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Chrysin loaded nanostructured lipid carriers (NLCs) triggers apoptosis in MCF-7 cancer cells by inhibiting the Nrf2 pathway
Accepted Manuscript Title: Chrysin loaded nanostructured lipid carriers (NLCs) triggers apoptosis in MCF-7 cancer cells by inhibiting the Nrf2 pathway Authors: Mehdi Sabzichi, Jamal Mohammadian, Roya Bazzaz, Mohammad Bagher Pirouzpanah, Maghsod Shaaker, Hamed Hamishehkar, Hadi Chavoshi, Roya Salehi, Nasser Samadi PII: DOI: Reference:
S1359-5113(17)30210-6 http://dx.doi.org/doi:10.1016/j.procbio.2017.05.024 PRBI 11053
To appear in:
Process Biochemistry
Received date: Revised date: Accepted date:
6-2-2017 16-4-2017 20-5-2017
Please cite this article as: Sabzichi Mehdi, Mohammadian Jamal, Bazzaz Roya, Pirouzpanah Mohammad Bagher, Shaaker Maghsod, Hamishehkar Hamed, Chavoshi Hadi, Salehi Roya, Samadi Nasser.Chrysin loaded nanostructured lipid carriers (NLCs) triggers apoptosis in MCF-7 cancer cells by inhibiting the Nrf2 pathway.Process Biochemistry http://dx.doi.org/10.1016/j.procbio.2017.05.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Chrysin loaded nanostructured lipid carriers (NLCs) triggers apoptosis in MCF-7 cancer cells by inhibiting the Nrf2 pathway
Mehdi Sabzichia †, Jamal Mohammadianb, †, Roya Bazzazc, MohammadBagher Pirouzpanahd, Maghsod Shaaker c, Hamed Hamishehkara, Hadi Chavoshic, Roya Salehia, *, Nasser Samadib, *
a
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
b
School of advanced medical science, Tabriz University of Medical Sciences, Tabriz, Iran
c
Hematology and Oncology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
d
Stem cell and Regenerative Medicine Institute, Tabriz University of Medical Sciences, Tabriz, Iran
* Corresponding authors: Roya Salehi: Assistant Professor of Pharmaceutical and Medical Nanotechnology, School of Advanced Medical Science, Tabriz University of Medical Sciences, Tabriz, IRAN. Nasser Samadi: Associate Professor of Clinical Biochemistry; Department of Biochemistry & Clinical Laboratory, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, IRAN. Daneshgah St. Tabriz-Iran. P.O. Box: 51656-65811
E-mail address: [email protected] [email protected] Running Title: Chrysin enhances doxorubicin effect in MCF-7 cancer cells
Graphical abstract
Highlights •
Chrysin increases the efficacy of Dox by altering the cell cycle distribution of MCF-7 cells.
•
NLCs can be considered as potential delivery systems.
•
Chrysin-loaded NLCs had synergistic effects on cellular uptake of Dox.
•
Chrysin inhibited Nrf2 pathway, which was associated with high percentages of apoptosis.
Abstract We investigated the role of chrysin as an effective adjuvant along with nanostructured lipid carriers (NLCs) to increase the cytotoxicity of doxorubicin (Dox) in MCF-7 breast cancer cells. The prepared formulation was characterized from point of view scanning electron microscope (SEM), size & zeta potential. Cellular uptake and cytotoxicity of nanoparticles were examined by fluorescent microscopy and MTT assay, respectively. Flow cytometry and real-time PCR were
used to understand the molecular mechanism of Nrf2 and related downregulating genes. The average size of the nanoparticles was 105 ± 2 nm, which was confirmed by SEM. Our results demonstrated that incubation of the cells with chrysin-loaded NLCs enhanced the percentage of apoptosis from 21.11 ±5.72% to 27 ±3.13% (p<0.05). Furthermore, the population of cancer cells in the sub-G1 phase increased up to 12 ± 2.1% compared to untreated cells (p<0.05). mRNA expression levels of Nrf2, NQO1, HO1, and MRP1 exhibited a significant decrease compared to the control group (p<0.05). Our findings recommend that chrysin delivery along with NLCs would enhance the efficacy of Dox by exerting inhibitory effects against drug efflux pumps and drug detoxification enzymes.
Chemical compounds: Chrysin (PubChem CID: 5281607); Doxorubicin (PubChem CID: 31703); Glyceryl Behenate (PubChem CID: 5362585); Dimethyl Sulfoxide (PubChem CID: 679); MTT formazan (PubChem CID: 16218671); Miglyol (PubChem CID: 53471835).
Abbreviations: RPMI, Roswell Park Memorial Institute medium; FBS, Fetal Bovine Serum; MCF7, Michigan Cancer Foundation-7; SEM, scanning electron microscopy; MTT, Tetrazolium, 2(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-, bromide; EE, Entrapment Efficiency; RT-PCR, Real Time polymerase chain reaction; DAPI,4',6-diamidino-2-phenylindole; PI, propidium iodide; FITC, Annexin V-fluorescein isothiocyanate; Dox, Doxorubicin.
Keywords: Chrysin; Doxorubicin; NLCs, MCF-7; Cytotoxicity
1. Introduction Breast cancer has become one of the major causes of death among women in the world along with cardiovascular disease [1]. Despite great attempts to reach advanced protocols in cancer treatment, chemotherapy with doxorubicin (Dox) continues to remain one of the fundamental therapeutic strategies. Dox is widely used because of its strong cytotoxic properties against
cancer cells through the induction of apoptosis, disruption of cell cycle progression, and interference toward topoisomerase-II enzyme [2]. Nevertheless, many unwanted side effects of Dox to normal healthy tissues and the acquired resistance to this drug in many patients have caused a decline in the final outcome of Dox efficacy [3, 4]. Therefore, applying new adjuvant with an advance drug delivery system is considered an alternative to conventional methods, which would be evaluated efficacy of chemotherapy [5-8]. Nuclear factor-E2-related factor 2 (Nrf2) has emerged as a key molecule that plays a pivotal role in controlling homeostasis in normal and cancer cells [9, 10]. Overexpression of Nrf2 is reported in many cancer cells and is recognized as one of the master transcription factors as it regulates three major classes of genes including drug transporters such as multi-drug resistance (MDR) and MDR-associated proteins (MRP1), anti-oxidants such as heme oxygenase1(HO1), and phase II detoxification enzymes such as NQO1 [11, 12]. Therefore, the inhibition of Nrf2 in cancer tissue is considered a novel strategy that sensitizes tumor cells to chemotherapy agents and prevents efflux and rapid detoxification of drugs in cancer tissues [13, 14]. Recently, chrysin (5, 7-dihydroxyflavone) has been introduced as a flavonoid that possesses anti-inflammatory, anti-oxidants, antidiarrheal, and anti-cancer properties [15, 16]. The molecular mechanism of chrysin has not yet been identified. A study reported chrysin that inhibited cell growth in MDA-MB-231 through the PPAR alpha signal pathway [17]. Another study demonstrated that chrysin suppresses cell proliferation through the activation of caspase superfamily and inactivation of PKB [18]. However, only a few studies have reported that chrysin can be considered a potent Nrf2 inhibitor [19-21]. In addition, similar to polyphenols, chrysin has a low bioavailability, which is a bottleneck for application in pharmaceutical sciences [22]. In this study, we formulated chrysin in nanostructured lipid carriers (NLCs) as the second generation of lipid-based carriers to enhance the bioavailability of drugs and sustained release of this formulation in tumor microenvironments [23, 24]. This delivery system has recently been evaluated for cancer therapy because of many advantages such as biocompatibility to biological systems, high entrapment efficiency, and controlled release of drugs [25, 26]. These properties have transformed NLC systems into effective carriers, which can open new approaches in cancer delivery [27, 28]. The purpose of this study was to investigate the role of chrysin with NLCs in
Dox-induced apoptosis in MCF-7 breast cancer cells. Moreover, the underlying mechanism of cell cycle progression was evaluated. Finally, we aimed to investigate the role of Nrf2 and downstream gene expression in Dox-mediated apoptosis in cancer cells.
2. Materials and methods Chrysin, Dox, fetal bovine serum (FBS), and tetrazolium salt (MTT) were procured from SigmaAldrich (Steinheim, Germany). Compritol® ATO 888 was purchased from Gattefossè (Saint Periest Cedex, France). Rhodamine B was obtained from (Merck Chemicals Germany). Roswell Park Memorial Institute (RPMI) 1640 medium, glutamine, streptomycin, penicillin G and dimethyl sulfoxide (DMSO) were obtained from Invitrogen Life Technologies (Auckland, New Zealand). Annexin V-fluorescein isothiocyanate (FITC), propidium iodide (PI) were purchased from (E-bioscience, USA) and primers from MWG Biotech (Ebersberg, Germany). The human breast cancer (MCF-7) cell line was procured from National Cell Bank, (Pasteur institute, Iran).
2.1. Preparation of NLCs containing chrysin NLCs were prepared by modified hot homogenization technique associated with the ultrasonication method [29]. Briefly, chrysin was solubilized in Miglyol and the mixture was added into melted compritol. Then, 1% poloxamer as surfactant was added drop wise into the lipid phase under homogenization at 20000 rpm (Heidolph, Germany) for 20 min. Then the sample was immersed into a water bath and sonicated for 5 min. The formed hot oil in Water (o/w) nano-emulsion was cooled at ambient temperature for lipid phase recrystallization, and finally the NLC was produced [30].
2.2. Determination of chrysin entrapment efficiency To determine the entrapment efficiency (EE) % of chrysin, we employed UV-Visible spectrophotometer (UV160-shimadzo -Japan). Unloaded chrysin was calculated according to calibration curve for 0.1-1 mg/ml. UV absorbance for chrysin was measured at ƛmax = 328 nm following the Beer-Lambert law in a range of 0.1-1.5. At first, 500 µL of the formulation was
added to the upper chamber of Amicon® centrifugal filter (Millipore, Germany) and centrifuged at 5,000 rpm (Beckman, Spain) for 25 min. Then, the amount of chrysin in the lower chamber was calculated according to the absorbance of the calibration curve. The following equation was applied to determine the percentage of EE:
EE(% , w / w)
winitialdrug _ w free drug winitialdrug
2.3. Characterization and optimization of chrysin-loaded NLCs Chrysin-loaded NLCs were evaluated for nanoparticle dimensions by the photon correlation spectroscopy technique using a particle diameter instrument (WING Sald 2101 SHIMADZU JAPAN) after appropriate dilution through deionized water. The surface morphology of the nanoparticles was determined by scanning electron microscope (SEM) (MV2300, Vega Tescan, Czech Republic). To determine the zeta potential, the NLC solution was diluted with phosphatebuffered saline (PBS) and was measured using Zeta Sizer ZS (Malvern Instruments, UK). 2.4. Cellular internalization experiment Cellular uptake was studied to understand the role of nanoparticle structure for the internalization of chrysin into cancer cells. To follow the fate of chrysin, Rhodamine B as a reporter dye, was incorporated into NLCs (0.4% w/w according to the weight of the lipids). The Amicon® tube was used to purify of the chrysin-loaded NLCs from unloaded rhodamine B. The free rhodamine B was passed through a filter and collected in the lower chamber of the Amicon® tube. The upper chamber containing fluorescent NLCs was diluted with PBS and centrifuged twice. MCF-7 cells (4×105 per well) were incubated in six–well plates that were typically covered with 12 mm coverslips. Cell uptake of the formulation was followed by fluorescent microscopy (Olympus, Japan) during various times interval (15–120 minutes). To quantify chrysin uptake, cancer cells were incubated with the formulation for 60 min. Then cells were collected and washed with PBS, and finally, florescent dye excitation was detected using FACS Calibur (flow cytometer; BD Biosciences, San Jose, CA).
2.5. In vitro Cytotoxicity Study The MCF-7 breast cancer cells (1.4×104 cells/ well) were maintained in RPMI medium and incubated at 37 °C in 5% CO2 as growth phase condition. The inhibitory effect of blank NLCs, chrysin, and chrysin-NLCs on cell growth and their cytotoxic effects were evaluated after treatment with different concentrations from 5 up to 40 μM. The cells were substituted with 200 μL fresh media containing 20 μL of MTT solution (2 mg/ml in PBS) and incubated for additional 4 h at 37˚C. Finally, the absorbance was measured at 570 nm after shaking for 20 min using a microplate ELISA reader (ELX 800, Biotek, USA)[31].
2.6. In vitro Apoptosis Assay To detect early and late apoptosis cells and distinguish these cells from necrosis cells, we analyzed the cells using= Annexin V/ FITC apoptosis detection kit (E-bioscience, USA). The cells (4×105) of each well were incubated in medium containing free chrysin (20 µM) and chrysinloaded NLCs at the same concentration of chrysin at 37°C for 24 h. MCF-7 cells were harvested through trypsinization and re-suspended in PBS. Lastly, annexin V/FITC along with PI were added and monitored by flow cytometry.
2.7. Cell cycle distribution To understand the molecular mechanism of cell death and detection rates of sub-G1 population, MCF-7 breast cancer cells were seeded at an initial density of 4×105 per well, and after treatment with the above mentioned conditions for 24 h, then the cells were harvested by trypsinization, washed using PBS (pH=7.4), re-suspended in ethanol, and incubated overnight at room temperature. Cells were then exposed to PI and ribonucleaseA for 25 min. Cell cycle alteration was evaluated using a flow cytometer.
2.8. DAPI Staining for Nuclear Morphology Study MCF-7 breast cancer cells (4×105 per well) were seeded in six-well plates and covered with 12 mm coverslips. Cells at sufficient confluence (70%) were treated with chrysin, dox, chrysin
loaded NLCs and combination of chrysin and dox for 24 h; then, the cells were washed with PBS, fixed using paraformaldehyde (4%) for 20 min, permeabilized using 0.1 % (w/v) Triton X100 for 15 min, and stained with DAPI dye for 15 min. Finally, the coverslips were washed with PBS. Triplicate samples were prepared for each treatment, and at least 300 cells were counted in random fields for each sample. Identification of apoptotic morphological alteration of cancer cells was investigated under a fluorescence microscope.
2.9. RNA extraction and real-time PCR The total RNA of MCF-7 cancer cells was isolated by Trizol according to the manufacturer’s instructions [32]. The amount of total of RNA was determined by optical density measurement (A260/A280 ratio) on Nano Drop 1000 Spectrophotometer (Wilmington, DE, USA). cDNA was produced using the First-Strand Synthesis kit (Vivantis, USA) following the manufacturer’s instructions. Real-time (RT)-PCR was performed using the SYBR Green Master Mix and evaluated on a Roche lightcycler96 (Germany). Specific primers were as follows: Nrf2 (5’ACTCCCAGGTTGCCCAC-3’ CTCGCCTCATGCGTTTTTG-3’
and and
5’-GTAGCCGAAGAAACCTCATTGTC-3’),
NQO1
(5’-
5'-CCCCTAATCTGACCTCGTTCAT-3’),
MRP1
(5’-
ATGACCAGGTATGCCTATTATTAC-3’ and 5’-CACATCAAACCAGCCTATCTC-3’), and HO1 (5’ACGGCTTCAAGCTGGTGATG-3’ and 5’- TGCAGCTCTTCTGGGAAGTAG-3’). All samples were performed in triplicates. Interpretation of the data was quantitated using the ΔΔCt method with efficiency correction by Pfaffl technique and the cycle threshold values were standardized with respect to GAPDH expression.
2.10 Statistical analysis Results were evaluated as the mean ± SD. One-way ANOVA was performed to determine acceptable differences between examination groups and control groups. P value < 0.05 was considered significant (Graph Pad V6.0 Software Inc., San Diego, CA, USA). 3. Results
3.1. Characterization of chrysin-loaded NLCs Surface morphology of chrysin-loaded NLC was determined by SEM images which confirmed a spherical shape (Fig.1a). Particle size measured by DLS was 55 to 300 nm (Fig. 1b). The new formulation was further characterized to determine the zeta potential. The surface charge of the particle showed a negative value of -11 mV at the original pH 7.4 (Fig. 1c). Table 1 demonstrates the effect of types of lipid carriers and surfactant amounts on nanoparticle size. The particle diameter of NLCs formulated with compritol was smaller than that of NLCs prepared with Precirol. In addition, increasing the surfactant amount from 80 to 140 mg caused a decrease in the particle size.
3.2. Cytotoxicity of chrysin-loaded NLCs Cytotoxicity analysis using MTT assay was performed to determine the effect of chrysin-loaded NLCs on MCF-7 cells after 24 and 48 h of incubation with desired concentrations of Dox (0.25 to 5 µM), chrysin (5 to 40 µM), and formulation (5 to 40 µM). As shown in (Figs.2a & b), the IC50 values for Dox, chrysin, and chrysin-loaded NLCs were 0.82, 20 ± 0.05, and 17.3 ± 0.04 µM, respectively. Chrysin-loaded NLCs induced the cytotoxicity of MCF-7 cells more effectively than chrysin only treatment (p<0.05). There were no significant differences between cancer cells incubated with NLCs alone and untreated cells (p>0.05). This demonstrated the biocompatibility and low cellular toxicity of chrysin-loaded NLCs (Fig.2c). 3.2.1. Cell morphology analysis by fluorescent microscopy The alteration of cancer cell morphology to apoptotic body by DAPI staining confirmed that NLC delivery system increased the apoptosis, which was also observed by light microscopy. As shown in (Fig.3a&b), chromatin fragmentation and cell wall shrinkage increased and the intensity of DAPI dye decreased, which indicates the degradation of cancer cells. 3.2.2. Cellular Uptake of Chrysin-loaded NLCs Cellular uptake was quantified to determine the cell permeation of chrysin due to enhanced endocytosis, which resulted in a positive correlation between lipid nanoparticles and cancer cell
membrane. The fluorescence intensity of chrysin in MCF-7 cells significantly increased from 20 to 120 min, representing the continuous accumulation of chrysin into the cells and showing that the chrysin-loaded NLCs were internalized in a time-dependent manner (Fig3.c). To further verify this, quantitative analysis of cellular uptake by flow cytometry was performed. It was demonstrated that compared with chrysin alone (64%), chrysin-loaded NLCs (84%) intensified the percentage of internalization into cells (Fig.4). 3.3. Chrysin-loaded NLCs induced apoptosis in MCF-7 cancer cells To determine the proportion of apoptotic cells, we used annexin V along with PI staining. The results revealed that NLCs alone showed slight apoptosis compared with untreated cells (p<0.05) (Figs.5a and b). Chrysin (20 µM) and Dox (0.01 µM) showed a cell population of 22.5 ± 1.4% and 19.1 ± 2.3%, respectively, in the apoptotic phase (Figs.5c and d). Chrysin-loaded NLCs increased the cell population in apoptotic phase up to 27 ±3.45% compared with that of chrysin alone (p<0.05) (Fig.5e). Furthermore, chrysin-loaded NLCs decreased necrosis rates from 15.11% to 8.9%. The percentage of apoptotic cells increased up to 37± 2.4% when MCF-7 cells were incubated with chrysin-loaded NLCs plus Dox (Fig.5j), while chrysin plus Dox exhibited 31% apoptotic cells (Fig.5f).
3.4. Effect of Chrysin-loaded NLCs on Cell Cycle Regulation Similar to the controls, incubating MCF-7 breast cancer cells with NLCs alone indicated no apoptotic cells (Fig.6a, b). When the cells were exposed to chrysin (20 µM) and Dox (0.01 µM), the percentage of apoptosis was 7.7 ± 0.4% and 2 ± 0.2%, respectively (Fig. 6c, d). Treating cells with chrysin-loaded NLCs for 24 h caused 27 ± 2.76% apoptosis, which was accompanied by 28± 0.7% G2/M arrest (Fig.6f). Applying Dox with chrysin-loaded NLCs enhanced the percentage of cells in sub-G1 population up to 19±3.11 (Fig.6j). Incubation of MCF-7 cells with chrysin-loaded NLCs increased the population of cells in G0/G1 phase up to 44% compared with the 38% with chrysin alone. DAPI staining confirmed our observation from apoptosis analysis, whereby the highest percentage of cell death (chromatin degradation and cell wall shrinkage) was observed in the cells exposed to chrysin-loaded NLCs plus Dox (Fig.3b).
3.5. RT-PCR analysis of Nrf2, MRP1, NQO1, and HO1 To understand the role of Nrf2 and downstream genes in controlling apoptosis in cancer cells, RT-PCR was performed. Applying chrysin-loaded NLCs caused the downregulation of Nrf2 compared with NLC or chrysin alone (p<0.001). In addition, NQO1 mRNA expression was dramatically downregulated in MCF-7 cells treated with chrysin-loaded NLCs compared with other groups (p<0.01) (Fig.7). Furthermore, chrysin and chrysin-loaded NLCs markedly decreased HO1 mRNA gene expression levels (p<0.05).
4. Discussion Recently, chrysin and its derivatives have been known to exert anti-cancer properties, which are reported in numerous studies [33-37]. Several molecular pathways such as NF-ƙB, Nrf2, and AMPK signal pathways have confirmed that chrysin can contribute to the initiation of apoptosis in cancer cells [38-40]. In this research, we addressed NLC technology can potentially enrich the anti-cancer activity of chrysin and enhance Dox efficacy in MCF-7 cells. Experimental evidence indicated that delivery of chrysin with NLCs induced apoptosis and altered cell cycle distribution in MCF-7 cells in a dose-dependent manner (Figs.5&6). Chrysin formulated in NLCs increased the percentage of late apoptosis cells up to 20% (Fig.5e), while cells in necrosis phase decreased to 8.9%. This result demonstrated that chrysin-loaded NLCs could enhance apoptotic pathway in cancer cells better than chrysin alone (Fig.5c). Cell cycle distribution confirmed apoptotic results. Chrysin-loaded NLCs caused the accumulation of cells in the sub-G1 region up to 12% compared with chrysin alone (just 7.8%). This data suggested that chrysin-loaded NLCs possess more toxicity than chrysin alone and show selectivity toxicity, with an increase in population of cells in G0/G1 phase. In addition, chrysin-loaded NLCs altered the percentage of cell population from G2/M to G0/G1 phase. This result suggests that chrysin interrupted DNA replication, while chrysin-loaded NLCs district mitochondrial member and initiate intrinsic apoptotic pathway. It was also observed that Nrf2 signal pathway was involved in apoptosis (Fig.7). RT-PCR results confirmed that the expression levels of Nrf2 downstream genes, including MRP1, NQO1, and HO1, significantly decreased after treating cancer cells with chrysin and chrysin-loaded NLCs compared with the control. The results of this study were consistent with those of Gao et al.
who reported that chrysin enhanced the sensitivity of BEL-7402 cells to Dox [41]. We believe that the formulation of chrysin into nanoparticles improved internalization of drugs to cancer cells through endocytosis. To better understand this claim, the cellular uptake of chrysin alone and chrysin-loaded NLCs (Figs. 3&4) was examined by using rhodamine B dye. Fluorescent microscopy results showed that the cancer cell uptake of chrysin-loaded NLCs was enhanced during different intervals of times between 20 to 120 min. The lipid structure of the formulation caused these particles to interact with the cell membrane, and because of the high biocompatibility of nanoparticles and lipid membrane fluidity, the formulation acquired superiority to chrysin alone in cell internalization. Zheng H et al demonstrated that mPEG chrysin nanoparticle presented significant capability for uptake by hepG2 cancer cells through endocytosis [42]. In another study, Feng Y et al. showed that chrysin-G directly caused rigidity and adhesion force to diminish in cancer cells by destroying the cell membrane [43]. Facilitation of chrysin accumulation in the cancer cells dramatically was associated with a decrease in the rate of cell growth inhibition dosage (Fig.2b). Our data indicated that chrysin at 20 µM inhibited cell proliferation in MCF-7 cells up to 50% (Fig.2b). Interestingly, it was observed that chrysinloaded NLCs significantly decreased cell viability up to 36% (Fig.2c). In addition, compared with chrysin alone, chrysin-loaded NLCs markedly sensitized cancer cells to Dox. We believed that chrysin, as a Nrf2 inhibitor, caused selectively low levels of expression in Nrf2 downstream genes including ATP-binding cassettes and phase II detoxification enzymes. This inhibitory effect of chrysin induces apoptotic pathway in cancer cells with the minimum concentration of Dox compared with treating cells with a high and toxic dosage of Dox. Cha et al. demonstrated the downregulation of Nrf2-induced apoptotic pathway through Bcl-xl in papillary thyroid cancer cells. In addition, Ko et al. showed the incubation of human stomach carcinoma cells with shikonin activated cell apoptosis through the Nrf2-P53 signal pathway [44, 45]. Increase in the rate of early and late apoptosis in cancer cells, which were incubated with chrysin-loaded NLCs confirmed our cytotoxicity results. To investigate this more thoroughly, DAPI staining was performed. Alteration in the morphology of nuclei justified high apoptotic rates in cells incubated with chrysin-loaded NLCs. We also report that treatment of cancer cells with chrysin and Dox altered cell cycle progression along with increased sub-G1 population (Fig.6). This
biological behavior of chrysin can be explained because of the production of narrow and uniform particles in the range of 55-120 nm (Fig.1a). These uniform and colloidal surfaces of nanoparticles were also observed by SEM (Fig.1b). In this study, poloxamer407 was applied as a surfactant to reach a stable and ideal range of particle size. Poloxamer 407 is recognized as a hydrophilic nonionic surfactant (HLB value=22) that was employed as formulation stabilizer. Increase in the surfactant concentration was accompanied with decrease in particle size patterns (Table1). Results of this study were consistent with those of Pezeshki A et al., which demonstrated the role of surfactant in NLC formulations [46]. Furthermore, Kovacevic A et al. showed that non-ionic surfactants could improve the physical stability, size, and structure of particles [47]. We believe that poloxamer 407, at optimum concentration, prevented the formulation from aggregation and provided small particles, which consequently exhibited optimized biological behavior in cancer cells.
5. Conclusion Chrysin inhibited Nrf2 and its downstream genes and induced apoptosis in MCF-7 breast cancer cells. NLCs, as a new generation of lipid-based drug delivery system, improved the efficacy of chrysin in the sensitization of cancer cells to a chemotherapy agent, Dox. Taken together, the results of the present study lead to the following noteworthy conclusions: (I) Chrysin increased the efficacy of Dox by altering cell cycle distribution in MCF-7 cells; (II) Chrysin-loaded NLCs had synergistic effects on cellular uptake of Dox; and (III) Chrysin inhibited Nrf2 pathway, which was associated with high percentages of apoptosis. The effects of NLC technology on the induction of apoptosis should be further investigated in an animal model and through clinical trials.
Conflict of interest The authors declare that there are no conflicts of interest
Acknowledgements The authors were financially supported by a grant (NO: 94.128) from Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
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[42] H. Zheng, S. Li, Y. Pu, Y. Lai, B. He, Z. Gu, Nanoparticles generated by PEG-Chrysin conjugates for efficient anticancer drug delivery, European Journal of Pharmaceutics and Biopharmaceutics 87(3) (2014) 454-460. [43] H.-Y. Cha, B.-S. Lee, J.W. Chang, J.K. Park, J.H. Han, Y.-S. Kim, Y.S. Shin, H.K. Byeon, C.-H. Kim, Downregulation of Nrf2 by the combination of TRAIL and Valproic acid induces apoptotic cell death of TRAIL-resistant papillary thyroid cancer cells via suppression of Bcl-xL, Cancer letters 372(1) (2016) 65-74. [44] H. Ko, S.-J. Kim, S.H. Shim, H. Chang, C.H. Ha, Shikonin Induces Apoptotic Cell Death via Regulation of p53 and Nrf2 in AGS Human Stomach Carcinoma Cells, Biomolecules & therapeutics 24(5) (2016) 501. [45] F. Yang, H. Jin, J. Pi, J.-h. Jiang, L. Liu, H.-h. Bai, P.-h. Yang, J.-Y. Cai, Anti-tumor activity evaluation of novel chrysin–organogermanium (IV) complex in MCF-7 cells, Bioorganic & medicinal chemistry letters 23(20) (2013) 5544-5551. [46] A. Pezeshki, B. Ghanbarzadeh, M. Mohammadi, I. Fathollahi, H. Hamishehkar, Encapsulation of vitamin A palmitate in nanostructured lipid carrier (NLC)-effect of surfactant concentration on the formulation properties, Advanced pharmaceutical bulletin 4(Suppl 2) (2014) 563. [47] A. Kovacevic, S. Savic, G. Vuleta, R. Müller, C.M. Keck, Polyhydroxy surfactants for the formulation of lipid nanoparticles (SLN and NLC): effects on size, physical stability and particle matrix structure, International journal of pharmaceutics 406(1) (2011) 163-172.
Figure legends: Fig1. Physical characterization of the chrysin-loaded Nanostructured lipid carriers (NLCs). a) Scanning electron microscope image of chrysin-loaded NLCs; Scan number: 0697, b) Size distribution, and c) Zeta potential.
Fig2. Inhibition the effect of Doxorubicin (Dox), chrysin, and chrysin-loaded nanostructured lipid carriers (NLCs) on the growth of MCF-7 (breast cancer cell line). Cells were treated with concentrations of 0.2 to 5 µM Dox, 5 to 40 µM chrysin, and chrysin-loaded NLCs with the same concentration of chrysin. a) and b): IC50 of Dox, chrysin, and chrysin-loaded NLCs by MTT assay. c) Chrysin-loaded NLCs had more cytotoxicity effects than chrysin alone. The results were calculated as the mean ± SD (n=3) *p<0.05. Sub-toxic concentration of Dox (0.01 µM) was applied along with chrysin and chrysin-loaded NLCs to investigate the cytotoxicity behavior of the NLC formulation in cancer cells.
Fig3. Light imaging, red and blue fluorescence microscopy images of MCF-7 cancer cells incubated with chrysin, Dox, and chrysin-loaded NLCs. a) Light microscopy image of MCF- 7 (breast cancer cells), b) MCF-7 cells were tracked with 2 mg/ml DAPI solution for 6 min. c) Investigation of cellular uptake by rhodamine B incorporated into nanostructured lipid carriers (NLCs) between 20 to 120-min time intervals confirmed the penetrability and retention of chrysin-loaded NLCs in MCF-7 cells.
Fig4. Fluorescence intensity of fluorescein Rhodamin-B permeated into MCF-7 cells after treatment with chrysin and chrysin-loaded NLCs. a) Untreated cells, b) Chrysin-Rhodamin-B, and c) Chrysin-loaded NLCs-Rhodamin-B.
Fig5. Chrysin-loaded nanostructured lipid carriers (NLCs) increased the rates of apoptosis of MCF-7 (breast cancer cells). a) Untreated group as a negative control, b) NLCs, c) chrysin (as a positive control), d) doxorubicin (Dox), e) chrysin-loaded NLCs, f) chrysin-Dox, and j) chrysinloaded NLCs + Dox.
Fig6. Effects of chrysin-loaded nanostructured lipid carriers (NLCs) on cell cycle distribution of MCF-7 cells (breast cancer cells). a) Untreated group as a negative control, b) Nano-void (Blank),
c) chrysin (as a positive control), d) Doxorubicin (Dox), e) chrysin + Dox, f) chrysin-loaded NLCs, and j) chrysin-loaded NLCs + Dox.
Fig7. Effects of chrysin-loaded nanostructured lipid carriers (NLCs) on the mRNA expression pattern of Nrf2, MRP1, NQO1, and HO1 genes. The results were considered as the mean ± standard deviation (n=3). *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 1
Fig 2
Fig 3
Fig 4
Fig 5
Fig 6
Fig 7
Table 1: Preparation and characterization of chrysin-loaded NLCs. Formulation
Lipid
Oil
(g)
a
Size
(mg)
(nm)
PDIa
EEb
LCc
(%)
(%)
F1
Precirol
0.25
Miglyol
80
114 ± 7
0.63
60.4 ± 2.1
7.2 ± 0.3
F2
Precirol
0.25
Octyl
85
107 ± 4
0.52
74.6 ± 5.2
11 ± 0.4
F3
Precirol
0.20
Octyl
95
104 ± 4
0.40
64.3 ± 5.0
9.3 ± 1.1
F4
Compritol 0.25
Miglyol
100
91 ± 4
0.37
66.9 ± 4.1
10.5 ± 1.3
F5
Compritol 0.25
Miglyol
110
78 ± 3
0.25
70.2 ± 2.5
12.4 ± 1.6
F6
Compritol 0.20
Miglyol
115
73 ± 4
0.27
70.4 ± 1.1
14.7 ± 1.5
F7
Compritol 0.20
Miglyol
120
77 ± 3
0.21
69.8 ± 3.2
13.1 ± 0.7
F8
Compritol 0.15
Miglyol
140
68 ± 3
0.22
71.6 ± 1.4
12.8 ± 1.1
F9
Compritol 0.12
Octyl
145
84 ± 3
0.31
62.7 ± 2.0
10.4 ± 1.4
F10
Compritol 0.10
Octyl
160
95 ± 3
0.46
72.2 ± 3.2
11 ± 0.5
Polydispersity index Encapsulation efficiency cLoading capacity b
Poloxamer
S1359-5113(17)30210-6 http://dx.doi.org/doi:10.1016/j.procbio.2017.05.024 PRBI 11053
To appear in:
Process Biochemistry
Received date: Revised date: Accepted date:
6-2-2017 16-4-2017 20-5-2017
Please cite this article as: Sabzichi Mehdi, Mohammadian Jamal, Bazzaz Roya, Pirouzpanah Mohammad Bagher, Shaaker Maghsod, Hamishehkar Hamed, Chavoshi Hadi, Salehi Roya, Samadi Nasser.Chrysin loaded nanostructured lipid carriers (NLCs) triggers apoptosis in MCF-7 cancer cells by inhibiting the Nrf2 pathway.Process Biochemistry http://dx.doi.org/10.1016/j.procbio.2017.05.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Chrysin loaded nanostructured lipid carriers (NLCs) triggers apoptosis in MCF-7 cancer cells by inhibiting the Nrf2 pathway
Mehdi Sabzichia †, Jamal Mohammadianb, †, Roya Bazzazc, MohammadBagher Pirouzpanahd, Maghsod Shaaker c, Hamed Hamishehkara, Hadi Chavoshic, Roya Salehia, *, Nasser Samadib, *
a
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
b
School of advanced medical science, Tabriz University of Medical Sciences, Tabriz, Iran
c
Hematology and Oncology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
d
Stem cell and Regenerative Medicine Institute, Tabriz University of Medical Sciences, Tabriz, Iran
* Corresponding authors: Roya Salehi: Assistant Professor of Pharmaceutical and Medical Nanotechnology, School of Advanced Medical Science, Tabriz University of Medical Sciences, Tabriz, IRAN. Nasser Samadi: Associate Professor of Clinical Biochemistry; Department of Biochemistry & Clinical Laboratory, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, IRAN. Daneshgah St. Tabriz-Iran. P.O. Box: 51656-65811
E-mail address: [email protected] [email protected] Running Title: Chrysin enhances doxorubicin effect in MCF-7 cancer cells
Graphical abstract
Highlights •
Chrysin increases the efficacy of Dox by altering the cell cycle distribution of MCF-7 cells.
•
NLCs can be considered as potential delivery systems.
•
Chrysin-loaded NLCs had synergistic effects on cellular uptake of Dox.
•
Chrysin inhibited Nrf2 pathway, which was associated with high percentages of apoptosis.
Abstract We investigated the role of chrysin as an effective adjuvant along with nanostructured lipid carriers (NLCs) to increase the cytotoxicity of doxorubicin (Dox) in MCF-7 breast cancer cells. The prepared formulation was characterized from point of view scanning electron microscope (SEM), size & zeta potential. Cellular uptake and cytotoxicity of nanoparticles were examined by fluorescent microscopy and MTT assay, respectively. Flow cytometry and real-time PCR were
used to understand the molecular mechanism of Nrf2 and related downregulating genes. The average size of the nanoparticles was 105 ± 2 nm, which was confirmed by SEM. Our results demonstrated that incubation of the cells with chrysin-loaded NLCs enhanced the percentage of apoptosis from 21.11 ±5.72% to 27 ±3.13% (p<0.05). Furthermore, the population of cancer cells in the sub-G1 phase increased up to 12 ± 2.1% compared to untreated cells (p<0.05). mRNA expression levels of Nrf2, NQO1, HO1, and MRP1 exhibited a significant decrease compared to the control group (p<0.05). Our findings recommend that chrysin delivery along with NLCs would enhance the efficacy of Dox by exerting inhibitory effects against drug efflux pumps and drug detoxification enzymes.
Chemical compounds: Chrysin (PubChem CID: 5281607); Doxorubicin (PubChem CID: 31703); Glyceryl Behenate (PubChem CID: 5362585); Dimethyl Sulfoxide (PubChem CID: 679); MTT formazan (PubChem CID: 16218671); Miglyol (PubChem CID: 53471835).
Abbreviations: RPMI, Roswell Park Memorial Institute medium; FBS, Fetal Bovine Serum; MCF7, Michigan Cancer Foundation-7; SEM, scanning electron microscopy; MTT, Tetrazolium, 2(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-, bromide; EE, Entrapment Efficiency; RT-PCR, Real Time polymerase chain reaction; DAPI,4',6-diamidino-2-phenylindole; PI, propidium iodide; FITC, Annexin V-fluorescein isothiocyanate; Dox, Doxorubicin.
Keywords: Chrysin; Doxorubicin; NLCs, MCF-7; Cytotoxicity
1. Introduction Breast cancer has become one of the major causes of death among women in the world along with cardiovascular disease [1]. Despite great attempts to reach advanced protocols in cancer treatment, chemotherapy with doxorubicin (Dox) continues to remain one of the fundamental therapeutic strategies. Dox is widely used because of its strong cytotoxic properties against
cancer cells through the induction of apoptosis, disruption of cell cycle progression, and interference toward topoisomerase-II enzyme [2]. Nevertheless, many unwanted side effects of Dox to normal healthy tissues and the acquired resistance to this drug in many patients have caused a decline in the final outcome of Dox efficacy [3, 4]. Therefore, applying new adjuvant with an advance drug delivery system is considered an alternative to conventional methods, which would be evaluated efficacy of chemotherapy [5-8]. Nuclear factor-E2-related factor 2 (Nrf2) has emerged as a key molecule that plays a pivotal role in controlling homeostasis in normal and cancer cells [9, 10]. Overexpression of Nrf2 is reported in many cancer cells and is recognized as one of the master transcription factors as it regulates three major classes of genes including drug transporters such as multi-drug resistance (MDR) and MDR-associated proteins (MRP1), anti-oxidants such as heme oxygenase1(HO1), and phase II detoxification enzymes such as NQO1 [11, 12]. Therefore, the inhibition of Nrf2 in cancer tissue is considered a novel strategy that sensitizes tumor cells to chemotherapy agents and prevents efflux and rapid detoxification of drugs in cancer tissues [13, 14]. Recently, chrysin (5, 7-dihydroxyflavone) has been introduced as a flavonoid that possesses anti-inflammatory, anti-oxidants, antidiarrheal, and anti-cancer properties [15, 16]. The molecular mechanism of chrysin has not yet been identified. A study reported chrysin that inhibited cell growth in MDA-MB-231 through the PPAR alpha signal pathway [17]. Another study demonstrated that chrysin suppresses cell proliferation through the activation of caspase superfamily and inactivation of PKB [18]. However, only a few studies have reported that chrysin can be considered a potent Nrf2 inhibitor [19-21]. In addition, similar to polyphenols, chrysin has a low bioavailability, which is a bottleneck for application in pharmaceutical sciences [22]. In this study, we formulated chrysin in nanostructured lipid carriers (NLCs) as the second generation of lipid-based carriers to enhance the bioavailability of drugs and sustained release of this formulation in tumor microenvironments [23, 24]. This delivery system has recently been evaluated for cancer therapy because of many advantages such as biocompatibility to biological systems, high entrapment efficiency, and controlled release of drugs [25, 26]. These properties have transformed NLC systems into effective carriers, which can open new approaches in cancer delivery [27, 28]. The purpose of this study was to investigate the role of chrysin with NLCs in
Dox-induced apoptosis in MCF-7 breast cancer cells. Moreover, the underlying mechanism of cell cycle progression was evaluated. Finally, we aimed to investigate the role of Nrf2 and downstream gene expression in Dox-mediated apoptosis in cancer cells.
2. Materials and methods Chrysin, Dox, fetal bovine serum (FBS), and tetrazolium salt (MTT) were procured from SigmaAldrich (Steinheim, Germany). Compritol® ATO 888 was purchased from Gattefossè (Saint Periest Cedex, France). Rhodamine B was obtained from (Merck Chemicals Germany). Roswell Park Memorial Institute (RPMI) 1640 medium, glutamine, streptomycin, penicillin G and dimethyl sulfoxide (DMSO) were obtained from Invitrogen Life Technologies (Auckland, New Zealand). Annexin V-fluorescein isothiocyanate (FITC), propidium iodide (PI) were purchased from (E-bioscience, USA) and primers from MWG Biotech (Ebersberg, Germany). The human breast cancer (MCF-7) cell line was procured from National Cell Bank, (Pasteur institute, Iran).
2.1. Preparation of NLCs containing chrysin NLCs were prepared by modified hot homogenization technique associated with the ultrasonication method [29]. Briefly, chrysin was solubilized in Miglyol and the mixture was added into melted compritol. Then, 1% poloxamer as surfactant was added drop wise into the lipid phase under homogenization at 20000 rpm (Heidolph, Germany) for 20 min. Then the sample was immersed into a water bath and sonicated for 5 min. The formed hot oil in Water (o/w) nano-emulsion was cooled at ambient temperature for lipid phase recrystallization, and finally the NLC was produced [30].
2.2. Determination of chrysin entrapment efficiency To determine the entrapment efficiency (EE) % of chrysin, we employed UV-Visible spectrophotometer (UV160-shimadzo -Japan). Unloaded chrysin was calculated according to calibration curve for 0.1-1 mg/ml. UV absorbance for chrysin was measured at ƛmax = 328 nm following the Beer-Lambert law in a range of 0.1-1.5. At first, 500 µL of the formulation was
added to the upper chamber of Amicon® centrifugal filter (Millipore, Germany) and centrifuged at 5,000 rpm (Beckman, Spain) for 25 min. Then, the amount of chrysin in the lower chamber was calculated according to the absorbance of the calibration curve. The following equation was applied to determine the percentage of EE:
EE(% , w / w)
winitialdrug _ w free drug winitialdrug
2.3. Characterization and optimization of chrysin-loaded NLCs Chrysin-loaded NLCs were evaluated for nanoparticle dimensions by the photon correlation spectroscopy technique using a particle diameter instrument (WING Sald 2101 SHIMADZU JAPAN) after appropriate dilution through deionized water. The surface morphology of the nanoparticles was determined by scanning electron microscope (SEM) (MV2300, Vega Tescan, Czech Republic). To determine the zeta potential, the NLC solution was diluted with phosphatebuffered saline (PBS) and was measured using Zeta Sizer ZS (Malvern Instruments, UK). 2.4. Cellular internalization experiment Cellular uptake was studied to understand the role of nanoparticle structure for the internalization of chrysin into cancer cells. To follow the fate of chrysin, Rhodamine B as a reporter dye, was incorporated into NLCs (0.4% w/w according to the weight of the lipids). The Amicon® tube was used to purify of the chrysin-loaded NLCs from unloaded rhodamine B. The free rhodamine B was passed through a filter and collected in the lower chamber of the Amicon® tube. The upper chamber containing fluorescent NLCs was diluted with PBS and centrifuged twice. MCF-7 cells (4×105 per well) were incubated in six–well plates that were typically covered with 12 mm coverslips. Cell uptake of the formulation was followed by fluorescent microscopy (Olympus, Japan) during various times interval (15–120 minutes). To quantify chrysin uptake, cancer cells were incubated with the formulation for 60 min. Then cells were collected and washed with PBS, and finally, florescent dye excitation was detected using FACS Calibur (flow cytometer; BD Biosciences, San Jose, CA).
2.5. In vitro Cytotoxicity Study The MCF-7 breast cancer cells (1.4×104 cells/ well) were maintained in RPMI medium and incubated at 37 °C in 5% CO2 as growth phase condition. The inhibitory effect of blank NLCs, chrysin, and chrysin-NLCs on cell growth and their cytotoxic effects were evaluated after treatment with different concentrations from 5 up to 40 μM. The cells were substituted with 200 μL fresh media containing 20 μL of MTT solution (2 mg/ml in PBS) and incubated for additional 4 h at 37˚C. Finally, the absorbance was measured at 570 nm after shaking for 20 min using a microplate ELISA reader (ELX 800, Biotek, USA)[31].
2.6. In vitro Apoptosis Assay To detect early and late apoptosis cells and distinguish these cells from necrosis cells, we analyzed the cells using= Annexin V/ FITC apoptosis detection kit (E-bioscience, USA). The cells (4×105) of each well were incubated in medium containing free chrysin (20 µM) and chrysinloaded NLCs at the same concentration of chrysin at 37°C for 24 h. MCF-7 cells were harvested through trypsinization and re-suspended in PBS. Lastly, annexin V/FITC along with PI were added and monitored by flow cytometry.
2.7. Cell cycle distribution To understand the molecular mechanism of cell death and detection rates of sub-G1 population, MCF-7 breast cancer cells were seeded at an initial density of 4×105 per well, and after treatment with the above mentioned conditions for 24 h, then the cells were harvested by trypsinization, washed using PBS (pH=7.4), re-suspended in ethanol, and incubated overnight at room temperature. Cells were then exposed to PI and ribonucleaseA for 25 min. Cell cycle alteration was evaluated using a flow cytometer.
2.8. DAPI Staining for Nuclear Morphology Study MCF-7 breast cancer cells (4×105 per well) were seeded in six-well plates and covered with 12 mm coverslips. Cells at sufficient confluence (70%) were treated with chrysin, dox, chrysin
loaded NLCs and combination of chrysin and dox for 24 h; then, the cells were washed with PBS, fixed using paraformaldehyde (4%) for 20 min, permeabilized using 0.1 % (w/v) Triton X100 for 15 min, and stained with DAPI dye for 15 min. Finally, the coverslips were washed with PBS. Triplicate samples were prepared for each treatment, and at least 300 cells were counted in random fields for each sample. Identification of apoptotic morphological alteration of cancer cells was investigated under a fluorescence microscope.
2.9. RNA extraction and real-time PCR The total RNA of MCF-7 cancer cells was isolated by Trizol according to the manufacturer’s instructions [32]. The amount of total of RNA was determined by optical density measurement (A260/A280 ratio) on Nano Drop 1000 Spectrophotometer (Wilmington, DE, USA). cDNA was produced using the First-Strand Synthesis kit (Vivantis, USA) following the manufacturer’s instructions. Real-time (RT)-PCR was performed using the SYBR Green Master Mix and evaluated on a Roche lightcycler96 (Germany). Specific primers were as follows: Nrf2 (5’ACTCCCAGGTTGCCCAC-3’ CTCGCCTCATGCGTTTTTG-3’
and and
5’-GTAGCCGAAGAAACCTCATTGTC-3’),
NQO1
(5’-
5'-CCCCTAATCTGACCTCGTTCAT-3’),
MRP1
(5’-
ATGACCAGGTATGCCTATTATTAC-3’ and 5’-CACATCAAACCAGCCTATCTC-3’), and HO1 (5’ACGGCTTCAAGCTGGTGATG-3’ and 5’- TGCAGCTCTTCTGGGAAGTAG-3’). All samples were performed in triplicates. Interpretation of the data was quantitated using the ΔΔCt method with efficiency correction by Pfaffl technique and the cycle threshold values were standardized with respect to GAPDH expression.
2.10 Statistical analysis Results were evaluated as the mean ± SD. One-way ANOVA was performed to determine acceptable differences between examination groups and control groups. P value < 0.05 was considered significant (Graph Pad V6.0 Software Inc., San Diego, CA, USA). 3. Results
3.1. Characterization of chrysin-loaded NLCs Surface morphology of chrysin-loaded NLC was determined by SEM images which confirmed a spherical shape (Fig.1a). Particle size measured by DLS was 55 to 300 nm (Fig. 1b). The new formulation was further characterized to determine the zeta potential. The surface charge of the particle showed a negative value of -11 mV at the original pH 7.4 (Fig. 1c). Table 1 demonstrates the effect of types of lipid carriers and surfactant amounts on nanoparticle size. The particle diameter of NLCs formulated with compritol was smaller than that of NLCs prepared with Precirol. In addition, increasing the surfactant amount from 80 to 140 mg caused a decrease in the particle size.
3.2. Cytotoxicity of chrysin-loaded NLCs Cytotoxicity analysis using MTT assay was performed to determine the effect of chrysin-loaded NLCs on MCF-7 cells after 24 and 48 h of incubation with desired concentrations of Dox (0.25 to 5 µM), chrysin (5 to 40 µM), and formulation (5 to 40 µM). As shown in (Figs.2a & b), the IC50 values for Dox, chrysin, and chrysin-loaded NLCs were 0.82, 20 ± 0.05, and 17.3 ± 0.04 µM, respectively. Chrysin-loaded NLCs induced the cytotoxicity of MCF-7 cells more effectively than chrysin only treatment (p<0.05). There were no significant differences between cancer cells incubated with NLCs alone and untreated cells (p>0.05). This demonstrated the biocompatibility and low cellular toxicity of chrysin-loaded NLCs (Fig.2c). 3.2.1. Cell morphology analysis by fluorescent microscopy The alteration of cancer cell morphology to apoptotic body by DAPI staining confirmed that NLC delivery system increased the apoptosis, which was also observed by light microscopy. As shown in (Fig.3a&b), chromatin fragmentation and cell wall shrinkage increased and the intensity of DAPI dye decreased, which indicates the degradation of cancer cells. 3.2.2. Cellular Uptake of Chrysin-loaded NLCs Cellular uptake was quantified to determine the cell permeation of chrysin due to enhanced endocytosis, which resulted in a positive correlation between lipid nanoparticles and cancer cell
membrane. The fluorescence intensity of chrysin in MCF-7 cells significantly increased from 20 to 120 min, representing the continuous accumulation of chrysin into the cells and showing that the chrysin-loaded NLCs were internalized in a time-dependent manner (Fig3.c). To further verify this, quantitative analysis of cellular uptake by flow cytometry was performed. It was demonstrated that compared with chrysin alone (64%), chrysin-loaded NLCs (84%) intensified the percentage of internalization into cells (Fig.4). 3.3. Chrysin-loaded NLCs induced apoptosis in MCF-7 cancer cells To determine the proportion of apoptotic cells, we used annexin V along with PI staining. The results revealed that NLCs alone showed slight apoptosis compared with untreated cells (p<0.05) (Figs.5a and b). Chrysin (20 µM) and Dox (0.01 µM) showed a cell population of 22.5 ± 1.4% and 19.1 ± 2.3%, respectively, in the apoptotic phase (Figs.5c and d). Chrysin-loaded NLCs increased the cell population in apoptotic phase up to 27 ±3.45% compared with that of chrysin alone (p<0.05) (Fig.5e). Furthermore, chrysin-loaded NLCs decreased necrosis rates from 15.11% to 8.9%. The percentage of apoptotic cells increased up to 37± 2.4% when MCF-7 cells were incubated with chrysin-loaded NLCs plus Dox (Fig.5j), while chrysin plus Dox exhibited 31% apoptotic cells (Fig.5f).
3.4. Effect of Chrysin-loaded NLCs on Cell Cycle Regulation Similar to the controls, incubating MCF-7 breast cancer cells with NLCs alone indicated no apoptotic cells (Fig.6a, b). When the cells were exposed to chrysin (20 µM) and Dox (0.01 µM), the percentage of apoptosis was 7.7 ± 0.4% and 2 ± 0.2%, respectively (Fig. 6c, d). Treating cells with chrysin-loaded NLCs for 24 h caused 27 ± 2.76% apoptosis, which was accompanied by 28± 0.7% G2/M arrest (Fig.6f). Applying Dox with chrysin-loaded NLCs enhanced the percentage of cells in sub-G1 population up to 19±3.11 (Fig.6j). Incubation of MCF-7 cells with chrysin-loaded NLCs increased the population of cells in G0/G1 phase up to 44% compared with the 38% with chrysin alone. DAPI staining confirmed our observation from apoptosis analysis, whereby the highest percentage of cell death (chromatin degradation and cell wall shrinkage) was observed in the cells exposed to chrysin-loaded NLCs plus Dox (Fig.3b).
3.5. RT-PCR analysis of Nrf2, MRP1, NQO1, and HO1 To understand the role of Nrf2 and downstream genes in controlling apoptosis in cancer cells, RT-PCR was performed. Applying chrysin-loaded NLCs caused the downregulation of Nrf2 compared with NLC or chrysin alone (p<0.001). In addition, NQO1 mRNA expression was dramatically downregulated in MCF-7 cells treated with chrysin-loaded NLCs compared with other groups (p<0.01) (Fig.7). Furthermore, chrysin and chrysin-loaded NLCs markedly decreased HO1 mRNA gene expression levels (p<0.05).
4. Discussion Recently, chrysin and its derivatives have been known to exert anti-cancer properties, which are reported in numerous studies [33-37]. Several molecular pathways such as NF-ƙB, Nrf2, and AMPK signal pathways have confirmed that chrysin can contribute to the initiation of apoptosis in cancer cells [38-40]. In this research, we addressed NLC technology can potentially enrich the anti-cancer activity of chrysin and enhance Dox efficacy in MCF-7 cells. Experimental evidence indicated that delivery of chrysin with NLCs induced apoptosis and altered cell cycle distribution in MCF-7 cells in a dose-dependent manner (Figs.5&6). Chrysin formulated in NLCs increased the percentage of late apoptosis cells up to 20% (Fig.5e), while cells in necrosis phase decreased to 8.9%. This result demonstrated that chrysin-loaded NLCs could enhance apoptotic pathway in cancer cells better than chrysin alone (Fig.5c). Cell cycle distribution confirmed apoptotic results. Chrysin-loaded NLCs caused the accumulation of cells in the sub-G1 region up to 12% compared with chrysin alone (just 7.8%). This data suggested that chrysin-loaded NLCs possess more toxicity than chrysin alone and show selectivity toxicity, with an increase in population of cells in G0/G1 phase. In addition, chrysin-loaded NLCs altered the percentage of cell population from G2/M to G0/G1 phase. This result suggests that chrysin interrupted DNA replication, while chrysin-loaded NLCs district mitochondrial member and initiate intrinsic apoptotic pathway. It was also observed that Nrf2 signal pathway was involved in apoptosis (Fig.7). RT-PCR results confirmed that the expression levels of Nrf2 downstream genes, including MRP1, NQO1, and HO1, significantly decreased after treating cancer cells with chrysin and chrysin-loaded NLCs compared with the control. The results of this study were consistent with those of Gao et al.
who reported that chrysin enhanced the sensitivity of BEL-7402 cells to Dox [41]. We believe that the formulation of chrysin into nanoparticles improved internalization of drugs to cancer cells through endocytosis. To better understand this claim, the cellular uptake of chrysin alone and chrysin-loaded NLCs (Figs. 3&4) was examined by using rhodamine B dye. Fluorescent microscopy results showed that the cancer cell uptake of chrysin-loaded NLCs was enhanced during different intervals of times between 20 to 120 min. The lipid structure of the formulation caused these particles to interact with the cell membrane, and because of the high biocompatibility of nanoparticles and lipid membrane fluidity, the formulation acquired superiority to chrysin alone in cell internalization. Zheng H et al demonstrated that mPEG chrysin nanoparticle presented significant capability for uptake by hepG2 cancer cells through endocytosis [42]. In another study, Feng Y et al. showed that chrysin-G directly caused rigidity and adhesion force to diminish in cancer cells by destroying the cell membrane [43]. Facilitation of chrysin accumulation in the cancer cells dramatically was associated with a decrease in the rate of cell growth inhibition dosage (Fig.2b). Our data indicated that chrysin at 20 µM inhibited cell proliferation in MCF-7 cells up to 50% (Fig.2b). Interestingly, it was observed that chrysinloaded NLCs significantly decreased cell viability up to 36% (Fig.2c). In addition, compared with chrysin alone, chrysin-loaded NLCs markedly sensitized cancer cells to Dox. We believed that chrysin, as a Nrf2 inhibitor, caused selectively low levels of expression in Nrf2 downstream genes including ATP-binding cassettes and phase II detoxification enzymes. This inhibitory effect of chrysin induces apoptotic pathway in cancer cells with the minimum concentration of Dox compared with treating cells with a high and toxic dosage of Dox. Cha et al. demonstrated the downregulation of Nrf2-induced apoptotic pathway through Bcl-xl in papillary thyroid cancer cells. In addition, Ko et al. showed the incubation of human stomach carcinoma cells with shikonin activated cell apoptosis through the Nrf2-P53 signal pathway [44, 45]. Increase in the rate of early and late apoptosis in cancer cells, which were incubated with chrysin-loaded NLCs confirmed our cytotoxicity results. To investigate this more thoroughly, DAPI staining was performed. Alteration in the morphology of nuclei justified high apoptotic rates in cells incubated with chrysin-loaded NLCs. We also report that treatment of cancer cells with chrysin and Dox altered cell cycle progression along with increased sub-G1 population (Fig.6). This
biological behavior of chrysin can be explained because of the production of narrow and uniform particles in the range of 55-120 nm (Fig.1a). These uniform and colloidal surfaces of nanoparticles were also observed by SEM (Fig.1b). In this study, poloxamer407 was applied as a surfactant to reach a stable and ideal range of particle size. Poloxamer 407 is recognized as a hydrophilic nonionic surfactant (HLB value=22) that was employed as formulation stabilizer. Increase in the surfactant concentration was accompanied with decrease in particle size patterns (Table1). Results of this study were consistent with those of Pezeshki A et al., which demonstrated the role of surfactant in NLC formulations [46]. Furthermore, Kovacevic A et al. showed that non-ionic surfactants could improve the physical stability, size, and structure of particles [47]. We believe that poloxamer 407, at optimum concentration, prevented the formulation from aggregation and provided small particles, which consequently exhibited optimized biological behavior in cancer cells.
5. Conclusion Chrysin inhibited Nrf2 and its downstream genes and induced apoptosis in MCF-7 breast cancer cells. NLCs, as a new generation of lipid-based drug delivery system, improved the efficacy of chrysin in the sensitization of cancer cells to a chemotherapy agent, Dox. Taken together, the results of the present study lead to the following noteworthy conclusions: (I) Chrysin increased the efficacy of Dox by altering cell cycle distribution in MCF-7 cells; (II) Chrysin-loaded NLCs had synergistic effects on cellular uptake of Dox; and (III) Chrysin inhibited Nrf2 pathway, which was associated with high percentages of apoptosis. The effects of NLC technology on the induction of apoptosis should be further investigated in an animal model and through clinical trials.
Conflict of interest The authors declare that there are no conflicts of interest
Acknowledgements The authors were financially supported by a grant (NO: 94.128) from Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
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Figure legends: Fig1. Physical characterization of the chrysin-loaded Nanostructured lipid carriers (NLCs). a) Scanning electron microscope image of chrysin-loaded NLCs; Scan number: 0697, b) Size distribution, and c) Zeta potential.
Fig2. Inhibition the effect of Doxorubicin (Dox), chrysin, and chrysin-loaded nanostructured lipid carriers (NLCs) on the growth of MCF-7 (breast cancer cell line). Cells were treated with concentrations of 0.2 to 5 µM Dox, 5 to 40 µM chrysin, and chrysin-loaded NLCs with the same concentration of chrysin. a) and b): IC50 of Dox, chrysin, and chrysin-loaded NLCs by MTT assay. c) Chrysin-loaded NLCs had more cytotoxicity effects than chrysin alone. The results were calculated as the mean ± SD (n=3) *p<0.05. Sub-toxic concentration of Dox (0.01 µM) was applied along with chrysin and chrysin-loaded NLCs to investigate the cytotoxicity behavior of the NLC formulation in cancer cells.
Fig3. Light imaging, red and blue fluorescence microscopy images of MCF-7 cancer cells incubated with chrysin, Dox, and chrysin-loaded NLCs. a) Light microscopy image of MCF- 7 (breast cancer cells), b) MCF-7 cells were tracked with 2 mg/ml DAPI solution for 6 min. c) Investigation of cellular uptake by rhodamine B incorporated into nanostructured lipid carriers (NLCs) between 20 to 120-min time intervals confirmed the penetrability and retention of chrysin-loaded NLCs in MCF-7 cells.
Fig4. Fluorescence intensity of fluorescein Rhodamin-B permeated into MCF-7 cells after treatment with chrysin and chrysin-loaded NLCs. a) Untreated cells, b) Chrysin-Rhodamin-B, and c) Chrysin-loaded NLCs-Rhodamin-B.
Fig5. Chrysin-loaded nanostructured lipid carriers (NLCs) increased the rates of apoptosis of MCF-7 (breast cancer cells). a) Untreated group as a negative control, b) NLCs, c) chrysin (as a positive control), d) doxorubicin (Dox), e) chrysin-loaded NLCs, f) chrysin-Dox, and j) chrysinloaded NLCs + Dox.
Fig6. Effects of chrysin-loaded nanostructured lipid carriers (NLCs) on cell cycle distribution of MCF-7 cells (breast cancer cells). a) Untreated group as a negative control, b) Nano-void (Blank),
c) chrysin (as a positive control), d) Doxorubicin (Dox), e) chrysin + Dox, f) chrysin-loaded NLCs, and j) chrysin-loaded NLCs + Dox.
Fig7. Effects of chrysin-loaded nanostructured lipid carriers (NLCs) on the mRNA expression pattern of Nrf2, MRP1, NQO1, and HO1 genes. The results were considered as the mean ± standard deviation (n=3). *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 1
Fig 2
Fig 3
Fig 4
Fig 5
Fig 6
Fig 7
Table 1: Preparation and characterization of chrysin-loaded NLCs. Formulation
Lipid
Oil
(g)
a
Size
(mg)
(nm)
PDIa
EEb
LCc
(%)
(%)
F1
Precirol
0.25
Miglyol
80
114 ± 7
0.63
60.4 ± 2.1
7.2 ± 0.3
F2
Precirol
0.25
Octyl
85
107 ± 4
0.52
74.6 ± 5.2
11 ± 0.4
F3
Precirol
0.20
Octyl
95
104 ± 4
0.40
64.3 ± 5.0
9.3 ± 1.1
F4
Compritol 0.25
Miglyol
100
91 ± 4
0.37
66.9 ± 4.1
10.5 ± 1.3
F5
Compritol 0.25
Miglyol
110
78 ± 3
0.25
70.2 ± 2.5
12.4 ± 1.6
F6
Compritol 0.20
Miglyol
115
73 ± 4
0.27
70.4 ± 1.1
14.7 ± 1.5
F7
Compritol 0.20
Miglyol
120
77 ± 3
0.21
69.8 ± 3.2
13.1 ± 0.7
F8
Compritol 0.15
Miglyol
140
68 ± 3
0.22
71.6 ± 1.4
12.8 ± 1.1
F9
Compritol 0.12
Octyl
145
84 ± 3
0.31
62.7 ± 2.0
10.4 ± 1.4
F10
Compritol 0.10
Octyl
160
95 ± 3
0.46
72.2 ± 3.2
11 ± 0.5
Polydispersity index Encapsulation efficiency cLoading capacity b
Poloxamer