut). Juglone is also found in the hickory tree (Carya ovata), Proteaceae, Caesalpiniaceae, and Fabaceae [10]. Juglone is a dark orange-brown compound formed through the shikimic acid pathway [11]. Juglone content in black walnut (Juglans nigra) husk varied between 1.54 and 1.66 mg/g [12]. Juglone content in walnut leaves varied between 44.55 mg and 205.12 mg per gram fresh weight [13]. Thakur and Cahallan [14] analyzed leave samples from 1121 trees and reported juglone content in the range of 13.1–1556.0 mg/100 g dry weight.

Juglone is cytotoxic against a wide range of human cancer cell lines including ovarian cancer cells [15], breast cancer cells [16], gastric cancer [17], lung cancer [18], prostate cancer cells [19,20](9), colon cancer cells [21,22], cervical cancer cells [23,24], and KRAS-wild type BxPC-3 and K-Ras and TP53 mutated PANC-1 pancreatic cancer [25]. Recently, we reported on the anti-angiogenic activity of juglone on K-ras mutated MIA Paca-2 pancreatic cancer cells [26].

Juglone down-regulated the Akt–HIF–1α and VEGF signaling pathways and inhibits angiogenesis.

1.1. In MIA Paca-2 pancreatic cancer in vitro

Thymoquinone (2-Isopropyl-5-methyl-1,4-benzoquinone, TQ) is the principle active component of Nigella sativa seeds’ oil. Nigella sativa belongs to Ranunculaceae family. TQ has been used in traditional medicine for the treatment of dysentery, asthma, gastrointestinal diseases, hypertension, and obesity [27]. TQ can enhance the anti-cancer potential of and reduce the toxicity several chemotherapeutic agents such as gemcitabine [28]. . Several in vitro and animal studies have shown that TQ can inhibit the growth of pancreatic, breast, colon, prostate, lung, and hematological malignancies [29,30]. The primary objectives of this research were to examine the effect of juglone alone or in combination with TQ on pancreatic MIA PaCa-2 pancreatic cancer cells which have with K-Ras and p53 mutations. Comparison was made on the effect of juglone on the viability of PANC-1 cells which respectively express mutant K-Ras and p53 protein, and BxPC-3 which express wild-type K-Ras and p53 mutation.

2. Materials and methods

2.1. Reagents

Juglone, thiazolyl blue tetrazolium bromide (MTT), crystal violet, dimethyl sulfoxide (DMSO), Annexin V-FITC Apoptosis detection kit, and thymoquinone were purchased from Sigma-Aldrich (St. Louis, MO); Trypsin EDTA, Dulbecco’s Modified Eagle Medium (DMEM), Hoechst 3342, Phosphate Buffer Saline (PBS), penicillin streptomycin were obtained from Invitrogen (Carlsbad, CA). Fetal bovine serum was from Atlanta Biologicals (Flowery Branch, GA).

2.2. Cell lines and culture conditions

MIA Paca-2, PANC-1 and BxPC-3 were obtained from ATCC (the American Type Culture Collection) As recommended by ATCC, the cell lines were maintained in culture in DMEM supplemented with 10% fetal bovine serum, penicillin and streptomycin and grown at 37 °C in a humidified incubator with 5% CO₂ until 70–80% confluence. After reaching confluence, it was split at the ratio of 1:3 to1:8. The growth medium was changed 2–3 times a week and 0.25% trypsin EDTA was used to detach the cells for subculture.

2.3. Screening of cytotoxicity

Stock solution of juglone or Thymoquinone was prepared in DMSO, the final concentration of DMSO when in culture was < 0.1%. In order to determine the dose- and time-dependent cytotoxicity of juglone, MIA Paca-2 cells (passage 9–12, BxPC-3 cells (passage 3–6), or PANC-1 cells (passage 59–63) were plated at a cell density of 5 × 10³ cells/well in a 96-well plate. After seeding overnight, cells were treated with juglone and incubated for 4 h or 24 h. Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay. Cytotoxicity of juglone at 24 h was also tested against BxPC-3 and PANC-1.

After required incubation, 20 μl of MTT (5 mg/ml) was added and incubated for 4 h. Formazan product formed from MTT was dissolved with addition of DMSO and absorbance was recorded at 590 nm. The number of viable cells is directly proportional to the absorbance at 590 nm. All experiments were performed in triplicates. Cell viability results are reported as percentage of control. Data collected from cytotoxicity assay were used to calculate the IC₅₀ value for juglone.

2.4. Morphological changes

Juglone-treated MIA Paca-2 cells were observed under inverted microscope and images were recorded at 10X magnification. To observe the change in nuclear morphology, cells treated with or without juglone were first fixed with 4% (v/v) formaldehyde for 10 min at RT, washed with PBS and stained with Hoechst 3342 (1 μg/ml) for at least 15 min. The nuclear morphology was observed with a fluorescent microscope at 40X magnification.

2.5. Long term survival colony formation assay

The method of Woolston et al. was used [31]. Logarithmically growing MIA Paca-2 cells were plated at a density of 3.5 × 10⁵ cells per well in a 6-well plate. After allowing the cells to attach properly, cells were treated with juglone for 6 h. After treatment, juglone-treated and untreated viable MIA Paca-2 cells (2 × 10³) were immediately replated in a 6- well plate with fresh medium containing no treatments. Cells were incubated at 37 °C in a humidified incubator with 5% CO₂. The plates were undisturbed during the entire incubation period and cells were allowed to grow until colonies consisting of more than 50 cells were seen, which was equivalent to about 12 days for MIA Paca-2 cells.

After colonies were formed, growth media was aspirated and cells were washed with PBS. Colonies were fixed with sufficient volume of methanol: saline (1:1) to cover the colonies and the methanol: saline solution was removed after 15 min. Methanol (100%) was added to the colonies and after 15 min colonies were stained with 0.5% crystal violet solution for at least 30 min at RT. Crystal violet solution was aspirated and colonies were washed carefully with an indirect flow of water. Plates were inverted and allowed to dry at RT. Experiments were conducted in triplicates. Cell colonies containing at least more than 50 cells were counted manually. Retained crystal violet by colonies was dissolved with 33% acetic acid, optical density was read and results from juglone-treated colonies were compared to colonies formed with untreated MIA Paca-2 cells.

2.6. Detection of apoptosis by annexin V staining and propidium iodide labeling

To confirm apoptosis, the translocation and externalization of phospholipid phosphatidylserine from cytoplasm to extracellular surface was detected by Annexin V staining and PI staining [32]. Cells were seeded in 6-well plates at a cell density of 350,000 cells/well. After treatment with juglone for 24 h, cells were collected with 0.25% trypsin, centrifuged at 1000 rpm for 5 min and re-suspended in the supplied binding buffer. Cells were stained with Annexin V-FITC following the manufacturer’s protocol (Sigma-Aldrich). Fluorescein isothiocyanate (FITC)-conjugated Annexin V was added to the cell suspension at a ratio of 1:100 and propidium iodide (PI) at 1:50. Cells were incubated for at least 10 min in the dark. Stained cells were analyzed by flow cytometry.

To further evaluate apoptosis, MIA Paca-2 cells (20,000 cells per well) were seeded in 96-well plates and after 24 h the cells were treated with different concentrations of juglone. To determine whether cell death occurred by necroptosis, MIA Paca-2 cells were treated with 10 μM juglone in absence or presence of necrostatin-1 (Nec-1, 20 μM), a RIPK1 inhibitor, for 24 h. Then, the cells were stained with propidium iodide (PI, 1 μg/ml) for 30 min at 37 °C. Lactate dehydrogenase (LDH) released in the culture medium was measured with the CYtoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega, Madison, WI).

To determine whether cell death occurred by ferroptosis, MIA Paca-2 cells (20,000 cells per well) were treated with 10 μM of juglone with or without the addition of 20 μM of ferrostatin for 24 h. The cells were stained with 1 μg/ml of propidium iodide for 30 min at 37 °C. Lactate dehydrogenase (LDH) released in the culture medium was measured with the CYtoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega, Madison, WI).

2.7. Drug combinatorial effect of juglone with thymoquinone

The multi-drug effect was evaluated using the combination index method of Chou and Talalay [33,34]. In order to determine whether Jug had an additive, synergistic or antagonistic effect with TQ first the IC₅₀ for each compound had to be established. MTT Assay as described above was performed using TQ to determine its IC₅₀. The dose range that would be used for compound combination was designed as explained by Chou and Talalay [34]. A total of six different concentration combinations (at least two data points above and below the IC₅₀ and a zero concentration) with three replicates per condition were used. ‘Constant combination ratio’ was used where two compounds were mixed simultaneously at their respective IC₅₀ value and dilutions were made accordingly. For that, serial dilution of both compounds was performed yielding 4, 2, 1, 0.5, 0.25, 0.125 times the IC₅₀ of each compound and MIA Paca-2 cells were treated at the respective concentrations. After 24 h of incubation, MTT assay was performed as described earlier.

The affected fraction of cells after treatment was then calculated and the dose-effect curves for juglone or TQ alone and for two-compound combinations juglone-TQ were created. Isobolograms were made using the values for affected fraction of cells and combination index (CI) was also determined using CompuSyn software. The CI value correlates with the effect of combination treatment. A CI of < 0.9 is considered synergistic, a CI of ⩾ 0.9 or ⩽ 1.1 is considered additive, and a CI of > 1.1 is considered antagonistic [34].

The formula for CI for two drug combinations [33,35] is:

$$\text{CI}=\frac{{\text{C}}_{\text{Jug},50}}{{\text{IC}}_{50,\text{Jug}}}+\frac{{\text{C}}_{\text{TQ},50}}{{\text{IC}}_{50,\text{TQ}}}$$

where C_{Jug, 50} = Corresponding concentration of juglone at the IC₅₀ level of compound combination C _{TQ, 50} = Corresponding concentration of TQ at the IC₅₀ level of compound combination

$${\text{IC}}_{50,\text{Jug}}\text{or}{\text{IC}}_{50,\text{TQ}}={\text{IC}}_{50}\text{for juglone or TQ alone}$$

2.8. Statistical analysis

All the experiments were conducted in triplicates. Results are expressed as means ± SD of experiments. Analysis of variance (ANOVA) was conducted to examine the differences between treatments followed by Tukey’s analysis using SAS software (Cary, NC). A P-value of < 0.05 was considered to be statistically significant and indicated with different letters.

3. Results

3.1. Effect of juglone on pancreatic cancer cell viability

To determine the effect of juglone in various types of human pancreatic cancer cells, MIA Paca-2, BXPC-3, and Panc-1 cells were used. Cancer cells were treated with equimolar (10 μM) concentration of juglone for 24 h. Juglone significantly inhibited the growth and proliferation of all of the pancreatic cancer cells investigated (Fig. 1A). However, the inhibitory effect of juglone was higher in MIA Paca-2 cells than BXPC-3 or Panc-1. Among these pancreatic cancer cell lines MIA Paca-2 cells contains high level of mutant KRAS whereas BxPC-3 has the wild type KRAS [36,37]. Therefore, remaining experiments to achieve the outlined aims of this study were done using MIA Paca-2 cells.

Fig. 1.

A. Comparative inhibitory effect of juglone (+) at equimolar concentration (10 μM) on the proliferation of various types of pancreatic cancer cells. B. Concentration and time dependent inhibition of juglone on the growth of MIA Paca-2 cells.

Juglone exhibited cytotoxic effect on MIA Paca-2 cells in a concentration- and a time-dependent manner (Fig. 1B). From here on, MIA Paca-2 will be the only cell line investigated. In order to calculate the IC₅₀ value of juglone in MIA Paca-2 cells, MTT assay was performed. Cells were treated with at least seven different concentrations of juglone in a range of 0.5 μM to 20 μM for 24 h. Juglone significantly inhibited the proliferation of MIA Paca-2 cells in a dose-dependent manner.

3.2. Dose-effect curve of juglone or thymoquinone on MIA Paca-2 cells

Fractions of affected cells at various concentrations were calculated and a dose effect curve was created (Fig. 2A). The IC₅₀ value of juglone on MIA Paca-2 cells was calculated using the concentration effect relationship and reported to be 5.05 μM. These data indicate that juglone exerts concentration- and time-dependent growth inhibitory effect on pancreatic cancer cells. IC₅₀ value for thymoquinone was found to be 24.15 μM (Fig. 2B).

Fig. 2.

A. Dose-effect curve of juglone on MIA Paca-2 cells on the basis of the results from the MTT Assay. B. Dose-effect curve of thymoquinone against MIA Paca-2 cells on the basis of the results from MTT Assay.

3.3. Juglone induced morphological changes in MIA Paca-2 pancreatic cancer cells

Results indicate that treatment with juglone controlled the proliferation of pancreatic cancer cells (Fig. 3A). Morphological changes such as cell shrinkage, nuclear condensation and formation of apoptotic bodies are observed in apoptotic cells. Chromatin condensation is one of the hallmarks of apoptosis. Untreated cancer cells had normal morphology, and possessed cell to cell contacts, but when treated with juglone, the cells shrunk and rounded up, an indication that cells underwent apoptosis (Fig. 3A).

Fig. 3.

A. Morphological Changes in juglone-treated MIA Paca-2 cells. Images were recorded at 10X magnification. B. Changes in cellular and nuclear morphology of MIA Paca-2 cells upon treatment with juglone. MIA Paca-2 cells were treated for 24 h and then fixed and stained with DNA binding dye Hoechst 3342 (1 μg/ml). Images were captured with a white field confocal microscope. Differential interference contrast (DIC) gray scale images of cells with blue nuclei in blue color are shown. Small, condensed and fragmented nuclei was observed in juglone-treated cells. Juglone-treated cells lost their cellular integrity and appeared smaller and less dense than the untreated MIA Paca-2 cells. Arrows indicate chromatin condensation and apoptotic bodies. Scale bar represents 10 μM. C. Suppressive effect of juglone on clonogenic ability of MIA Paca-2 on cells. A. Representative images of colony formation assay. MIA Paca-2 cells were treated with juglone for at least 6 h and the treated cells were plated and incubated for 12 days. Formed colonies were stained with crystal violet. B. Retained CV by the colonies were dissolved in 33% acetic acid and optical density was measured at 590 nm and reported in terms of control. C. Colonies were also manually counted and reported.

The nucleus of cells treated with or without juglone for 24 h were also observed by Hoechst staining with a fluorescent microscope. Results of nuclear morphology indicate that the untreated cancer cells had intact nuclear architecture whereas presence of small, condensed and bright nuclei and apoptotic bodies were seen in juglone-treated MIA Paca-2 cells due to the cytotoxic effect of juglone (Fig. 3B).

3.4. Effect of juglone on colony forming ability of MIA Paca-2 pancreatic cancer cells

Clonogenic assay is widely used to assess the sensitivity of cancer cells to anti-cancer drugs. To determine long term effect of juglone on survival and proliferation, clonogenic assay was performed. Results of colony formation assay show that there was a dose-dependent inhibition of juglone on clonogenicity of pancreatic cancer cells (Fig. 3C). Formed colonies were stained with crystal violet (Fig. 3C). Quantification of the colonies formed was done either manually or using a spectrophotometer. Retained crystal violet in the colonies was dissolved using 33% acetic acid, optical density was measured, and results were expressed as percentage of control (Fig. 3C). Colonies were also counted manually and the number of colonies formed are presented in Fig. 5C. MIA Paca-2 cells treated with juglone at 5 and 10 μM for 6 h significantly lost the ability to form colonies.

Fig. 5.

Graphical representation of combinatorial effect of juglone and Thymoquinone. A. Juglone exerts an antagonistic relationship with thymoquinone at lower concentrations. Isobologram was created with Compusyn software. The blue, red and green line represents line of additivity at which there is 10%, 20% and 50% effect on the cells respectively. Points lying above the line of additivity indicate antagonistic effect. B. Juglone exerts an additive and synergistic relationship with thymoquinone at higher concentrations. Isobologram was created with Compusyn software. The blue, red and green line represents line of additivity at which there is 75%, 90% and 95% effect on the cells respectively. Points lying below the line of additivity indicate synergistic effect and points lying on the equivalence line indicate additive effect.

3.5. Juglone induced apoptosis of MIA Paca-2 pancreatic cancer cells

Apoptotic cell death was identified by double staining with FITC-conjugated Annexin V and PI using flow cytometry (Fig. 4). In a live cell, the phosphatidylserine (PS) molecules are located in the inner surface of the cell membrane and are not accessible to the Annexin molecules for binding. Externalization of the PS molecules from inner to outer membrane in cells is a very common feature of cells in the early stage of apoptosis. Annexin V specifically binds to the PS molecules, and when conjugated to fluorescent dye like FITC, makes it possible for detecting apoptosis with a flow cytometer. When cells die, they also lose membrane integrity and permeability in the plasma membrane mostly occurs in the late stage of apoptosis. Dyes such as propidium iodide that only binds to the nucleic acids of cells get access into the cell only during the late stage of apoptosis when the membrane of the cells are permeable. Therefore, labeling cell with Annexin V and PI distinguishes and confirms if cells are viable or are in an early or a late stage of apoptosis.

Fig. 4.

Juglone induces apoptosis in MIA Paca-2 cells. Cells were treated with juglone as indicated and then stained with annexin V-FITC and PI. A. Results from flow cytometry analysis are presented in a dot-plot graph. B. Percentage of apoptotic cells (in the early and late stage of apoptosis). C. Cell death by ferroptosis.

MIA Paca-2 cells were treated with juglone at 1, 5 or 10 μM concentrations and co-stained with Annexin V and PI. Results from flow cytometry are presented in dot-plot graph in Fig. 4A. The graph is divided into four quadrants; Lower Left (LL), Lower Right (LR), Upper left (UL) and Upper Right (UR). The LL quadrant represents Annexin V negative and PI negative cell population, the LR quadrant represents Annexin V positive and PI negative cell population and UR quadrant represents both Annexin V and PI positive cell population. Cells in the early phase of apoptosis are located in the Lower Right (LR) quadrant (Annexin V^+ve/PI^−ve), those in the late stage of apoptosis in the Upper Right (UR) quadrant (Annexin V^+ve/PI^+ve), and viable cells (Annexin V^−ve/PI^−ve) in the Lower Left (LL) quadrant (Fig. 4A). The results show that apoptosis was observed in juglone-treated MIA Paca-2 cells within 24 h (Fig. 4).

Results show that when MIA Paca-2 cells were treated with increasing concentration of juglone, the percentage of cells in the early and late stage of apoptosis increased simultaneously. At 1 μM of juglone, about 42.31% of cells were in the early stage of apoptosis, about 35.92% cells in the late stage of apoptosis and about 20.13% cells were viable. When cells were treated with 5 μM of juglone, about 52.09% of cells were in the early stage of apoptosis, about 68.06% cells in the late stage of apoptosis and about 2.44% cells were viable. At 10 μM juglone, most of the cells were in the late stage of apoptosis with ~14.63% in the early stage of apoptosis, about 85.05% in the late stage of apoptosis and only about 1.16% cells were viable. These data strongly show that upon treatment with juglone MIA Paca-2 cells underwent apoptosis.

Treatment of MIA Paca-2 cells with juglone in the presence of necrostatin did not fully block juglone killing of cells at 24 h (Fig. 4C). Treatment of MIA Paca-2 cells with the ferroptosis inhibitor ferrostatin-1 at 20 μM resulted in full suppression of cell death at 24 h treatment with juglone (Fig. 4C). Treatment of MIA PaCa-2 cells with deferoxamine almost completely protected the pancreatic cancer cells against the cytotoxic effects of 10 μM juglone. The effect of juglone on mitochondrial potential was not investigated.

3.6. Combinatorial effect of juglone and thymoquinone on MIA-Paca-2 cells

Combination therapy is mostly used in the drug development process to target multi oncogenic markers and avoid drug resistance. In this experimental design, the effect of the combination of juglone and TQ on MIA Paca-2 cells was also evaluated. When two or more drugs are combined, there are three possible outcomes with treatment; synergy, additive effect, or antagonist effect. To determine the drug combination effect, first IC₅₀ value of each compound was determined by MTT assay. IC₅₀ value for juglone was found to be 5 μM. From the dose effect curve (Fig. 2A), IC₅₀ value for TQ was found to be 24.15 μM. After determining the IC₅₀ values, juglone and thymoquinone were mixed at a constant ratio and combination index was determined at each concentration using Compusyn software.

The effect of juglone in combination with TQ is summarized in Tables 1–3. The IC₅₀ value for juglone and TQ in combination was found to be 24.75 μM, which was higher than juglone or TQ alone. At concentrations where 10, 20 or 50% of cells were affected, there existed a moderate antagonistic relationship between juglone and TQ (Table 2.) as indicated by the combination index (CI) value determined by the Compusyn software. At concentrations that affected 75% and 90% of cells, there were nearly an additive effect with CI value of 1.09249 and 0.92391, respectively. Moderate synergism was only seen at concentration where 95% of cells were affected, and the corresponding concentration of juglone and TQ at that combination was 40.90 μM and 511.19 μM respectively.

Table 1.

IC₅₀ values for juglone, thymoquinone and their combination in human pancreatic cancer MIA Paca-2 cells.

Drug	IC50 value (μM)	m	r
Juglone	5.27353	0.89120	0.99055
Thymoquinone	24.1519	0.80316	0.99713
Juglone + Thymoquinone	24.7545	0.94838	0.92373

Table 3.

Assessment of combinatorial effect of juglone and thymoquinone at Fa: 0.75, 0.90 and 0.95. Combinatorial index was determined by Compusyn software.

Fa	CI Value	Total Dose	Juglone (μM)	Thymoquinone (μM)
0.75	1.09	78.84	5.84	73.00
0.90	0.92	251.09	18.60	232.49
0.95	0.83	552.08	40.90	511.19

Table 2.

Assessment of combinatorial effect of juglone and thymoquinone at Fa: 0.1, 0.2 and 0.5. Combination index was determined by Compusyn software.

Fa	CI Value	Total Dose	Juglone (μM)	Thymoquinone (μM)
0.1	1.84625	2.44049	0.18078	2.25971
0.2	1.61803	5.73887	0.42510	5.31377
0.5	1.29674	24.75447	1.83367	22.9208

The graphical representation of combinatorial effect of juglone and TQ are also presented via isobolograms (Fig. 5A and B) that were created using Compusyn software. The nature of interaction between two drugs can be evaluated by isobologram analysis as well in a two coordinate plot. The x and y coordinates in an isobologram represent the concentrations of individual drugs required to produce a defined effect. Respective line connecting two data points in two different axes is the line of additivity. Points lying below the line of equivalency or line of additivity in the isobologram indicate synergistic effect between two drugs and points above the line of equivalency represent antagonistic effect [38]. The resulting isobolograms in our study show that juglone and TQ act antagonistically at most of the concentrations and only exert some synergy at very high concentration.

4. Discussion

Dietary bioactive compounds such as resveratrol [39], sulforaphane [40], epigallocatechingallate [41] inhibit the growth and proliferation of pancreatic cancer cells in various in vitro and in vivo experiments. Juglone has been investigated against KRAS-wild or KRAS- and TP53-mutated pancreatic cancer cells but not against the KRAS-mutated such as MIA Paca-2 cells [42].

In this study, the effect of juglone on the proliferation and apoptosis of KRAS-mutated human pancreatic cancer MIA Paca-2 cells was analyzed. The IC₅₀ value of juglone in pancreatic cancer MIA Paca-2 cells was determined to be 5.27 μM at 24 h. The IC₅₀ value for juglone on ovarian cancer SKOV3 cells was 30.13 μM at 24 h [7]. Juglone has also shown to be effective against melanoma cells in a dose- and time-dependent manner. The IC₅₀ for juglone on B16F1 melanoma cells at 1, 24, and 48 h was 9.9 μM, 7.46 μM, and 6.92 μM, respectively [43]. Very high intracellular production of ROS was linked to the mode of action of juglone mediated apoptosis in melanoma cells. These studies with juglone on various cancer models show that juglone reacts in different ways depending upon the type of cancer and therefore it is very critical to establish the IC₅₀ values of juglone for every cancer cell line.

In this study we also investigated whether juglone induced apoptosis in pancreatic cancer cells and the apoptotic cell death mode. The morphological changes that occur during apoptosis are cell shrinkage, membrane blebbing, chromatin condensation and nuclear fragmentation [44]. Results in Fig. 3 clearly show that cells treated with juglone stained positive with annexin V and PI. As cells undergoing apoptosis have condensed chromatin, fluorescent dye such as Hoechst is known to stain the nucleus of apoptotic cells more brightly than that of the normal cells [45]. Results of juglone on MIA Paca-2 cells show bright fluorescence in nucleus of apoptotic cells (Fig. 3B). Morphological changes, cell shrinkage, translocation of phosphatidylserine molecules were observed in MIA Paca-2 cells exposed to juglone at concentration range of 1–10 μM. Similar concentration of juglone increased annexin V binding in juglone-treated erythrocytes [46] and cancer cells [47]. Juglone induced MIA Paca-2 cell death by ferroptosis. To determine whether cell death occurred by ferroptosis, Mia Paca-2 cells (20,000 cells per well) were treated with 10 μM of juglone with or without the addition of 20 μM of ferrostatin for 24 h. The reaction can be reversed by deferoxamine.

Colony formation assay was used to determine juglone associated long term inhibition of the proliferation of MIA Paca-2 cells. As demonstrated in Fig. 3C., there was a significant reduction in the number of colonies formed in juglone-treated pancreatic cancer cells in comparison to the untreated MIA Paca-2 cells. These findings are in line with other reports of juglone treatment in other types of cancer. Juglone at μM reduce colony formation in MCF-7 cells [48].

Drug combination studies was also carried out using juglone and TQ, the principal bioactive component of the volatile oil of Nigella sativa also known as black seed. Each of these compounds was demonstrated to be cytotoxic to MIA Paca-2 cells individually. Based on their cytotoxicity, we hypothesized that combination treatment of MIA Paca-2 cells with juglone and TQ would result in synergistic cytotoxicity. Several in-vitro and in-vivo studies have reported synergistic action of TQ in conjunction with other phytochemicals such as EGCG against prostate cancer [49], lycopene against cervical cancer [50], gemcitabine against pancreatic cancer [27], or doxorubicin against breast cancer cells [51]. However, our results demonstrated that TQ in combination with juglone, did not exert favorable synergistic relationship against pancreatic cancer cells. TQ acts as an anti-oxidant (free radical scavenger) at lower concentrations and at higher concentrations acts as a pro-oxidant [52], which might be one of the possible mechanisms responsible for the antagonistic effect between juglone and TQ. Juglone-induced cytotoxicity in cancer cells often involves production of reactive oxygen species through redox activation [53]. There was no favorable synergistic relationship at low concentration combination of juglone and thymoquinone. Synergy between juglone and TQ was only seen at very high dose combination where physiological concentrations of each dietary bioactive compound could be very difficult to achieve.

The anti-cancer effects of juglone have been analyzed in vivo in animal models. .Juglone at 200 ppm reduced the occurrence of multiple intestinal tumors in azoxymethane-induced colon cancer rats [54]. When LnCap tumor cells bearing mice were intra-peritoneal injected with 40 μg of juglone once a week for 4 weeks, there was a significant reduction of tumor size and volume with no significant reduction in weight of the mice [19]. These studies suggest that juglone can be effective and safe in vivo.

5. Conclusion

This study also investigated, using an analysis of isobolograms, the type and degree of interactions between juglone and thymoquinone on MIA PaCa-2 pancreatic cancer cells. The IC₅₀ value for juglone and TQ in combination was found to be 24.75 μM, which was higher than juglone or TQ alone. Juglone alone killed Mia Paca-2 cells by ferroptosis. At concentrations where 10, 20 or 50% of cells were affected, there existed a moderate antagonistic relationship between juglone and TQ as indicated by the combination index (CI) value determined by the Compusyn software. An additive effect between juglone and TQ was found at concentrations that affected 75% and 90% of cells. Moderate synergism was only seen at concentration where 95% of cells were affected.