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. 2021 Jul 26;6(30):20042–20052. doi: 10.1021/acsomega.1c03080

Bioactivity Studies of Hesperidin and XAV939

Ahmed E Fazary †,‡,*, Mohammad Y Alfaifi §, Serag Eldin I Elbehairi §,∥, Mohamed E Amer ⊥, Mohamed S M Nasr #, Tamer M M Abuamara #, Doaa A Badr †, Yi-Hsu Ju ∇, Aly F Mohamed ○,*
PMCID: PMC8340382  PMID: 34368589

Abstract

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The present work aimed to evaluate the reactivity of natural bioflavonoid hesperidin (HSP) and synthetically derived XAV939 (XAV) against human hepatocellular carcinoma (HepG2), human breast cancer (MDA-MB231) cancer cell lines, and related molecular and pathological profiles. Data recorded revealed that the cytotoxic potential of the tested products was found to be cell type- and concentration-dependent. The half-maximal inhibitory concentration (IC50) value of the HSP-XAV mixture against MDA-MB231 was significantly decreased in the case of using the HSP-XAV mixture against the HepG2 cell line. Also, there was a significant upregulation of the phosphotumor suppressor protein gene (P53) and proapoptotic genes such as B-cell lymphoma-associated X-protein (Bax, CK, and Caspase-3), while antiapoptotic gene B-cell lymphoma (Bcl-2) was significantly downregulated compared with the untreated cell control. The cell cycle analysis demonstrated that DNA accumulation was detected mainly during the G2/M phase of the cell cycle accompanied with the elevated reactive oxygen species level in the treatment of HepG2 and MDA-MB231 cell lines by the HSP-XAV mixture, more significantly than that in the case of cell control. Finally, our finding suggests that both HSP and XAV939 and their mixture may offer an alternative in human liver and breast cancer therapy.

1. Introduction

Cancer is a disease recognized by uncontrolled cell division to make a malignant tumor that can invade cells and tissues in a process called metastasis.1 This is the second main cause of death worldwide, causing 9.6 million deaths.2,3 Cancer has various types, and liver cancer is the fourth cause of death followed by breast cancer in the United States.4 Liver cancer has two types; the first one is primary liver cancer that starts in liver cells such as hepatocellular carcinoma that mainly affects the adults. The second one is cancer from another site metastasized to the liver.5 The treatment of primary liver cancer was linked to the stage of the disease (ablation, surgery, transarterial chemoembolisation (TACE), liver transplant, and palliative).5 Meanwhile, the treatment of the second type was controlled using chemotherapy, liver surgery, radiation therapy, or targeted drug therapy.6,7

Additionally, breast cancer, the most common cancer affecting women worldwide, was located in lobules, connective tissues, or ducts.8 Treatment of breast cancer depends on the cancer stage and type.9 A literature research work has shown that many natural products can induce cancer cell death.10 Most fruits and vegetables contain flavonoids. For example, citrus contains carotenoids, limonoids, and both glycone and glycoside flavonoids.11 Hesperetin and naringenin are the most significant phytochemical glycone flavanones that have anti-inflammatory, antiproliferative, antiallergic, antiviral, and anticancer activities.12 The active citrus flavonane glycoside HSP ((2S)-3′,5-dihydroxy-4′-methoxy-7-[α-l-rhamnopyranosyl-(1 → 6)-β-d-glucopyranosyloxy]flavan-4-one) found in oranges and lemons (5,7,3-trihydroxy-4-methoxyflavanones-7-rhamnoglucoside; Scheme 1) was found to be toxic to cancer cells.13 A previous research proved the anticancer effect of citrus flavonoids as limonin and its glucosides in the process of inhibition proliferation of colon adenocarcinoma (SW480) and neuroblastoma (SH-SY5Y) cell lines.14 Furthermore, HSP has an effect in apoptotic cell death of cancer cells.15 It is known that the apoptosis mechanism has intrinsic and extrinsic pathways in which the intrinsic pathway is controlled by mitochondria, which are both anti-apoptotic (BclXL/Bcl2) and proapoptotic (Bax/Bak) and are members of regulatory proteins maintaining mitochondrial membrane potential (MMP).15,16 Through apoptotic stimulation, MMP is diminished and releases many proapoptotic members such as endonuclease G, cytochrome c, Smac/Diablo, and caspase cascade cleavaging cellular protein, leading to cell death.16 Meanwhile, the extrinsic pathway happens when different death signals and ligands bind to death membrane receptors.15,16 In addition, HSP fundamentally triggers plasma membrane blebs, shrinking cells, and cell detachment showing an anticancer liver effect and induced apoptosis in HepG2 cells.17 The previous research results that were observed on the treated HepG2 cells are endoplasmic reticulum swelling, mitochondria swelling, cytoplasmic vacuolization, and uncondensed chromatin.17,18

Scheme 1. Chemical Structures of Hesperidin (A) and XAV939 (B) Organic Molecules.

Scheme 1

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In addition to XAV939 (3,5,7,8-tetrahydro-2-4-(trifluoromethyl)phenyl-4H-thiopyrano-4,3-dpyrimidin-4-one), Scheme 1 shows a small molecule that inhibits the overexpression of tankyrase 1/2 enzymes (TNKS1 and TNKS2) during cancer tumor growth through the inhibition of the Wnt/β-catenin signaling pathway associated with tumor metastasis and progression.19 Also, it was used previously in the treatment of small cell lung cancer (SCLC) and in the suppression of metastasis inhibiting breast cancer stem cells.20 Additionally, there is an important signaling pathway that controls apoptosis and migration called Wnt stimulating tumor formation.19,21 The regulatory factor of Wnt/β-catenin signaling is tankyrase (TNKS) that binds to the telomerase repeat binding sequences and is considered as one of the families of proteins including tankyrase 1/2 enzymes (TNKS1 and TNKS2) that is responsible for polyADP-ribosylation.22 Furthermore, a previous study proved that TNKS knockdown is responsible for decreasing tumor formation and cell proliferation in lung cancer cell lines.23 Recently, XAV939 is considered as an enzyme inhibitor factor to both TNKS1 and TNKS2 proteins by inhibiting the transcription by β-cetanin in the Wnt protein signaling pathway, in which after the binding of β-catenin with TNKS, the XAV939 molecule was able to induce β-catenin phosphorylation and neglect the action of axin in the destruction complex,24 resulting in the inhibition of the Wnt protein signaling pathway.25,26

2. Materials and Methods

2.1. Cell Culture

Human hepatocellular carcinoma (HepG2) and human breast cancer (MDA-MB231) cell lines were kindly supplied by the Cell Culture Department of the Egyptian Organiztion for Biological Products and Vaccines (VACSERA Holding Company, Giza, Egypt). Liver and breast cancer cell lines were cultured in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS), 25 mM sodium bicarbonate, 20 mM HEPES, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were incubated at 37 °C in a 5% CO2 atmosphere humidified incubator (Jouan, France) according to previous reports.27,28 Cells need to be submerged in an isotonic fluid (Dulbecco’s minimal essential medium (DMEM) supplemented with 5% fetal bovine serum, penicillin (100 units/mL), streptomycin (100 μg/mL), 2 mM glutamine, and 0.3% glucose) that has similar concentration of solute molecules as inside the cell to survive. The liquid media were buffered using bicarbonate buffer to sustain a compatible physiological pH in a 5% CO2 incubator providing nutritious benefits to the incubated cells. A humidified 5% CO2 incubator was closed to grow cell cultures and to keep the same conditions as inside the human body and to prevent the pH inside the cells from becoming either alkaline or acidic, which both prevent cell growth. CO2 levels inside a CO2 incubator were determined with exact optical nondispersive infrared sensors.

2.2. Cytotoxicity Assay (MTT Assay)

The cytotoxicity assay was performed to decide the effective dose of both HSP and XAV939 relative to their concentration, as described previously.29,30 Briefly, HePG2 and MDA-MB231 were seeded in 96-well microtiter plates (5 × 103 cells/well). Cells were incubated using both HSP and XAV939 drugs in sole and mixed forms in different two-fold serial dilutions for 24 h at 37 °C. Untreated cells functioned as negative control. After the incubation, the detached cells were washed out with phosphate buffered saline (PBS). MTT (0.05 mL) dissolved in PBS solutions (0.5 mg/mL) was added to the wells. Then, plates were incubated at 37 °C for 4 h and DMSO (50 μL) was added to dissolve the developed formazan crystals. The treated plates were read at 570 nm using an ELISA plate reader (ELX-800, BioTek, USA), and the absorbance was recorded. Viability % was calculated by the equation viability % = (mean OD of test dilution/mean OD of negative control) × 100. The inhibitory concentration (or IC50) of cell viability for both drugs used in sole and mixed forms was measured using MasterPlex 2010 software.

2.3. Microscopic Examination

Detached and adhered cells were collected post-treatment using trypsinization, as previously described.29 Pelleted cells were resuspended in PBS, and about 50 μL of it was dispended in a clean ethanol washed glass slide, air-desiccated, and fixed using methanol as an elementary step for cytological investigation. Ten microscopic fields of all groups were photomicrographed at a power of 1000× (oil immersion). This was done by a digital video camera (Canon, Japan), which was attached on a light microscope (Olympus BX60, Japan). Images were then transported to a processor system for more analysis. Field selection was built on the existence of the maximum number of apoptotic cells. The photomicrographs were estimated for the presence of morphological standards of apoptosis.

2.4. Reactive Oxygen Species Assay

In vitro quantitative determination of reactive oxygen species (ROS) concentrations in the treated tissue homogenate was conducted using the enzyme-linked immunosorbent assay (ELISA) where the microtiter plate provided was pre-coated with an antibody specific to ROS. Standards or samples were added to the proper microtiter plate wells. A biotin-conjugated polyclonal antibody preparation definite for ROS and horseradish peroxidase (HRP) conjugated avidin was added to each microplate well and incubated at 37 °C for 10 min. Tetramethylbenzidine (TMB) substrate solution was added to each well. The ELISA analytical biochemical technique of the MBS9718216 kit is based on ROS antibody–ROS antigen interactions and an HRP colorimetric detection system to identify ROS antigen targets in samples. The wells containing ROS, biotin-conjugated antibody, and enzyme-conjugated avidin showed a variation in color. The enzyme-substrate reaction was ended by adding 2% sulfuric acid solution, and the color alteration was detected spectrophotometrically at 450 nm. The concentration of ROS in the samples was measured by comparing the optical density of samples to the standard curve.

2.5. Expression of Apoptosis-Related Genes Using Real-Time PCR

The expression of apoptoic genes was conducted in accordance to the previuosly published work.31 RNA was extracted from the IC50 of both drugs treated and untreated breast and liver cancer cells and a mixture of XAV939 and HSP as well. This was post-treated for 24 h using an RNeasy mini kit (Qiagen, USA), according to the manufacturer’s instructions. The concentration of RNA was determined by means of a Beckman dual spectrophotometer (MN, USA). The expression levels of apoptosis-related genes Bax (F: 5′-ATG GAC GGG TCC GGG GAG CA-3′ and R: 5′-CCC AGT TGA AGT TGC CGT CA-3′), p53 (F: 5′-CCCCTCCTGGCCCCTGTCATCTTC-3′ and R: 5′-GCAGCGCCTCACAACCTCCGTCAT-3′), bcl-2 ( F: 5′-CCTGTG GAT GAC TGA GTA CC-3′ and R: 5′-GAGACA GCC AGG AGA AATCA-3′), MMP-1 (F: 5′-CTGGCCACAACTGCCAAATG-3′ and R: 5′-CTGTCCCTGAACAGCCCAGTACTTA-3′), Casp3 (F: 5′-TTC ATT ATT CAG GCC TGC CGA GG-3′ and R: 5′-TTC TGA CAG GCC ATG TCA TCC TCA-3′), and CYC (F: 5′-AGTGTTCCCAGTGCCACACCG-3′ and R: 5′-TCCTCTCCCCAGAATGATGCCTTT-3′) were determined using real-time polymerse chain reaction (RT-PCR). For cDNA synthesis, 10 mg of the extracted RNA from each sample was used by a high capacity cDNA reverse transcriptase kit (Applied Biosystems, California, USA). The acquired cDNA was subsequently amplified by using a Syber Green I PCR master kit (Fermentas, Lithuania) using a StepOne instrument (Applied Biosystems, California, USA) as follows: for the initial denaturation and activation enzyme, 1 cycle was applied for 10 min at 95 °C, then 40 cycles for 15 s at 95°, 20 s at 55 °C for the annealing step, and 30 s at 72 °C for the extension step. The change of the target genes was determined by using a relative standard curve method (ΔCt) to the mean critical threshold (Ct) of β-actin (F: 5′-GTGACATCCACACCCAGAGG-3′ and R: 5′-ACAGGATGTCAAAACTGCCC-3′) as the housekeeping gene.

2.6. Flow Cytometric Analysis

All adherent and detached cells were collected, harvested, and then washed two times with PBS. An Annexin V, FITC (fluorescein isothiocyanate) conjugate/propidium iodide (PI) apoptosis detection kit was used according to the manufacturer’s procedure (ab139418 propidium iodide flow cytometry kit/BD). After adding the Annexin V-FITC conjugate into the labeled tube for 5 min, a PI stain was also added and the tube was incubated for 10 min in the dark at 37 °C. Samples were investigated with scan flow cytometry (FAC), and the data were evaluated using the CellQuest analysis software program (Becton–Dickinson, USA).32,33

2.7. Determination of ROS Generation

The treated cell lines (HepG2 and MDA-MB231) were treated by HSP and XAV939 compounds for 24 h in sole and combined forms and at various concentrations (5–1200 μg/mL) for 4 h. Dichlorofluorescein diacetate (DCF-DA, Molecular Probes, Leiden, Netherlands) was added at a final concentration of 5 μM and incubated for 15 min at 37 °C before measuring the fluorescence intensity of dichlorofluorescein (DCF) using a flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).34 To identify the production of intracellular ROS, 2′,7′-dichlorofluorescin diacetate (DCF-DA) was used, as described earlier.35 Cells treated with HSP, XAV939 compounds, and a combination of them were harvested, washed, and resuspended in a 10 μM DCF-DA solution. Then, the cells were incubated at 37 °C for 20 min in the dark before being examined by a flow cytometer. The values were expressed as percentage of fluorescence intensity relative to the blank control cells. The marked cells were also mounted on the chamber slide with a mounting medium. The images were achieved on a fluorescence microscope (BioTek, VT, USA). To confirm whether intracellular ROS levels play any role in HSP, XAV939, and the combination-induced cytotoxicity, cells were pretreated with N-acetyl-l-cysteine (NAC, Sigma-Aldrich Chemical Co., London, UK).

2.8. Microscopic Examination

The pelleted cells were resuspended in PBS, and then 50 μL was smeared on a sterilized glass slide that was washed with ethanol and exposed to air to dry; then, it was fixed with methanol to prepare for the cytological examination. Ten microscopic fields of each slide were photomicrographed (1000×, oil immersion) by a digital video camera (Canon, Japan), which was attached on a light microscope (Olympus BX60, Japan). Images were then transported to a processer system for analysis. Field selection was built on the presence of the maximum number of apoptotic cells. The photomicrographs were evaluated for the presence of morphological criteria of apoptosis.

2.9. Statistical Analysis

All investigational experiments were performed independently three times. Experimental results were statistically examined by one-way analysis of alteration (ANOVA) and were obtainable as mean ±SD. The difference is measured statistically significant if p < 0.05.

3. Results and Discussion

Regarding the cytotoxic effect of the test drugs on breast (MDAMB-231) and liver (HepG2) cancer cell lines, it was noticed that XAV939 was significantly more toxic (P < 0.05) to both cell lines than hesperedin and the viability monitored was cell type- and concentration-dependent (Figure 1). Synergetic potential of both drugs was assessed on both cell lines, and it was noted that there was a synergetic potential in the case of XAV-HSP mix on MDAMB-231 compared to that of HepG2 (P < 0.05). Concurrently, the IC50 values were significantly decreased in the case of the XAV939 sole application to both cell lines (P < 0.05). Meanwhile, the use of XAV939 and hesperdine mix showed significant toxicity in MDA-MB231 cell lines compared to that in HepG2 cells (P < 0.05); there was a significant disagreement between IC50 values (P < 0.05) in the case of sole and mix applications of XAV939 compared to hesperdine (Figures 1 and 2).

Figure 1.

Figure 1

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Evaluation of cell viability percentage. (A) MDA-MB231 cells treated with HSP and XAV; (B) HepG2 cells treated with HSP and XAV; (C) MDA-MB231 and HepG2 cells treated with a mixture of both compounds (HSP and XAV).

Figure 2.

Figure 2

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Evaluation of the IC50 value of the tested compounds XAV939 (XAV) and hesperidin (HSP) in sole and combined forms (XAV + HSP) against MDA-MB231 and HepG2 cancer cell lines.

The toxicity of the test drugs against both liver and breast cancer cell lines was cell type-dependent, and the mode of action mainly affected the mitochondrial membrane where the MMP1 protein gene was significantly downregulated compared with that of cell control (P < 0.05). Also, there was an insignificant difference of MMP1 gene expression in the case of cell treatment with test drugs in the sole form. Also, Casp-3 was significantly upregulated and its upregulation was drug- and cell type-dependent where XAV939 was efficiently more effective on MDAMB-231 than HSP (P < 0.05). Meanwhile, HSP was more effective in a significant way on HepG2 cells than MDAMB-231 (P < 0.05). Also, both drugs (XAV939 and HSP mixture) were found to be significantly more effective than the sole form application (P < 0.05). The pro-apoptotic genes (P53/Bax) were found to be significantly upregulated compared with its values in untreated cell control.

In the meantime, the upregulation of the pro-apoptotic gene (P53) was found to be cell type-dependent. Bax, despite its significant upregulation compared with its control value, was found to have insignificantly changed in MDAMB-231 and HepG2 cell lines post-treated with both drugs (XAV939 and HSP), either solely or mixed with the anti-apoptotic gene (Bcl-2). It was downregulated under the effect of both drugs in both cell lines recording an insignificant difference (P > 0.05), while treatment with mixed drugs showed a significant synergetic potential (P < 0.05) compared with control and solely tested drugs (XAV939 and HSP) on MDAMB-231 and HepG2 cell lines (Figure 3). It was noticed that using the drug mixture and fluorouracil (5FU) as standard drugs showed a significant increment in the downregulation of both MMP1 and Bcl-2 genes compared with their values in control treated cells and in the sole application of each test drug (P < 0.05). Meanwhile, there was an insignificant difference of pro-apoptotic gene (P53, Bax, and Casp-3) expression in the case of the sole application of each tested drug (P > 0.052) to both cell lines. It was found that MDAMB-231 showed a more significantly elevated Caspase 3 gene upregulation than HepG2 cells, and XAV939/MDAMB-231 showed a more significantly elevated Caspase 3 gene upregulation than HSP/HepG2 (P < 0.05). Meanwhile, in the case of the Bax gene, both 5FU and (XAV939 and HSP) drug mixtures showed significant (P < 0.05) elevation.

Figure 3.

Figure 3

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Evaluation of pro- and anti-apoptotic gene regulations under the effect of the tested compounds XAV939 (XAV) and hesperidin (HSP) in a sole and combined form (XAV + HSP) treated MDA-MB231 and HepG2 cancer cell lines compared to using reference drug and non-treated control cancer cell lines.

The apoptotic profile of treated cells showed a significantly elevated apoptosis in the case of drug mix treatment of both cell lines compared with monovalent treated cells. Also, there was an insignificant difference among treated cells during the early apoptotic phase (P > 0.05). Meanwhile, there was a significant difference of late apoptotic cell percentage in using drug mix compared to the sole application of drugs (P < 0.05), and the necrotic cell percent was significantly elevated (P < 0.05) in the case of the XAV939 drug-treated MDAMB-231 cell line (Figure 4). These findings were in accordance with a previous study (Figure 4),36 showing that the HSP drug inhibited both HepG2 cancer cells and Hep3B cells as a normal liver cell line. Also, it was noted that cell viability was reduced post-treatment with an IC50 value of the 1 mM HSP drug for HepG2 cells, while the viability of other cells did not decrease efficiently (<70%) the effect on HepG2 cells. The results of our study were compared with another study about using XAV939 to investigate the potential molecular mechanisms that induced suppression of the viability of small cell lung cancer NCI-H446 cells.

Figure 4.

Figure 4

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Apoptotic phase percentage of cells in different phases of cell cycle for treatment involving the tested compounds XAV939 (XAV) and hesperidin (HSP) in a sole and combined form (XAV + HSP) treated MDA-MB231 and HepG2 cancer cell lines for 24 h.

The results of that study showed that XAV939 inhibited the viability of NCI-H446 cells in a dose-dependent manner; however, cisplatin as a positive control inhibited NCI-H446 cell viability in a time- and dose-dependent manner. The combination of XAV939 and cisplatin exhibited a slightly more pronounced inhibition of cell viability at an increased dose of the XAV939 drug.37 Moreover, the effect of the HSP drug on the proliferation of the MCF-7 cell line transfected with green fluorescence protein (GFP)/α-tubulin (MCF-7-GFP-Tubulin cells), androgen-independent prostate cancer cells (PC-3), and androgen-dependent lymph node carcinoma of the prostate (LNCaP) and human prostate (DU-145) cancer cells.38 The results showed the inhibition of breast cancer cell line proliferation (MCF-7-GFP-Tubulin cells). Also, it inhibited the proliferation of LNCaP cancer cells and testosterone produced from cell proliferation. Also, the HSP drug did not significantly decrease cell proliferation of two hormone-independent prostate cancer cells (DU-145 and PC-3). As shown in our results, HSP could inhibit the proliferation of breast cancer cells. Furthermore, results of previous studies agreed that the dysfunctions of Wnt, Notch, and Hedgehog pathways are apparent in different tumor types and malignancies. Also, some discussions about the disregulation of Wnt/β-catenin signaling that occurs in human breast cancer as a target of Wnt/β-catenin signaling with this inhibitor represent a promising strategy to suppress metastasis.39

Regarding the cytotoxic effect of anticancer tested drugs (XAV939 and HSP) in the sole or combined form, it was noticed that the drugs showed oxidative stress (in μM/mL), in which ROS was significantly elevated in sole and in combined tested drug-treated cells compared with its level in untreated cell control. Additionally, the combined drugs showed a significant synergetic activity of drug toxicity compared to that in the drug sole form. In the meantime, the 5-fluorouracil (5FU) standard drug showed an insignificant (P > 0.05) change of the ROS level compared with the combined drug-treated cells (Figure 5). Nature-derived products have drawn attention for their anticancer potential and safety for normal cells during the last few decades.40−47 It has been reported that extracts from Scutellaria baicalensis, Citrus aurantium, and Lonicera japonica showed anticancer activity toward liver, lung, and human gastric cancers.40−47 There were also reports that some small molecule compounds are effective and have little or no side effects, and they target a specific pathway.45−48 So, in this study, HSP, a citrus fruit flavonoid derivative, has been evaluated as an anti-carcinogenic, anti-inflammatory, and antioxidant agent.49,50 Another study proved that there was an inverse relationship between the HSP (flavonoids) content and the risk of cancer induction.51 Furthermore, XAV939, a small molecule, targeted a specific pathway that inhibits TNKS.52 A previous study proved that XAV939 suppressed the viability of breast cancer cells and colon cancer cells by targeting the naturally inhibiting Wnt signaling pathway.53 The effect of XAV939 was investigated for the effective treatment of small cell lung cancer cells (SCLC) affecting the cell cycle inducing cell apoptosis via the repression of endogenous tankyrases (TNKS1 and TNKS2).54 In the present study, the MTT assay was performed to confirm that HSP and XAV939 could effectively inhibit MDA-MB231 and HepG2 cancer cell proliferation in a dose-dependent manner. Also, the synergetic potential of tested drugs was considered. In this study, the measuring of ROS concentration was done regarding the cytotoxic effect of anticancer drugs in sole or in combined forms. It indicated that XAV939 and HSP drugs showed an oxidative stress (μM/mL). It possessed a significant elevated ROS level in sole and in combined form-treated cells associated with that in the untreated cell control. Also, it was shown that the combination of both drugs showed a significant ROS level compared with the sole form-treated cells. In the meantime, the reference drug 5-fluorouracil (5FU) showed an insignificant change in the ROS level compared with the combined drug-treated cells (p < 0.05), while the combination form showed a significantly elevated ROS level compared to the ROS level of 5FU-treated cells (p < 0.05) (Figure 5).

Figure 5.

Figure 5

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Effect of the tested compounds XAV939 (XAV) and hesperidin (HSP) in sole and combined forms (XAV + HSP) post 24 h treatment of MDA-MB231 and HepG2 cancer cells on generated ROS. Generation post-treatment was measured spectrofluorimetrically for 24 h versus control cells and reference drug 5-fluorouracil (5FU).

A number of studies indicating the improved levels of ROS are related to the stimulation of apoptosis in cancer cells.55−61 Chemotherapy induces the oxidative stress in patients. After administration of epirubicin, elevated levels of oxidants have been detected in the circulation of patients.56,57 A previous study reported that botanicals showed in vitro and in vivo anticancer activity, resulting from the induction of reactive oxygen species (ROS) in cancer cells with a high ROS content.58 Furthermore, in our study, the 5FU reference drug was used as control to compare the effect of using XAV939 and HSP in single form on MDA-MB231 and HepG2 cells with the effect of 5FU on the same cell lines. Previous studies have demonstrated that intracellular ROS plays a key role in 5FU-mediated anti-tumor effects.59,60 However, there is a study about ROS levels after using 5FU and ultrasound in sole and in combined forms. They monitored intracellular ROS levels, and after comparing with the control group, it was found that the relative green fluorescence concentration of ROS increased 3-fold in the US alone group and 7-fold in the 5FU group alone; nevertheless, the fluorescence intensities of the 5FU treatment improved significantly by 14-fold (p < 0.05).61 Another study supported the presented data regarding the anticancer potential of target drugs despite their use of low-intensity ultrasound-enhanced 5FU against hepatocellular carcinoma cells.62 The results of this study include the effect of using low-intensity ultrasound (US) (1.1 MHz, 1.0 W/cm2, 10% duty cycle) to boost the uptake of 5FU. Reactive oxygen species (ROS) and DNA damage generation were induced under the effect of 5FU. The increment of ROS leads to the upregulation of p53 and Bax levels accompanied with the downregulation of the Bcl-2 gene. The development of ROS generation and the activation of the apoptosis-associated proteins excite the collapse of mitochondrial membrane potential (MMP), release cytochrome c (CYC) from mitochondria into cytosol, and activate the mitochondria-caspase pathway and cell apoptosis. In our study, toxicity of the tested drugs against both liver and breast cancer cell lines was less than that of fluorouracil (5FU), which was sufficiently effective against HepG2 in a significant way compared with the single forms of HSP and XAV939 and effective in an insignificant way compared with the use of the HSP and XAV939 mixture. The present results agree with a study that was conducted to evaluate the monoclonal antibody used as an anticancer against the HepG2 cell line, examined cell cycling, flow cytometry, and mRNA expression of factors involved in apoptosis and paraptosis in monoclonal antibodies against the hepatocellular carcinoma cell line (Hep88 mAb) and treated HepG2 cells using real-time PCR (Figure 6).31

Figure 6.

Figure 6

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Evaluation of cell cycle and induction of apoptosis. (A) Control MDA-MB231 cells; (B) control HepG2 cells; (C) MDA-MB231 cells treated with HSP; (D) HepG2 cells treated with HSP; (E) MDA-MB231 cells treated with XAV; (F) HepG2 cells treated with XAV; (G) MDA-MB231 cells treated with the XAV + HSP mixture; (H) HepG2 cells treated with the XAV + HSP mixture. FL2A: cell cycle distribution was analyzed by PI staining and determined using FlowJo 7.6 software.

The cell cycle examination established a growth inhibitory activity associated with G2/M cell cycle arrest. In addition, Hep88 mAb induced a significant increase in apoptotic cell populations in dose- and time-dependent mode. Also, the mRNA expression proposed that the process triggered by Hep88 mAb involved the upregulation of the tumor suppressor P53 gene, pro-apoptotic Bax, Cathepsin B, damage of mitochondria membrane potential, and the release of cytochrome c (CYC), CASP-3, and CASP-9, with a reduction of anti-apoptotic Bcl-2, confirming paraptosis and apoptosis automated cell death.

Our results represent new visions into the molecular mechanisms essentially in the anti-cancer properties of Hep88 mAb in liver cancer cell lines (Figure 7). However, some data63 supported our results despite their evaluation of the anticancer potential of Ginnalin-A (GA) on the breast cancer cell line, namely, MCF-7 and MDA-MB-231. The expression of genes was important in confirming apoptosis evaluated by qPCR including CASP3, CASP7, CASP8, CASP9, BCL2, BAX, CYCS, MMP1, FAS, and P53. Conferring to the results, a significant increment was observed in the expression of CASP3, CASP8, CASP9, CYCS, FAS, and P53 genes in MDA-MB-231. Meanwhile, in MCF-7 cells, the expression of CASP9 and P53 genes was significantly improved, while the expression of the MMP1 and BCL2 genes was significantly decreased compared with the control group expression profile that may be attributed to the mode of action of the tested drugs in the regulation of important apoptotic gene expression in breast cancer cells.

Figure 7.

Figure 7

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Evaluation of apoptotic cell profiles using Annexin V-FITC/PI staining for apoptosis. (A) Control MDA-MB231 cells; (B) control HepG2 cells; (C) MDA-MB231 cells treated with HSP; (D) HepG2 cells treated with HSP; (E) MDA-MB231 cells treated with XAV; (F) HepG2 cells treated with XAV; (G) MDA-MB231 cells treated with the XAV + HSP mixture; (H) HepG2 cells treated with the XAV + HSP mixture. PI: propidium iodide; FITC: fluorescein isothiocyanate.

Histopathological examination of controlled MDA-MB231 cells showed almost rounded, hyperchromatic, and condensed nuclei. The cellular outline was almost regular without evidence of any folding in cellular or nuclear membranes (Figure 8A). After 24 h post-treatment with IC50 concentration of HSP, the treated cells showed the presence of apoptotic features such as cellular and nuclear shrinkage, an irregular cell membrane, and apoptotic bodies. Meanwhile, after 24 h post-treatment with IC50 concentration of XAV939, some of the treated cells showed necrotic features. The cells were swollen with mixed euchromatin and heterochromatin and ruptured cell membranes (Figure 8B). Other cells revealed apoptotic features of shrunken cells and nuclei, peripheral margination of chromatin, and prominent nucleolus. In addition, apoptotic bodies could be seen (Figure 8C). After 24 h post-treatment with IC50 concentration of the mixture of XAV939 and HSP molecules, the treated cells showed apoptotic features of shrunken cells, an irregular cell membrane, membrane blebbing, and apoptotic bodies (Figure 8D–G). The necrotic cell could also be detected (Figure 8H). The controlled HepG2 cells showed regular, hyperchromatic, and condensed nuclei. The cellular outline was also regular without evidence of any folding in the cellular or nuclear membrane. Cellular and nuclear pleomorphism could also be detected (Figure 8I). After 24 h post-treatment with IC50 concentration of HSP, HepG2 cells showed secondary necrosis (Figure 8J), necrotic cell debris, and apoptotic bodies (Figure 8K). After 24 h post-treatment with IC50 concentration of XAV939, again, HepG2 showed shrunken apoptotic cells with an irregular cell membrane and membrane blebbing (Figure 8I), apoptotic bodies (Figure 8L), and necrotic cell debris (Figure 8M). After 24 h post-treatment with IC50 concentration of the XAV939 and HSP mixture, the cells showed apoptotic features of cellular and nuclear shrinkage (Figure 8N), an irregular cell membrane (Figure 8O), membrane blebbing (Figure 8P), and apoptotic bodies (Figure 8Q).

Figure 8.

Figure 8

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Histopathological photomicrographs. (A) Photomicrograph: non-treated MDA-MB231 cancer cells. (B–D) Photomicrographs: MDA-MB231 cancer cells treated with hesperidin (HSP). (E–G) Photomicrographs: MDA-MB231 cancer cells treated with XAV939 (XAV). (H–J) Photomicrographs: MDA-MB231 cancer cells treated with a combined form of hesperidin and XAV939 (HSP + XAV). (K) Photomicrograph: non-treated HepG2 cancer cells. (L–M) Photomicrographs: HepG2 cancer cells treated with hesperidin (HSP). (P–Q) Photomicrographs: HepG2 cancer cells treated with XAV939 (XAV). (R, S) HepG2 cancer cells treated with a combined form of hesperidin and XAV939 (HSP + XAV). The photomicrograph (B) showed small shrunken apoptotic cells with shrunken nuclei (yellow arrows) with an irregular cell membrane (green arrow). Meanwhile, (C) the photomicrograph showed small shrunken apoptotic cells with shrunken nuclei (yellow arrows). (D–G) Photomicrographs showed small shrunken apoptotic cells with shrunken nuclei (yellow arrow) and apoptotic bodies (green arrow). The photomicrograph (E) showed swollen necrotic cells with mixed euchromatin and heterochromatin (yellow arrows) and ruptured cell membranes (green arrows). Likewise, the photomicrograph (F) showed shrunken apoptotic cells with peripheral condensation of chromatin (yellow arrow) and prominent nucleolus (green arrow). The photomicrograph (H) showed shrunken apoptotic cells (yellow arrows), apoptotic bodies (green arrow), and necrotic cell debris (black arrow). The photomicrograph (I) showed shrunken apoptotic cells (yellow arrows) and apoptotic bodies (green arrows). (J) Photomicrograph showed shrunken apoptotic cells (yellow arrows) with an irregular cell membrane (black arrow) and membrane blebbing (red arrow) and apoptotic bodies (green arrows). Meanwhile, (L) the photomicrograph showed a secondary necrosis where the cells were swollen with nuclear fragmentation (yellow arrow). (M) Photomicrograph showed necrotic cell debris (green arrows) and apoptotic bodies (Yellow arrows). Also, (N) the photomicrograph showed a shrunken apoptotic cell with an irregular cell membrane (yellow arrow) and membrane blebbing (green arrow), apoptotic bodies (black arrow), and necrotic cell debris (red arrow). In the meantime, (O) the photomicrograph showed apoptotic bodies (red arrows). (P) Photomicrograph showed shrunken apoptotic cells (red arrows) with an irregular cell membrane (green arrow), membrane blebbing (yellow arrow), and fragmented nucleus (orange arrow). In panel (Q), photomicrograph shrunken apoptotic cells (red arrows) with irregular cell membrane (green arrow), membrane blebbing (yellow arrow), and apoptotic bodies (black arrows) were shown.

In the present study, HepG2 and MDA-MB231 cell line cycle profiling was performed to monitor cell cycle arrest in addition to apoptotic percentage post-treatment with XAV939 and HSP drugs in sole and combination forms. The results regarding DNA distribution suggest that cell division arrest was performed mainly during the G2/M phase. Also, it was revealed that MDA-MB231 cells treated with XAV939 and HSP and XAV939 mixture forms have a significantly higher arrest percentage than the rest of the formulae used (P < 0.5). Concurrently, there was a significant elevated arrest during the G0/G1 type phase post-treatment of MDA-MB231 with XAV939 cells and the HSP-XAV939 mixture treated HepG2 cells associated with control. Also, MDA-MB 231 cells treated with the HSP-XAV939 mixture compounds insignificantly elevated arrest compared to control (P > 0.05) (Figure 8).

At the same time, cell arrest was accompanied with cell apoptosis that was the highest in the case of HepG2 and MDA-MB231 cells treated with XAV939. At the same time the use of the HSP-XAV939 tested drug mixture in both HepG2 and MDA-MB231 cells showed an extremely elevated total apoptosis compared to cell control (P < 0.001). The ratios of early, late, and necrotic cell values of the same pattern to total arrest are shown in Figure 8. Results from this study agree with the flow cytometry results showing that HSP has the ability to interrupt cell cycle progression in human osteosarcoma MG-63 cells, and raising the doses of HSP led to G2/M phase cell cycle capture. The ratio of G2/M cells improved from around 30% in untreated cells to 35, 46, and 72% in 5, 50, and 150 μM HSP-treated cells, individually. This was accompanied by a simultaneous reduction in the G0/G1 cell population as the HSP concentration was increased to 150 μM. The ratio of cells in altered cell cycle phases was determined using a FACSCalibur flow cytometer using CellQuest 3.3 software (BD Biosciences).64 In addition, another study65 supports that XAV939 obviously induced cell apoptosis of the small cell lung cancer SCLC cell line H446 by growing the proportion of cells in the G0/G1 phase, indicating the inhibition of the cell cycle and showing that XAV939 inhibited the viability of the NCI-H446 SCLC cell line by inducing cell apoptosis via the Wnt signaling pathway.

4. Concluding Remarks and Research Highlights

It can be conlcuded that both XAV939 and hespiridin are potentially active against the cancer cell lines studied. The usage of a combined form showed a synergetic activity against breast and liver cancer cell lines inducing the upregulation of pro-apoptotic genes and downregulation of anti-apoptotic genes, and the upregulation was more enhanced in the case of the application of combined forms than the sole one. Also, DNA accumulation was noticed throughout the G2/M phase of cell cycle arrest. ROS concentration was supported in the case of a XAV939/HSP mixture application. The histopathological profile indicated apoptotic, necrotic, and nuclear fragmentation features confirming the anticancer potential. The in vivo test of both XAV939 and hespiridin against diverse types of cancer cell lines was recommended. Evaluation of the test drug histological cytotyoxicity effect on normal tissues should be done. Also, different formulations of test drugs for targeting application and evaluation of sole and combined forms and sole drug formulation relative to the route of application should be performed.

Author Contributions

All authors read and approved the final manuscript. A.F.M., A.E.F., and D.A.B. were major contributors in writing the manuscript, processing the analytical data, and designing and leading this research. M.E.A., M.S.M.N., and T.M.M.A. designed and performed the experiments. M.Y.A. and S.E.I.E. at King Khalid University supported this work. Professor Y.-H.J. made the final editing and proofreading of the revised manuscript.

The authors declare no competing financial interest.

Notes

The authors extend their appreciation to the Deanship of Scientific Research at King Khalid University for supporting this work through the research group program under grant number R.G.P. 1/113/42.

Notes

All of the data presented in the current study are available from the corresponding authors upon reasonable request.

Notes

The authors declare that they take into their consideration all ethical requirements when deriving cell lines from human tissues in accordance with the ethical standards of the Research Ethics Council, Academy of Scientific Research and Technology (ASRT), Cairo, Egypt, and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.66

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