Zingerone Nanotetramer Strengthened the Polypharmacological

Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Zingerone Nanotetramer Strengthened the Polypharmacological Efficacy of Zingerone on Human Hepatoma Cell Lines Mei-Lang Kung,†,‡‡ Pei-Ying Lin,†,‡‡ Shih-Tsung Huang,‡,§ Ming-Hong Tai,‡,§,∥,⊥ Shu-Ling Hsieh,# Chih-Chung Wu,¶ Bi-Wen Yeh,∇,○ Wen-Jeng Wu,∇,○,⧫ and Shuchen Hsieh*,†,⊗

ACS Appl. Mater. Interfaces Downloaded from pubs.acs.org by TULANE UNIV on 12/19/18. For personal use only.

†

Department of Chemistry, ‡Doctoral Degree Program in Marine Biotechnology, ∥Institute of Biomedical Sciences, and ⊥Center for Neuroscience, National Sun Yat-Sen University, Kaohsiung 80424, Taiwan § Doctoral Degree Program in Marine Biotechnology, Academia Sinica, Taipei 11529, Taiwan # Department of Seafood Science, National Kaohsiung University of Science and Technology, Kaohsiung 81157, Taiwan ¶ Department of Food and Nutrition, Providence University, Taichung 43301, Taiwan ∇ Department of Urology, Kaohsiung Medical University Hospital, ○Department of Urology, School of Medicine, College of Medicine, ⧫Department of Urology, Kaohsiung Municipal Ta-Tung Hospital, and ⊗School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 80708, Taiwan S Supporting Information *

ABSTRACT: We base this study on the concept of drug repositioning to reconstitute the natural product of zingerone as zingerone nanoparticles (zingerone NPs) through a one-pot synthesized process. The as-fabricated zingerone NPs were characterized; they possessed a particle size of 1.42 ± 0.67 nm and a reconstituted structure of zingerone nanotetramer. We further validate the effects of zingerone NPs on the antitumor activity and investigate the relative underlying mechanisms on the human hepatoma SK-Hep-1 and Huh7 cell lines. Our results demonstrated that zingerone NPs significantly inhibit Akt activity and NFκB expression as well as activate the caspases cascade signaling pathway which are involved in the antiproliferation, antitumorigenicity, disturbing cell cycle progression, and induction of DNA damage as well as cell apoptosis. These findings were promising to provide a “Nano-chemoprevention” strategy in future cancer therapeutics and medical and clinical applications. KEYWORDS: zingerone nanoparticles, nanotetramer, polypharmacological efficacy, chemoprevention, human hepatoma cells

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INTRODUCTION Nanomedicine is a rapidly growing field that incorporates materials technology with medical applications and is being used to develop novel and effective treatments for a wide range of conditions such as illnesses, infections, and cancer therapeutics. Cancer and cancer-associated complications are the leading causes of death worldwide. Fortunately, the recent advances in ameliorated the methodologies and materials for many types of cancers are conduct to significant improvements in patient prognosis and quality of life.1,2 Plants and their derivatives have been used throughout history for treating illnesses and diseases. Even today, despite modern chemical synthesis technology, phytochemicals continue to be the sources and inspirations for the novel and effective drugs for treatment of a wide range of ailments.3,4 Furthermore, their natural origins may make them more easily accepted by patients in some cultures where use of natural and herbal remedies is common. Thus, natural products and their derivatives continue to be a major area of exploration for new © XXXX American Chemical Society

and improved chemopreventive drugs. Chemoprevention is defined that uses natural, synthetic, and/or biological chemical agents to reverse, suppress, and prevent carcinogenic progression.5 This strategy is emboldened by recent successes in cancer prevention clinical trials in high-risk populations.6 As research efforts continue, an increasing variety of materials such as natural and herbal constituents are being tested and explored for cancer prevention efficacy.7,8 So far, many phytochemicals and herbal bioactive compounds including phenolics and flavonoids agents, taxanes (paclitaxel), curcumins, and alkaloids have been identified and characterized their excellent chemopreventive properties and anticancer activities.9−11 For examples, Curcumin has demonstrated a wide range of anti-oxidative, anti-inflammatory, and antiproliferative activities and strong cancer preventive activity Received: August 23, 2018 Accepted: December 7, 2018

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DOI: 10.1021/acsami.8b14559 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 1. Characterization of zingerone NPs. (A) AFM topographical image of zingerone NPs deposited on a silicon wafer (20 × 20 μm2 scan). (B) TEM image of zingerone NPs. (C) Size histogram of zingerone NPs from AFM analysis is 1.42 ± 0.67 nm. (D) UV absorption spectra of zingerone and zingerone NPs, respectively. The absorption peak of zingerone NPs was observed around 350 nm in the UV−vis spectra. The inset photographs show zingerone (left) and zingerone NPs (right) solutions by visible light, respectively.

against a wide range of tumor cells.11,12 In addition, the organic phenolic-derived compounds in the ginger have shown their excellent efficiency in anticarcinogenic, antibacterial, antifungal, and anti-atherosclerotic activity.13 Therefore, natural herbal compounds have been believed to be more valuable and reliable for their superior therapeutic effects in many diseases and symptoms. However, the insufficient water solubility or low production remains a major obstruction for their further developments and clinical applications. Although many methodologies have been developed to improve the solubility issues, the decreased bioavailability and poor pharmacokinetics caused another new challenge. Recently, nanotechnology has offered the opportunities to overcome the solubility limitations. Because nanoparticles (NPs) possess novel physical and chemical properties as well as their small size (1−100 nm), shape, and composition, such particles can be further optimized for targeting specific sites within the body by functionalization or ligand attachment.14 Indeed, by facilitated with nanotechnological development, several methodologies such as nanocrystals, nanoemulsions, and polymeric micelles have been developed and to be validated their breakthrough the solubility limitations of herbal bioactive compounds and therefore acquired a good therapeutics efficiency. However, their complex manipulation processes are needed to be paid more concern and accurate assessment such as modification and/or functionalization, stability issue, potential flocculation and coalescence, as well as high fabrication cost.15−18 Carbon-based NPs (carbon dots) are carbonaceous NPs, which possess a small size (diameter below 10 nm) and have excellent physical, chemical, and optical properties.19 In addition, their superior advantages, such as low toxicity, water solubility, biocompatibility and excellent fluorescence, have attracted a greatly attentions for widely fields and

therefore make them good candidates for interdisciplinary applications including of bioimaging, drug delivery, cancer therapy and biosensing as well as chemical catalysis and analysis.20,21 Indeed, our previous studies have successively generated and reported several types of carbon dots, which derived from either available alkylalkoxysilane22,23 or natural products such as essential oil from peppermint plants24 and shrimp eggs,25 which have illustrated their intriguing and potential biofunctional application in bioimaging and biolabeling and even reveal an excellent antibacterial efficiency. In this study, we first utilize phytochemical compounds to synthesize a novel and carbon-based NP, which are corresponding to several indispensable elements such as abundant, environmental friendly, water solubility, bioefficiency, and low cost. Here, we choose the zingerone as the precursor for preparing this green nanoformulation. Zingerone is one of herbal nonvolatile and pungent compounds and is present in noteworthy amounts (∼9.25%) in ginger.26 Moreover, zingerone have been reported the potent effects on anti-oxidation, anti-inflammation, and anti-cancer in vivo and in vitro.13,26 Therefore, we develop a novel natural carbonbased zingerone NP through a one-pot synthesized method. The zingerone NPs are validated a small size of 1.42 ± 0.67 nm and composed of nanotetramer. Furthermore, these asfabricated zingerone NPs have shown their excellent chemopreventive effects of antiproliferation, tumorigenicity suppression (over 4-fold efficacy than raw zingerone), enhancing the DNA instability and damage, and induction of cell apoptosis in the human malignant hepatoma SK-Hep-1 and Huh7 cell lines. Moreover, the signaling mechanism studies demonstrated that downregulating the Akt/NFκB signaling and inhibiting the activation of caspase-cascade signaling pathways play critical roles in zingerone NPs-mediated anti-cell-proliferation, antitumorigenicity, and cell apoptosis. B


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Figure 2. Total ion chromatogram analysis for clarifying the constitutive structure of zingerone NPs. The total ion scan modes were used to detect the time scan and mass to charge ratio (m/z) of zingerone standard (A,B) and zingerone NPs (C,D), respectively. The ESI (+) MS/MS spectrum of peak I and peak II of zingerone NPs (C) was further analyzed and presented as (E,F), respectively. The squares with red dash line indicate the m/z values which corresponded to either peak I or peak II.

Figure 3. Zingerone NPs exhibit excellent cytotoxicity for malignant hepatoma SK-Hep-1 and Huh7 cell lines. Microscopy observation of cellular morphology changes with a filopodia-like protrusions structure formation in (A) SK-Hep-1 cells and (B) Huh7 cells during zingerone NPs administration at 24 h. Bar: 50 μm. Detection of the cellular viability impacts by zingerone and zingerone NPs treatment using MTT assay in both (C) SK-Hep-1 cells and (D) Huh7 cells at 24 h. Data are expressed as mean ± SEM of three experiments.

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RESULTS AND DISCUSSION Characterization and Structure Definition of Zingerone NPs. Here, we prepare the zingerone NPs through a onepot heat condensation reaction. The physical characterizations and chemical analysis of the as-generated zingerone NPs were next executed using atomic force microscopy (AFM), transmission electron microscopy (TEM), ultraviolet (UV), zeta potential, and liquid chromatography-mass spectrometry (LCMS) analysis. The AFM topographic image (Figure 1A) and TEM image (Figure 1B) were scanned and obtained by deposited zingerone NPs on the silicon wafer substrate. The

zingerone NPs show a spherical and monodispersed conformation. We determined the NP size distribution by measuring the height of 894 separate NPs and the performed histogram analysis of zingerone NPs has obtained an average size of 1.42 ± 0.67 nm (Figure 1C). The zeta potential of the zingerone NPs was −15.99 ± 0.23 mV in phosphate-buffered saline (PBS) solution. To further demonstrate the successive fabrication of zingerone NPs, we next detected the absorption spectrum from the self-assembly zingerone NPs. As compared to the colorless and transparency of zingerone solution, zingerone NPs own a clear and amber apparent. Moreover, C


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Figure 4. Zingerone NPs reveal high efficiency of antitumorigenicity for malignant hepatoma SK-Hep-1 and Huh7 cell lines in vitro. Cells were treated with various concentrations of zingerone and zingerone NPs for 7−10 days and then subjected to crystal violet staining. The effects of zingerone NPs on (A) SK-Hep-1 cells and (B) Huh7 cells were taken images and recorded using inverted microscopy. Quantitative measures of colony formation by counting the number of crystal violet positive cells for (C) SK-Hep-1 cells and (D) Huh7 cells, respectively. Colony survival activity was further determined by measuring the dissolved supernatant from crystal violet-stained colonies and/or cells of (E) SK-Hep-1 cells and (F) Huh7 cells using absorbance detection at 595 nm. All data are expressed as the mean ± SEM of three experiments.

the UV analysis results revealed that zingerone NPs show a clear absorption feature centered at 350 nm due to π−π* transition of the benzenoid rings27 and therefore demonstrated the generation of zingerone NPs (Figure 1D). Next, to clarify the constitutive structure of zingerone NPs, we used the LC-MS to analyze the ion scanning profiles. In the total ion scan mode, we used zingerone as a standard and observed a major peak at 24.32 min (Figure 2A), which corresponded to the major fragment (m/z 194) in ESI (+) MS/MS analysis (Figure 2B). We next detected the zingerone NP constituents and found that two more peaks were presented at 32.96 (peak I) and 33.77 min (peak II) besides the major peak at 24.32 min (Figure 2C). By analyzing the ESI (+) MS/MS mode of zingerone NPs, the major peak of m/z 194 was consistent with zingerone (Figure 2D). To further verify the m/z values of peak I and peak II, we analyzed several major ion fragments and obtained their m/z values, which respectively corresponded to 409.03, 216.95, 194.98, and 136.99 in peak I (Figure 2E) and 795.24, 409.03, 216.95, 194.98, and 136.99 in peak II (Figure 2F). On the basis of these results, we speculate that the constitutive structure of zingerone NPs is resulted from a hemiketal reaction between two zingerone compounds owing to the alcohol group condensated with the adjacent ketone group. This hemiketal reaction further polymerizes and finally exhibits a nanotetramer structure. Zingerone NPs Enhance the Raw Zingerone-Mediated Anti-Proliferation and Anti-Clonogenic Survival of Human Hepatoma SK-Hep-1 and Huh7 Cell Lines. Nanotechnology can improve the raw material activity via altering the size, constitutive structure, and increasing the surface area, which effectively enhance the interaction between NPs and target objectives. To further understand the biological activity of the as-synthesized zingerone NPs, we next validate and compare the cytotoxic effects of zingerone and zingerone NPs on two hepatoma SK-Hep-1 and Huh7 cell lines. Cells were treated with different concentrations (0, 25, 50, 100, 200, and 400 μM) of zingerone and zingerone NPs, respectively, for 24 h and the cell viability was further detected and recorded

using the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. As compared to zingerone treatment, both the SK-Hep-1 cells and Huh7 cells exhibited high sensitivity to zingerone NP treatment, which led to an obviously cell morphological changed and a significant inhibition of cell viability in a dose-dependent manner (Figure 3). In SK-Hep-1 cell, the zingerone NPs exposure (100 μM) induces a filopodia-like protrusions structure. Furthermore, the high dose of zingerone NPs (200 μM) treatment elicits more extending protrusion structures, cell round-up, and severe cell degradation/death (Figure 3A). In Huh7 cells, the filopodialike protrusion structures were easy noticeable at lower dose of zingerone NPs (50 μM) incubation. While with the increased doses of zingerone NP administration, the filopodia-like protrusion branches were getting more extension and cell body was round-up and become smaller and/or cell death. Finally, a severely cell death was occurred (Figure 3B). The cytotoxicity effects were further detected using MTT assay. A parallel experiment on zingerone was performed to compare with zingerone NP efficacy. Our results revealed that lower dose (50 μM) of zingerone NPs treatment induces a significant cell survival stress in either SK-Hep-1 cells (Figure 3C) or Huh7 cells (Figure 3D). Also, a remarkable viability impact was observed at 100 μM of zingerone NPs incubation in both cell lines as compared to zingerone treatment. The IC50 (median inhibition concentration) of zingerone NPs were determined as 175.1 ± 3.6 and 168.9 ± 14.6 μM in SK-Hep-1 cells and Huh7 cells, respectively. These data demonstrate that the zingerone NPs can significantly induce cellular morphology changes and enhance cell survival stress in both hepatoma SKHep-1 and Huh7 cell lines. Moreover, we have detected and assessed the effects of both zingerone and zingerone NPs on liver functions of animal models through detection of the food intake, liver weight, and GOT and GPT indexes. As shown in Table S1, our results indicate that both GOT and GPT index show no significant changes. These data suggest that the fabricated zingerone NPs are safe and have no liver toxicity. D


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Figure 5. Zingerone NPs-mediated antiproliferation in both hepatoma SK-Hep-1 and Huh7 cell lines. Both (A) SK-Hep-1 and (B) Huh7 cell lines were treated with various doses of zingerone and zingerone NPs for 24 h. Cells were next stained with Ki-67 antibody and the Ki 67 positive cells were evaluated using immunofluorescent staining. Nuclei were stained with DAPI. Scale bar = 50 μm. The Ki67 positive cells of SK-Hep-1 and Huh7 cells were further quantitated and recorded in (C,D), respectively. Each point is mean ± SEM of three experiments. *P < 0.05; **P < 0.001.

lower than those of control and zingerone groups (Figure S1C,D). These results strongly supported that zingerone NPs possess an excellent antitumorigenicity and imply their potential on tumor therapeutic efficacy. Moreover, the normal mice model (ICR mice) also demonstrated that zingerone NPs show no significant effects on mice food uptake and liver functions as compared to both control and zingerone groups (Table S1). To further evaluate the effects of zingerone NPs on cell proliferation, we next detect the expression of Ki67 protein, which is a proliferation marker and is used to validate the cell growth fraction in either tumor cells or other tissues. Cells were treated with various concentrations of zingerone and zingerone NPs for 24 h, and the Ki67 positive cells were recorded and analyzed using immunofluorescent assay. Our results indicated that zingerone NPs significantly inhibit cell proliferation by decreased Ki67 expression in both SK-Hep-1 and Huh7 cell lines. Moreover, the reduced nuclear size was also observed in zingerone NP treatment (especially over 100 μM treatment) (Figure 5A,B). In SK-Hep-1 cells, zingerone shows a limited dose-dependent stimulation of cell proliferation (8.3 ± 1.6, 8.7 ± 2.8, 9.8 ± 1.6, and 11.3 ± 3.1% at 0, 25, 50, and 100 μM of zingerone treatment, respectively), and the growth induction was discontinued at high dose of 200 μM treatment (7.0 ± 1.6%). As compared to zingerone treatment, zingerone NPs-induced proliferation inhibition was observed at 50 μM administration (7.0 ± 1.0%). A dramatic antiproliferation was found at higher doses of zingerone NPs treatment (4.6 ± 1.9 and 1.0 ± 1.7% at 100 and 200 μM treatment, respectively) (Figure 5C). For Huh7 cells, the zingerone NPs exhibit a significant anti-cell-proliferation in a dose-dependent manner (8.9 ± 3.4, 6.5 ± 2.3, 5.3 ± 0.8, 3.8 ± 0.7, and 1.2 ± 0.6% at concentrations of 0, 25, 50, 100, and 200 μM treatment, respectively) as compared to zingerone treatment (11.6 ± 2.5, 7.4 ± 0.3, 7.3 ± 2.8, and 5.2 ± 0.8% at 25, 50, 100, and 200 μM treatment, respectively) (Figure 5D). In addition, a BrdU incorporation assay also demonstrated that zingerone NPs significantly inhibit the BrdU incorporation in a

Inhibition of tumor cell proliferation is one of efficient strategies for antitumorigenesis. Tumorigenesis is composed of multiple oncogenic steps including sustaining proliferative signaling, against cell death, and modifying the microenvironment to enhance tumor cell immortality such as triggering angiogenesis, cell invasion, and distant metastasis.28 On the basis of the excellent anti-cell-growth efficiency of zingerone NPs, we next investigated the antitumorigenicity of zingerone NPs on both hepatoma SK-Hep-1 cells and Huh7 cells using colony formation assay. Cells were incubated with various doses of zingerone and zingerone NPs for 7−10 days, and the colony formation was monitored and recorded. Our results indicated that zingerone NPs reveal a superior suppression for colony formation in a dose-dependent manner as compared with zingerone treatment in both SK-Hep-1 and Huh7 cells (Figure 4A,B). Next, we calculated the colony numbers in both cell lines. Our data showed that zingerone NPs exhibit a markedly attenuation of colony formation (over 50%) at lower dose treatment (25 μM). Furthermore, an over 95% of colony inhibition was detected at 50 μM treatment and the overall colony inhibition was observed at doses over than 100 μM treatment in both SK-Hep-1 and Huh7 cells (Figure 4C,D). Furthermore, detection of the colony survival also obtained a consistent result with colony formation in both SK-Hep-1 and Huh7 cells (Figure 4E,F). These results also indicated that zingerone NPs elicit a dramatic anticlonogenic survival with an over 4-fold efficacy rather than zingerone. Moreover, we also detected the effects of zingerone NPs on antitumorigenicity in vivo using the Huh7 xenograft model in immunodeficiency NOD/SCID mice. Our results indicated that the tumor volume in zingerone NP-treated mice was significantly inhibited as compared to both the control and zingerone groups (Figure S1A). Interestingly, the weight gain of both zingerone and zingerone groups was increased as compared to control group (Figure S1B). This result is consistent with normal mice model analysis in Table S1. Moreover, our results also revealed that the tumors weights and sizes of zingerone NPs-treated mice were significantly E


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Figure 6. Zingerone NPs suppress Akt/NFκB-mediated cell survival signaling to inhibit cell proliferation. Cells were treated with zingerone and/or zingerone NPs for 24 h. (A,B) Akt activity and (C,D) protein expression levels of NFκB and IκBα were detected on SK-Hep-1 cells and Huh7 cells, respectively, using western blotting. In addition, cells were transiently cotransfected with the NFκB-driven luciferase vector and the internal control of Renilla reniformis luciferase reporter vector overnight. Cells were further administrated with zingerone and/or zingerone NPs for 24 h. The effects of both zingerone and zingerone NPs on NFκB promoter activity were determined on both (E) SK-Hep-1 and (F) Huh7 cell lines. The NFκBdriven luciferase activity was further assayed as described in materials and methods procedures. Data are expressed as mean ± SEM of three experiments.

In addition, the cell death occurrence is usually accompanied with the changes of cytoskeletal architectures and cell morphologies. Recently, studies have indicated that cells can transfer and/or exchange their cellular biomolecules, cytosolic materials and/or organelles, and pathogens into another target cells and/or neighbor cells through protruding their cell membrane with the long distance intercellular connections and bridgelike structures, termed tunneling nanotube (TNT).31 TNTs have been demonstrated that involved in many physiological and pathological processes including signal transduction, immune responses, apoptosis, cancer, and neuron disorders.32 In this present study, we find that zingerone NPs treatment elicited obviously filopodia-like protrusions and cytotoxicity in a dose-dependent manner (Figure 3A,B). Therefore, we believe that this cell morphological change with filopodia-like protrusion structures may play a role for zingerone NPs-mediated cytotoxicity. Indeed, TNTs involved in the modulation of cell death have been validated by transferred apoptotic biomolecules such as phosphatidylserine membrane and Fas ligand.33 However, the roles of zingerone NPs on TNT-mediated cell survival stress are needed to be investigated with more additional experiments. Altogether, the as-fabricated zingerone NPs have exhibited their excellent benefits of water-soluble, high stability, bioactivity, green, low cost, and tiny size. Moreover, zingerone NPs have first guaranteed their superior effects on antiproliferation and antitumorigenicity. Downregulation of Akt/NFκB Signaling Is Involved in Zingerone NPs-Mediated Anti-Cell-Proliferation and Anti-Cell-Growth. The tumor initiation and progression are associated with the interaction of many complexities of cellular cell signaling regulations, metabolites, gene expression, and so

dose-dependent manner (Figure S2). These results further demonstrated that zingerone NPs display the excellent anticell-proliferation in both hepatoma cell lines. Natural compounds including flavonoids, curcumin, and zingerone have been demonstrated their excellent chemopreventive effects in such as anti-inflammatory and antioxidative properties as well as reveal substantial anticarcinogenic and anti-angiogenesis activities.10,11,13,16 Unfortunately, their undesirable features including of low bioavailability, poorly soluble (or hydrophobic), and instability (or premature degradation) were limited their further applications in medical, health care, and cancer therapeutics.12,29 NPs possess excellent physicochemical properties and small sizes, which are believed that they can enter easily into cells with a high efficiency and therefore are agreed to be one of candidates as drug (nanomedicine), gene delivery vectors, bioimaging, and biolabeling.30 Here, we generate a novel carbon-based zingerone NP which derived from the nonvolatile and pungent compounds of ginger through the one-pot synthesize method. The active zingerone NPs own physiochemical characteristics of a tiny size of 1.42 ± 0.67 nm (Figure 1), a constitutive structure of nanotetramer (Figure 2) and excellent stability (the bioactive function can be maintained at least 7−10 days at 37 °C incubation, Figure 4). Furthermore, the as-fabricated zingerone NPs exhibit an over 4-fold efficacy on antiproliferation (Figure 3) and antitumorigenicity (Figure 4) in both hepatoma SKHep-1 and Huh7 cell lines as compared to the zingerone treatment. In addition, the Ki67 staining, an indicator for cell proliferation and growth, was also dramatically inhibited by zingerone NPs administration in a dose-dependent manner (Figure 5). F


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Table 1. Effects of Zingerone and Zingerone NPs on NFκB Activity Was Acquired from the Ratio of NFκB-Driven Luminescent Intensity and R. reniformis Luciferase Intensity conc. (μM)

zingerone

zingerone NPs

cell lines

0

25

50

100

200

25

50

100

200

SK-Hep-1 Huh7

100 ± 3.1 100 ± 3.9

99.9 ± 1.3 97.5 ± 3.1

107.2 ± 1.8 98.6 ± 3.1

97.9 ± 0.9 96.2 ± 1.9

94.2 ± 0.6 98.9 ± 0.3

86.7 ± 0.3a 93.4 ± 2.6

86.6 ± 2.6a 98.5 ± 6.0

86.5 ± 4.7a 83.6 ± 4.6a

NA NA

a

Statistically significant (P < 0.05); NA: not available.

Figure 7. Zingerone NPs interfere cell cycle progression and mediate cell apoptosis. Cells were treated with various concentrations of zingerone and/or zingerone NPs for 24 h and the histograms of cell cycle distribution of (A) SK-Hep-1 cells and (B) Huh7 cells were further analyzed by a flow cytometer. The vertical axis indicates the cell numbers; the horizontal axis indicates the PI stained strength. The cell cycle distribution of (C) SK-Hep-1 and (D) Huh7 cells was further analyzed and shown in bar graphs. The vertical numbers represent the cell population percentage in respective cell cycle phases of SubG0 (or apoptosis), G0/G1, S, and G2/M. These results shown are representative of at least three independent experiments and expressed as mean ± SEM. *P < 0.05; **P < 0.001.

forth. Activation of Akt- and Nuclear factor-κB (NF-κB)mediated signaling cascades and their critical roles in cell proliferation, survival, invasion, and apoptosis have been verified, which are also involved in tumorigenesis and many types of tumor malignancies.34,35 Therefore, we next elucidate the roles of Akt/NFκB signaling on zingerone NPs-mediated anti-proliferation using western blot analysis.

In SK-Hep-1 cells, our results revealed that zingerone treatment elicits the Ser 476 phosphorylation of Akt and slightly increased Akt activity (pAkt/Akt ratio), while zingerone NPs administration reveals a dramatic inhibition of Akt activity in a dose-dependent manner (Figure 6A). Furthermore, a considerable Akt activity dropped down at lower dose treatment (25 μM) has indicated that Huh7 cells G


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Figure 8. Zingerone NPs induce cell apoptosis in human hepatoma SK-Hep-1 and Huh7 cell lines. Both SK-Hep-1 (A) and Huh7 (B) cell lines were treated with various doses of zingerone and zingerone NPs for 24 h. Cell apoptosis was analyzed using TUNEL staining, and nuclei were stained with DAPI. Scale bar = 20 μm. The apoptotic positive cells (white arrow) of SK-Hep-1 and Huh7 cells were further quantitated and recorded in (C,D), respectively. Each point is mean ± SEM of three experiments. *P < 0.05; **P < 0.001.

downregulation of NFκB promoter activity in both SK-Hep-1 cells (14%) and Huh7 cells (17%). Additionally, owing to the high dose of zingerone NPs (200 μM) triggered a severely cell death and thus leading to an extremely luminescent inhibition in both NFκB and Renilla, and further the NFκB promoter activity is obtained a not available (NA) data in both SK-Hep1 cells and Huh7 cells (Table 1). These results suggest that the as-synthesized zingerone NPs reduced cell proliferation via inhibited Akt/NFκB signaling pathway. Additionally, our data also indicate that the zingerone NPs-mediated NFκB inhibition is through downregulation of the NFκB translation level rather than a transcription level. One of the strategies against cancer is to inhibit the cell growth associated-signaling pathways such as PI3K/Akt/ mTOR pathway34,36 and NFκB-mediated signaling.37 Akt inherits the upstream PI3K/PDK messages and regulates the activity of the downstream target proteins such as mTOR and NFκB to regulate the cell growth, proliferation, cell migration, and tumor metastasis.38 Therefore, inhibiting the activation and expression in both Akt and NFκB can achieve an advantageous effect against cancer. In this present study, we demonstrated that zingerone NPs display the excellent efficacy in cell proliferation inhibition and tumorigenicity suppression. Thus, the zingerone NPs blocking Akt/NFκB signaling and inhibiting its downstream effector of Ki67 protein are considered to play an essential role in zingerone NPs-mediated antiproliferation and antitumorigenicity in hepatoma cells. Zingerone NPs Inhibit the Progression of Cell Cycle and Induce Apoptotic Cell Death. To understand the mechanisms leading to loss of cell proliferation by zingerone NPs, flow cytometry analysis was performed to detect the effects of zingerone NPs on cell cycle distributions in both SKHep-1 cells and Huh7 cells (Figure 7A,B). In SK-Hep-1 cells, besides high dose of 200 μM treatment, zingerone induces no significant change for cell cycle distribution as compared to the control group. In addition, our data also showed that zingerone

are more susceptible to zingerone NPs treatment (Figure 6B). Next, we examine the effects of both zingerone and zingerone NPs on NFκB p65 protein expression and NFκB transcription activity. Our results revealed that the NFκB p65 expression is slightly upregulated at lower dose of zingerone treatment (25 μM) and then slowly downregulated with the dose increasing in SK-Hep-1 cells. Meanwhile, a significant attenuation of NFκB p65 expression was found in zingerone NPs administration in a dose-dependent manner. Furthermore, owing to IκBα is one of inhibitory subunits for NFκB p65 and the IκBα degradation is involved in NFκB p65 translocated into nuclear and activation. Thus, we also detected the effects of both zingerone and zingerone NPs on IκBα expression level. Our results indicated that zingerone significantly induces IκBα upregulation which is corresponded to NFκB p65 downregulation. Interestingly, a dramatically attenuated IκBα was found in zingerone NPs treatment in a dose-dependent manner, which is possibly associated with the zingerone NPs-mediated the getting worse of cell proliferation (Figure 6C). Moreover, the similar modulating effects of zingerone and zingerone NPs on both NFκB p65 and IκBα expression levels were also found in Huh7 cells (Figure 6D). To further understand the zingerone NPs contributed to NFκB activity regulation, both SK-Hep-1 and Huh7 cells were transient transfected with the NFκB-driven luciferase reporter vector and then administrated with either zingerone or zingerone NPs for 24 h. The NFκB-driven luciferase activities were assayed by using a Dual-Light kit in a luminometer. Our results revealed that only the higher dose of zingerone NPs treatment can induce a dramatic inhibition of luminescence intensity in both SK-Hep-1 cells (at least 200 μM treatment, Figure 6E) and Huh7 cells (at least 100 μM treatment, Figure 6F). We further analyzed the NFκB promoter activity, which was obtained from the luminescence intensity ratio of NFκBdriven luciferase and internal control Renilla luciferase. Our results showed that zingerone NPs induce a significant H


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Figure 9. Zingerone NPs enhanced DNA damage and induced cell apoptosis through activation of the caspase-cascade signal. The effects of zingerone NPs on DNA damage were validated through staining with a DNA damage maker-γH2AX on (A) SK-Hep-1 and (B) Huh7 cells using immunofluorescence assay. Meanwhile, the γH2AX protein expression levels of (C) SK-Hep-1 and (D) Huh7 cells were analyzed using western blotting. Furthermore, the effects of zingerone and zingerone NPs on the activity statuses of several caspases, such as caspase-8, caspase-9, caspase3, and PARP, on both (E) SK-Hep-1 and (F) Huh7 cells were achieved using western blotting assay. These results shown are representative of at least three independent experiments.

results suggest that lower dose (100 μM) of zingerone NPs would disturb cell cycle progression, which induces cell apoptosis and G1 and/or G2/M phase arrest. To validate the zingerone NPs-mediated cellular survival threaten through induction of cell apoptosis, the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was performed to demonstrate zingerone NPs-induced DNA damage and cell apoptosis. Our results demonstrated that zingerone NPs significantly increase the green signals which indicate the BrdU labeling for the DNA fragmentation level in a dose-dependent manner. Moreover, zingerone NPs can induce nuclear shrinkage/condensation and nuclear fragmentation at higher doses (100 and 200 μM) treatment which indicated that zingerone NPs-induced DNA damage and cell apoptosis in both hepatoma SK-Hep-1 (Figure 8A) and Huh7 cell lines (Figure 8B). Quantitative results also demonstrated that zingerone NPs significantly increase positive apoptotic cells in a dose-dependent manner in both hepatoma SK-Hep-1 (Figure 8C) and Huh7 cell lines (Figure 8D). These results validated that zingerone NPs trigger cytotoxicity via increased DNA damage and induced cell apoptosis. Cell cycle arrest is a cellular instinctual self-protective strategy in response to the external injury or threats. In this present study, we demonstrated that zingerone NPs reveal dose-dependent effects on cell cycle progression. Zingerone NPs-mediated S phase arrest (25−50 μM) supported that

induces limited effects on cell cycle, such as mild increase of SubG0 (0.5 vs 1.0%), decrease of G0/G1 phase (56.3 vs 45.7%), and increase of S phase (18.8 vs 27.2%). In contrast, zingerone NPs induce an increase of SubG0 phase in a dosedependent manner, which implies that zingerone NPs elicit a cell apoptosis. Furthermore, lower doses of zingerone NPs treatment (25 and 50 μM) elicit a significant decrease of G0/ G1 phase (48.8 and 37.8% respectively vs 57.1 and 54.7% respectively in zingerone treatment) and enhance the accumulation of S phase (22.9 and 34.6% respectively vs18.5 and 19.3% respectively in zingerone treatment). In addition, a higher dose of zingerone NPs (100 μM) administration can reverse the lower-dose zingerone NPs-mediated decrease of G0/G1 phase (61.1%) and increase of S phase (17.6%). Furthermore, the obviously declined G2/M phase was also observed in 100 μM of zingerone NPs treatment. In addition, the high dose of zingerone NPs (200 μM) treatment shows a dramatic inhibition of G0/G1 phase (32.9%) and S phase (13.9%) as well as a considerable increase of G2/M phase (44.5%) as compared to zingerone treatment (Figure 7C and Table S2). On the other hand, the effects of zingerone treatment on Huh7 cells are similar to those on SK-Hep-1 cells. Moreover, our results also indicated that the zingerone NPs induced the alterations of SubG0, G0/G1, and S phases (25 to 200 μM) in Huh7 cells, which is consistent with that of SK-Hep-1 cell. Unlike in SK-Hep-1 cells, high dose of zingerone NPs (200 μM) treatment in Huh7 cells does not induce the G2/M phase accumulation and instead directly promote cell apoptosis (Figure 7D and Table S3). These I


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caspases-cascade signaling, which is a critical signaling mechanism for cell apoptosis, were further investigated. Cells were treated with zingerone and/or zingerone NPs for 24 h and the protein expression profiles of caspase-8, caspase-9, and caspase-3 as well as the DNA fragmentation indicatorpoly(ADP-ribose) polymerase (PARP) were analyzed using western blot analysis. Our results showed that zingerone does not elicit obviously changes on caspases activity and PARP activation in SK-Hep-1 cell (Figure 9E), while higher dose of zingerone (>100 μM) treatment induced a slightly cleavage (activation) of caspase-9 and PARP in Huh7 cells (Figure 9F). Conversely, in SK-Hep-1 cells, zingerone NPs significantly stimulate the cleavage of caspase-8 in a dose dependent manner and generate an intermediate fragment of p43, which will be next cleaved into a smaller active fragment of p18. Next, the downstream candidates of caspase-8 including caspase-9 (p47) and caspase-3 (p34) also found to undergo an obvious cleavage and to generate active forms of p37 and p17/19 in caspase-9 and caspase-3, respectively, at higher dose treatment (>100 μM). Furthermore, it is also observed that zingerone NPs mediated PARP (p116) cleavage and generated the active fragment (p85) in a dose-dependent manner (Figure 9E). Additionally, zingerone NPs-triggered activation of caspases cascade signaling is also found in Huh7 cells. The dramatic cleavage of these caspases and PARP at the lowest dose of zingerone NPs (25 μM) treatment also supported that Huh7 cells are extremely susceptible to zingerone NPs treatment (Figure 9F). Altogether, these results suggest that zingerone NPs significantly elicit the genome instability and cytotoxicity via enhancing DNA damage and promoting caspases cascade signaling-mediated cell apoptosis. DNA damage, including that of double-strand breaks, which is triggered by the cells exposure to physical and/or chemical damage such as ionizing radiation and chemical agents, is accompanied with histone H2AX phosphorylation (γ-H2AX). The γ-H2AX therefore is an early sensitive biomarker of DNA damage.45,46 Moreover, γ-H2AX is an alarming factor and enables to recruit many check point proteins, which involved in DNA repair such as Mre11/Rad50/Nbs1 (MRN) complex, BRCA1, and 53BP1, to form a huge protein complex on the DNA damaged area and called “foci”.47,48 Furthermore, a wide distribution and high expression of γ-H2AX in nuclear termed pan-nuclear γ-H2AX was found to be highly associated with severe DNA damage and cell death.49 Indeed, high expression of γ-H2AX induced by ataxia-telangiectasia mutated kinase and c-jun-N-kinase has been suggested, which is involved in UV irradiation-mediated apoptosis.50,51 Here, we found that lower dose of zingerone NPs (50 μM) significantly elicited H2AX phosphorylation as compared to zingerone treatment. Further, an obvious γ-H2AX foci and the pan-nuclear γ-H2AX response (especially in Huh7 cells) were induced by higher dose of zingerone NPs (>100 μM) administration. These results indicate that zingerone NPs treatment induces the DNA damage and cell apoptosis. Indeed, our results have shown that higher dose of zingerone NPs administration elicited markable cellular survival threaten such as cell morphology deformation (Figure 1), cell cycle arrest (Figure 7), TUNEL staining apoptosis (Figure 8), and caspases activation (Figure 9), which suggested severe DNA damage and cell apoptosis. Cell apoptosis can be triggered by the family of cysteinyl aspartate-specific protease caspases that directly or indirectly

lower dose treatment induces a significant downregulation of cell viability, proliferation, and colony formation (Figures 3−5). In addition, the Akt activity and NFκB expression also obviously downregulated during lower dose of zingerone NPs treatment (Figure 6). Furthermore, higher doses of zingerone NPs administration (100−200 μM) elicit a fully dysfunction of Akt and NFκB (Figure 6) and enhance cell cycle arrest in either G1 phase (Huh7 cells) or G2/M phase (SK-Hep-1 cells), as well as caused DNA damage and/or cell apoptosis (Figure 8). Indeed, reports have demonstrated that either Akt or NFκB can interact with cell cycle regulators such as CDKs, cyclins, c-Myc, p21, p27, and so forth to comprehensively regulate cell cycle progression.39−41 For example, Akt is able to phosphorylate several proteins such as p21, p27, and GSK-3β to facilitate the G1-S phase transition.39 In addition, the S−G2 phase transition is also intimately associated with Akt-mediated CDK2 phosphorylation and their subcellular localizations.42 On the other hand, NFκB is a pleiotropic regulator in cellular physiological and pathological processes including cell proliferation, inflammation, tumorigenicity, and metastasis when cells were stimulated with a variety of stimuli and environmental challenges. NFκB therefore plays important roles in regulating cell cycle progression.40 For example, NFκB binds to cyclin D1 promoter and regulates cyclin D1 expression to trigger G1−S phase transition.43 Additionally, NFκB-induced CDK2 expression may involve in S−G2 phase transition.44 In this study, we have demonstrated that zingerone NPs exhibited an excellent power for antiproliferation and antitumorigenicity though abolished Akt- and NFκBmediated signaling. Moreover, our results also suggested that the zingerone NPs-elicited disturbing of cell cycle progression and cell apoptosis are associated with attenuation of Akt- and NFκB-mediated signaling. Zingerone NPs Enhance DNA Damage and Induce Cell Apoptosis by Triggered the Activation of CaspaseCascade Signaling. To further investigate whether zingerone NPs-induced cytotoxicity and cell apoptosis are caused by enhanced DNA damage and activated apoptotic signaling pathway, we first examined the expression level of DNA damage marker-phosphorylated histone H2AX (γ-H2AX) to validate the role of zingerone NPs-elicited DNA damage. Our results demonstrated that zingerone NPs significantly stimulate the upregulation of γ-H2AX in a dose-dependent manner as compared with the zingerone group in both SK-Hep-1 (Figure 9A) and Huh7 cells (Figure 9B) using the immunofluorescence assay. The obviously γ-H2AX-mediated nuclear foci characteristics of DNA double strain damage were observed at lower dose of zingerone NPs (50 μM) treatment, and the dramatically magnified γ-H2AX foci were exhibited at 100 μM of zingerone NPs treatment. Finally, a severe DNA damage was illustrated through a pan-nuclear γ-H2AX expression (whole nucleus filled with high intensity fluorescence) when the high dose of zingerone NPs (200 μM) treatment. Next, the western blotting assay also demonstrated that lower dose of zingerone NPs (50 μM) enhances an obviously γ-H2AX expression. Furthermore, higher than 100 μM of zingerone NPs treatment would accelerate a dramatic γ-H2AX generation in both SK-Hep-1 (Figure 9C) and Huh7 cells (Figure 9D). These results demonstrated that zingerone NPs induce DNA damage and enhance the genome instability to elicit cytotoxicity and cell apoptosis. To demonstrate the signaling mechanisms of zingerone NPsmediated cell apoptosis, a series of cysteine proteases-mediated J


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functionalization, has revealed its properties such as dramatic growth inhibition, tumorigenicity suppression, and cell apoptosis. Our study will provide an advance strategy for the design of more effective “Nano-chemoprevention” drugs in future cancer therapeutics and biomedical and clinical applications.

induce the DNA fragmentation, which is a hallmark of apoptosis.52 In general, there are two groups of apoptotic caspases that are verified including the initiator caspases such as caspase-8 and caspase-9 and the executioner caspases such as caspases-3. The activated initiator caspases can directly or indirectly elicit the activation of the executioner caspases,53,54 while executioner caspases can regulate feedback the initiator caspases. These activated caspases cascade signaling finally trigger caspase-3 activation. Caspase-3 therefore translocates into nuclear and cleaves or activates the PARP, which is considered as a guard for protection of the genomic stability, and therefore induce the DNA fragmentation and cell apoptosis.52,55 In the study, we have demonstrated that zingerone NPs elicited DNA damage and cell apoptosis. We also found that the dramatic cell apoptosis was accompanied with the pan-nuclear γ-H2AX expression and induced the activation of caspase-8/caspase-9/caspase-3/PARP cascade signaling during zingerone NPs treatment. In summary, we generate a novel carbon-based nanotetramer called zingerone NP, which precursor is derived from a bioactive, nonvolatile, and pungent compound of ginger. We verify that the zingerone NPs induce a significant cytotoxicity and colony inhibition. Our data demonstrated that zingerone NPs dramatically elicited antiproliferation, antitumorigenicity, and apoptosis through downregulation of the Akt/NFκB signaling mechanisms, DNA damage-mediated cell cycle arrest, and triggered cell apoptosis via activation of caspase-cascade signaling pathway (Figure 10). These outcomes indicate that

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MATERIALS AND METHODS

Zingerone NP Synthesis. Zingerone was purchased from SigmaAldrich and used without further purification. Zingerone was mixed with pure ethanol (2% w/v), and then a 50 mL aliquot of the solution was placed into thoroughly cleaned beaker and held at a constant temperature of 120 °C on a hot plate (Suntex Inc. Co., Taiwan, SH301) using heat stirring (1100 rpm) for 3−4 h in an ambient air atmosphere and then stir-cooled to room temperature. Finally, the NP (zingerone NPs) liquids were passed through a 0.45 μm polyvinylidene fluoride (PVDF) syringe filter (Millipore MillexHV). This final synthesis product is the zingerone NP stock solution, which then was diluted for further antiproliferation and tumorigenicity suppression tests. Atomic Force Microscopy. Samples for AFM were prepared by drop casting of the zingerone NPs solution (5 μL) onto a silicon wafer sample at room temperature and allowed them to dry. Topographic images were acquired in AC mode using an AFM (MFP-3D, Asylum Research, Santa Barbara, CA, USA) under ambient conditions. A silicon cantilever (Olympus AC240TS) with a nominal spring constant of 2 N m−1 was used for all images, with a scan rate of 0.7 Hz and an image resolution of 512 × 512 pixels. Transmission Electron Microscopy. TEM images of zingerone NPs were acquired by deposited NPs solution (0.2 μL) onto TEM 200 mesh copper grid (Ted Pella Inc., CA, USA) and allowed to dry. Images were further obtained using a JEOL JEM-2100 and operated at 200 kV. UV−Visible Spectroscopy. We pipetted samples (either zingerone or zingerone NPs) into the 1 cm path length quartz cell and then subjected to the UV−vis spectrophotometer (JASCO V-630, Japan). The obtained data of UV−vis absorption spectrum were then analyzed. Zeta Potential Analysis. We pipetted fresh zingerone NPs into the quartz cell and then measured the effective electric charge on the NP surface by zeta potential analysis (Delsa nano, zeta potential and submicron particle size analyzer, Beckman Coulter Inc., USA). Liquid Chromatography-Mass Spectrometry. The LC-MS system consisted of a Waters 2695 separation module for highperformance liquid chromatography with an outflow that was coupled to an electrospray (ESI) source and a Waters Micromass ZQ mass spectrometer (Waters Co., Milford, MA). Zingerone standard and zingerone NPs were eluted from a ZORBAX Eclipse XDB-C18 column (2.1 × 150 mm; particle size 3.5 μm) with an isocratic mobile phase (0.02% formic acid in H2O and 0.02% formic acid in methanol) at a flow rate of 0.2 mL/min. The mass spectrometer was operated in positive ion mode (ESI+: cone voltage 20 V). Cell Lines and Cell Culture. Human hepatoma cell lines of SKHep-1 and Huh7 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 100 IU/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine. These cells were incubated under humidified conditions in 95% air and 5% CO2 at 37 °C. Cytotoxicity Assay. Cell viability was measured by a quantitative colorimetric assay with MTT assay. Cells were seeded in a 96-well plate with a density of 2 × 104 cells/100 μL/well overnight and then treated with various concentrations of zingerone and zingerone NPs for 24 h. The cells were further incubated in adequate medium containing 0.5 mg/mL of MTT for 2 h at 37 °C. The formazan in viable cells was dissolved with dimethyl sulfoxide (100 μL/well) (Sigma-Aldrich, USA) and determined the optical densities using a microplate reader (BIO-RAD Benchmrk plus, France) at the absorption wavelength of 570 nm.

Figure 10. One-pot synthesized zingerone nanotetramer induces a severe DNA damage and upregulated caspase-cascade signaling to trigger cell apoptosis. Meanwhile, a cell survival signaling of Akt/ NFκB pathway was also downregulation to abolish cell proliferation. All these outcomes demonstrate that the zingerone NPs own excellent potential for anti-cell-proliferation, cell apoptosis, and tumorigenicity suppression on human hepatoma cells.

the novel synthesized zingerone NPs have the potential efficiency for chemoprevention and can be a potent nanomedicine for future anticancer strategies.

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CONCLUSIONS In this study, we developed novel natural carbon-based zingerone NPs via the one-pot synthesized process and validated a small size of 1.42 ± 0.67 nm as well as composed of nanotetramer through using AFM, TEM, UV, and LC-MS analysis. The zingerone NPs have shown their excellent chemopreventive effects such as antiproliferation, tumorigenicity suppression, enhanced the DNA instability and damage, as well as triggered cell apoptosis through downregulation of Akt/NFκB signaling mechanisms and activation of caspase-cascade signaling pathway on the human malignant hepatoma SK-Hep-1 and Huh7 cell lines. These results first investigated that the nanosized bioactivity component zingerone NPs, which is without any modification and/or K



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Colony Formation Assay. Cells were seeded (2000 cells per well) and incubated overnight. After cells were treated with zingerone and/or zingerone NPs for 7−10 days and next fixed with 4% paraformaldehyde for 20 min and stained with crystal violet (0.01% in 10% buffered formalin; Sigma, St. Louis, MO) for 30 min. For colony counting, the aggregates of more than 50 cells were scored as colonies. For determining colony survival efficiency, the colonies and/or cells which stained with crystal violet were redissolved with an adequate volume of crystal violet elution buffer (ethanol absolute 50% and acetic acid 0.1% in double-distilled water) and shaken for 15 min. The spectrometric absorbance at 595 nm was measured using a microplate reader (BioTek EON). Cell Cycle Analysis. Cells were treated with various doses of zingerone and/or zingerone NPs for 24 h. Cells were harvested and washed twice with PBS before fixation with ice-cold ethanol (70%) and then stored at −20 °C for overnight. Cells were washed twice with PBS before incubation with RNase A (10 μg/mL) and PI (50 μg/mL) for 60 min at RT. The DNA content of 20 000 events was analyzed by flow cytometry using a CytoFLEX Flow Cytometer (Beckman Coulter, Inc., USA) and CytExpert 2.0 software. Immunofluorescence Analysis and Terminal Deoxynucleotidyl TUNEL Assay. After cells were treated with zingerone and/or zingerone NPs for 24 h, cells were fixed with 4% paraformaldehyde for 20 min and permeability of 0.2% Triton X-100 for 15 min. Fixed cells were next incubated with the primary antibodies against Ki67 (NeoMarkers, Fremont, CA) and γ-H2AX (Santa Cruz Biotechnology). After further washes, the cells were incubated with the appropriate secondary IgG antibody conjugates of the Alexa Fluor 546 or Alexa Fluor 488 dyes (Invitrogen, Thermo Fisher Scientific) for 60 min at RT. To detect the cell apoptosis, after cells were executed with fixation and permeability, the cells were then examined the DNA-strand breaks during apoptosis by incubated with the TUNEL reaction mixture for 60 min at 37 °C using in Situ Cell Death Detection Kit (Fluorescein, ROCHE). The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min at RT. The immunostaining images were captured with a fluorescence microscope (Leica, DMIL, Germany), and the Ki67 positive cells and apoptosis-stained cells were then analyzed and counted at either 20× or 40× magnification. Western Blot Analysis. Cell lysates were separated with 8−12% SDS-PAGE and were transferred onto PVDF membranes. Following the blocking procedure, the membrane was then incubated with primary antibodies of phospho-Akt (S473), Akt, caspase-8, caspase-9, PARP (Cell Signaling Technology), active caspase-3 (CHEMICON International), caspase-3, NFκB p65, IκBα, γ-H2AX (Santa Cruz Biotechnology), and GAPDH antibody (Proteintech) for 1 h at room temperature or for overnight at 4 °C. After the HRP secondary antibody (Santa Cruz Biotechnology) administration, the signals were detected using ECL western blotting substrate (Promega) and exposed to X-ray film for autoradiography. NFκB Luciferase Activity Assay. The NFκB promotor activity was performed as previously described.56 Briefly, cells were seeded in a six-well plate for 80% confluence and then co-transfected with NFκB-driven luciferase vector and the R. reniformis luciferase reporter vector at a ratio of 1:1/10 using Lipofectamine 3000 (Invitrogen) for 5−6 h and then changed into complete medium and incubated for overnight. Cells were further administrated with zingerone and/or zingerone NPs for 24 h. The NFκB-driven luciferase activities in cells were determined using a Dual-Light kit (Promega, Madison, WI) in a luminometer (POLARstar+ OPTIMA microplate reader, BMG LABTECH) and normalized with that of R. reniformis luciferase according to manufacturer’s instructions. Statistical Analysis. Data are reported as mean ± SEM. The statistical significance of the data from the experiments or assays was determined using a two-tailed Student’s t test. Differences were considered significant at P < 0.05.

Research Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b14559. Zingerone NPs suppress tumor growth in Huh7 tumor xenograft model of NOD/SCID mice; zingerone NPs inhibit cell proliferation using BrdU incorporation assay; effects of zingerone and zingerone NPs; flow cytometric analysis of various doses treatment of zingerone and zingerone NPs in human hepatoma SK-Hep-1 cell lines; and further experimental data on materials and methods (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +886 7 5252000 ext. 3931. Fax: +886 7 525-3908. ORCID

Shuchen Hsieh: 0000-0001-5358-0836 Author Contributions ‡‡

M.-L.K and P.-Y.L. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Ministry of Science and Technology of Taiwan (MOST 106-2321-B-110-001-MY3, MOST 104-2628-M-110-001-MY3, and MOST 107-2113-M110-009).

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REFERENCES

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