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Biological and Medical Applications of Materials and Interfaces

Blocking Autophagic Flux Enhances Iron Oxide Nanoparticles Photothermal Therapeutic Efficiency in Cancer Treatment Xiaoqing Ren, Yiting Chen, Haibao Peng, Xiaoling Fang, Xiulei Zhang, Qinyue Chen, Xiaofei Wang, Wuli Yang, and Xianyi Sha ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b10167 • Publication Date (Web): 26 Jul 2018 Downloaded from http://pubs.acs.org on July 27, 2018

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ACS Applied Materials & Interfaces

Blocking Autophagic Flux Enhances Iron Oxide Nanoparticles Photothermal Therapeutic Efficiency in Cancer Treatment Xiaoqing Ren,†,‡ Yiting Chen,† Haibao Peng,§ Xiaoling Fang,† Xiulei Zhang,† Qinyue Chen,† Xiaofei Wang,† Wuli Yang,*,§,iD Xianyi Sha*,†,iD †

Key Laboratory of Smart Drug Delivery, Ministry of Education；Department of

Pharmaceutics, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, PR China ‡

Department of Pharmacy, Peking University Third Hospital, Beijing, 100191, PR

China §

State Key Laboratory of Molecular Engineering of Polymers & Department of

Macromolecular Science, Fudan University, 220 Handan Road, Shanghai 200433,PR China

ABSTRACT Autophagy is a conservative eukaryotic pathway which plays a crucial role in maintaining cellular homeostasis, and dysfunction of autophagy is usually associated with pathological conditions. Recently, emerging reports have stressed that various types of nanomaterials and therapeutic approaches interfere with cellular autophagy process, which has brought up concerns to their future biomedical applications. Here, we present a study elaborating the relationships between autophagy and iron oxide nanoparticle (IONP) mediated photothermal therapy in cancer treatment. Our results reveal that IONP photothermal effect could lead to autophagy induction in cancerous MCF-7 cells in a laser dose-dependent manner, and inhibition of autophagy would enhance the photothermal cell killing by increasing cell apoptosis. In an MCF-7 xenograft model, co-treatment of autophagy inhibitor and IONP under laser exposure could promote the tumor inhibition rate from 43.26% to 68.56%, and the tumor immunohistochemistry assay of microtubule-associated protein 1-light chain 3 (LC3) and TdT-mediated dUTP nick-end labeling (TUNEL) also demonstrates augmentation in both autophagosomes accumulation and apoptosis in vivo. This work helps us to ACS Paragon Plus Environment

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better understand the regulation of autophagy during IONP mediated photothermal therapy and provides us with a potential combination therapeutic approach of autophagy modulators and photothermal agents.

KEYWORDS Iron oxide nanoparticles Photothermal therapy MCF-7 cells Autophagy Apoptosis

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1. Introduction Autophagy is a highly conservative eukaryotic pathway in which damaged organelles, misfolded proteins and other extrinsic substances are engulfed into double-membrane autophagosomes and degraded and recycled later on.1-3 It plays a crucial role in maintaining cellular homeostasis, and dysfunction of autophagy is associated

with

pathological

conditions

such

as

bacterial

infections,

neurodegeneration and cancer.4,5 Recently, it was reported that a series of nanomaterials, such as quantum dots, dendrimers, silver nanoparticles, gold nanoparticles and iron oxide nanoparticles, could interfere with cellular autophagic process.6-11 And chemotherapeutic, radiation and magnetic hyperthermia have been discussed regarding their capabilities in modulating cell autophagy in the context of cancer treatment.12-15 However, the function of cellular autophagy in anti-tumor therapies is still controversial and needs to be further addressed.14-16 Photothermal therapy (PTT) is a newly developed anticancer strategy that utilizes photomediators transforming light into heat to induce local ablation of tumor and to achieve therapeutic outcomes.17,18 Due to its noninvasive nature and the ease of temporal and spatial control, PTT has garnered great interest among researchers in biomedical field. A variety of photomediators have been developed for this purpose. Iron oxide nanomaterials, owing to their good biocompatibility, low cytotoxicity, intrinsic capability of magnetic modulation and potential in magnetic contrast imaging (MRI), have been extensively studied for anticancer PTT applications.19,20 Chu group and Chen group first reported that different shaped iron oxide nanomaterials could be used as efficient photothermal agents for cancer cell ablation.21,22 Our group has also worked actively in this field. We demonstrated that iron oxide nanoparticles (IONP) of different sizes, ligands or surface properties could exhibit photothermal effect and could be utilized for all sorts of anticancer strategies.18, 23,24 However, the impact of photothermal IONP on cellular autophagic pathway has not been clarified. And, the role of autophagy playing in the process of IONP mediated photothermal treatment has also not yet been elucidated. The aim of this work is to try to fill in the gap between autophagy and IONP ACS Paragon Plus Environment


mediated photothermal anticancer treatment. In this study, after the construction and confirmation of its photothermal capability, the impact of IONP photothermal effect on cellular autophagy level in cancerous cells is investigated, and how autophagy acts in IONP mediated photothermal cell killing process is also studied with the help of autophagic inhibitors. Our data show that IONP upon laser irradiation can lead to significant autophagy induction in MCF-7 cells, and the inhibition of cellular autophagy by chloroquine (CQ) can enhance its anti-tumor photothermal cytotoxicity both in vitro and in vivo (Figure 1). These results can help us to better understand the regulation of autophagy during PTT and provide us with a potential combination cancer therapy of autophagy modulators and photothermal agents.

Figure 1. Schematic illustration of IONP’s photothermal effect induced cell autophagy induction and the inhibition of autophagy enhances IONP’s photothermal cytotoxicity.

2. Experimental section 2.1 Materials Iron chloride (FeCl3•6H2O), oleic acid, 1-octadecylene, hydroxy-propionic acid and chloroquine diphosphate (CQ) were purchased from Sigma-Aldrich LLC. Sodium oleate was purchased from TCl. 3-methyladenine (3-MA) were ordered from Merck ACS Paragon Plus Environment

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& Co., Inc. Hexane, ethanol, tetrahydrofuran, ammonium chloride and all the rest chemicals were bought from Shanghai Chemical Reagents Co., Shanghai, China. Dulbecco's Modified Eagle Media (DMEM) and feral bovine serum were bought from Gibco, USA. Other materials used were listed below. 2.2 Synthesis and phase transfer of IONP OA-IONP was synthesized by a well-established method with slight modifications.25 In brief, 10.8 g FeCl3•6H2O (40 mmol) and 36.5 g sodium oleate (120 mmol) were dissolved in a mixed solvent system containing 80 mL ethanol, 140 mL hexane and 60 mL deionized water. The resulting mixture was heated to 70 oC under continuous stirring and kept at this temperature for 4 h. After the reaction, the mixture was cooled to room temperature and the upper liquid layer was separated via a split funnel and washed three times with 30 mL deionized water. A waxy form of iron oleate was acquired after the complete evaporation of hexane. 7.2 g as-prepared iron oleate and 1.14 g oleic acid were then well dissolved again in 40 g 1-octadecylene (ODE) and the whole mixture was heated to 320 oC in nitrogen atmosphere for 1 h. After cooling, excessive amount of alcohol was added to precipitate the nanoparticles and the resultant hydrophobic OA-IONP was washed three times and collected by centrifugation. Since the product nanoparticles are hydrophobic, ligand exchange using hydroxy-propionic acid (DHCA) was conducted to render hydrophilicity to the OA-IONP26: 200 mg DHCA was dissolved in 24 mL tetrahydrofuran (THF) with mechanic stirring and was heated to 50 oC under argon atmosphere with condensation reflux. 80 mg of the hydrophobic OA-IONP dissolved in 4 mL THF was added dropwise into the above solution and reacted for 3 h. After complete reaction, the solution was cooled to room temperature and 3 mL of NaOH was added to precipitate the nanoparticles. The final product of hydrophilic IONP was washed and redispersed in water for future use. 2.3 Characterization and photothermal effect of as-synthesized IONP Transmission electron microscope (TEM) (model Tecnai G2 20 TWIN) and High resolution transmission electron microscope (HRTEM) were adopted to ACS Paragon Plus Environment


characterize as-synthesized IONP’s morphology and crystalline nature. Dynamic light scattering (DLS) using Zetasizer Nano ZS was employed to monitor the size and zeta potential of IONP in aqueous solutions. In TEM image, 25 random nanoparticles were measured manually to calculate the average particle size. Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) with the model of Atom Scan 2000 was used to determine the concentration of Fe ion in solutions. In order to determine the photothermal effect of as-synthesized IONP, 200 µL of IONP water solution of different iron concentrations were transferred into a clear 96-well plate and were illuminated by an 808 nm laser (spot size: 5 mm x 8 mm, Changchun New Industries Optoelectronics Technology Co.) at a power density of 3 W/cm2 for 480 s. During this process, the temperature elevation of IONP solutions under laser irradiation was monitored by a near infrared (NIR) camera (VarioCAM HR, InfraTec, Germany). 2.4 Cell culture MCF-7 cells (a human breast cancer cell line, ATCC) were obtained from the Cell Bank of Chinese Academy of Sciences, Shanghai, China. And GFP-LC3/MCF-7 cell line (stable GFP-LC3 expression) was kindly provided by Prof. Yonghua Yang from Fudan University, Shanghai, China. Both cell lines were cultured in DMEM (Gibco, USA) with 10% fetal bovine serum (FBS) and 1% of penicillin and streptomycin addition under a 5% CO2 atmosphere at 37 °C. 2.5 Cyto-ID staining MCF-7 cells were seeded in the confocal dish overnight and incubated with fresh medium (control) and IONP (50 µg Fe/mL) for 24 h. Cells with IONP photothermal treatments were firstly incubated with IONP for 2 h and then introduced with laser illumination at certain settings (808 nm, 3 W/cm2, 3 min and 5 min; L+ and L++). Before observation, the cells were washed and stained with CYTO-ID® Autophagy detection kit (ENZO life sciences, USA) to mark the autophagosomes and nucleus


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according to the vender’s instruction. Then the cells were fixed and observed via a confocal fluorescence microscope (LSM710, Zeiss, Germany). 2.6 Fluorescence microscope observation of GFP-LC3/MCF-7 cells GFP-LC3/MCF-7 cells were seeded into confocal dishes at a density of 1 x 104 cells/dish and incubated overnight. The cells were treated with blank medium, high dose of laser (808 nm, 3 W/cm2, 5 min; L++), CQ (20 µM) or IONP (50 µg Fe/mL) for 24 h before their fluorescence pictures were taken. For the samples with both nanoparticles and laser addition, GFP-LC3/MCF-7 cells were incubated with IONP 2 h before the laser irradiation (808 nm, 3 W/cm2, 3 min and 5 min were represented by L+ and L++), and the cells were cultured normally after the light treatment till pictured by fluorescence microscope. 2.7 Western blotting assay After treatment, cells were gently harvested with a cell scraper and lysed with a RIPA lysis buffer supplemented with PMSF and enzyme inhibitors (Beyotime Biotechnology, Shanghai, China). The protein concentrations were determined by Broadford assay with a commercial kit (Beyotime Biotechnology, Shanghai, China) and samples were equally loaded and separated on SDS-PAGE and then transferred to a PVDF membrane. After blocking with 5% BSA at room temperature for 2 h, the membrane was washed by TBST and incubated with primary antibodies (1:2000 dilution): LC3A/B (D3U4C) XP® Rabbit mAb (Cell Signaling Technology, Inc, MA, USA); Anti-SQSTM1/p62 antibody (EPR4844) (Abcam, Shanghai, China) at room temperature overnight. After washing, the membrane was incubated with the corresponding secondary antibody (anti-rabbit immunoglobin horseradish peroxidase) (1:5000 dilution, Jackson ImmunoResearch, USA) and finally detected via ImageQuant LAS 4000 system (GE Healthcare Life Sciences，USA). 2.8 Transmission electron microscope (TEM) observation MCF-7 cells were incubated with IONPs (50 µg Fe/mL) containing complete medium for 24 h. The cells in laser treatment group were irradiated by an 808 nm laser beam at the power density of 3 W/cm2 for 5 min after 2 h of the IONP addition



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and were cultured for another 22 h. The cells were washed, collected and fixed by 2.5% glutaraldehyde, dehydrated using increased concentrations of ethanol, cleared with propylene oxide and then embedded into Epon resin. The sample contained resin were sliced into thin films using a diamond knife on an ultracut microtome (Leica, Germany) and stained with uranyl acetate and lead citrate and observed through a JEM-1410 TEM (JEOL, Japan). 2.9 Photothermal cytotoxicity assay of IONP with autophagy inhibitors addition MCF-7 cells were seeded into 96 well plates at a cell density of 2 x 104 cells/well and incubated overnight. The culture medium were discarded and reagent contained fresh complete medium were added respectively: blank medium as control; IONP (50 µg Fe/mL); IONP/CQ (50 µg Fe/mL, CQ: 20 µM) and IONP/3-MA (50 µg Fe/mL, 3-MA: 2 mM). An 808 nm laser was introduced to the laser treatment groups at a density of 3 W/cm2 for 4 min. About 24 h after the light irradiation, cell viabilities were

evaluated

by

a

well-established

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) MTT assay and the data was collected by a micro plate reader (Synergy 2, Biotek, USA). 2.10 siRNA transfection siRNA against ATG5 (Sense #1: 5'- GACGUUGGUAACUGACAAATT-3', Anti-sense

#1:

5'-

UUUGUCAGUUACCAACGUCTT-3';

5'-GUCCAUCUAAGGAUGCAAUTT-3',

Anti-sense

Sense #2:

#2: 5'-

AUUGCAUCCUUAGAUGGACTT-3') and negative control siRNA were purchased from GenePharma Biotechnology (Shanghai, China). For the transfection, 1.5 µL siRNA (20 µM) were added into 500 µL Opti-MEM medium and were then mixed with 5 µL Lipofectamine RNAiMAX (Thermo Fisher Scientific Inc, MA, USA) in a 6-well plate and incubated for about 20 min at room temperature. Afterwards, approximately 30×104 MCF-7 cells were added into the mixed medium containing the siRNA. After 24 h incubation, the cells were harvested for further treatments and assays. 2.11 Flow cytometry assay To explore the photothermal and autophagy inhibitor enhanced photothermal cell ACS Paragon Plus Environment

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killing effect, the MCF-7 cells with different treatments were stained and assessed for apoptosis via flow cytometry (FCM) (FACSAria II, BD Biosciences, USA). In brief, cells were seeded and cultured overnight, and incubated with fresh complete medium (control), IONP (50 µg Fe/mL) and IONP/CQ (50 µg Fe/mL, CQ: 20 µM). Light treatment (808 nm, 3 W/cm2, 4 min) was introduced to the cells after a certain time of incubation and afterwards the cells were trypsinized and stained with Annexin V-FITC/PI apoptosis detection kit (KeyGEN BioTECH, China) accordingly and further analyzed via FCM. 2.12 Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining Both the cell samples and the tumor tissues were fixed using 4% formalin; the tumors were embedded and sliced before staining. Then, the samples were incubated and stained following the protocol of the commercial TUNEL FITC Apoptosis Detection Kit (Vazyme Biotech Co., Ltd, Jiangsu, China). The stained sample slices were then observed and imaged by a fluorescence microscope and the result were measured and calculated using ImageJ (ImageJ1.42, National Institutes of Health, Maryland, USA). 2.13 In vivo photothermal anti-tumor performance of IONP with autophagic inhibition Animals Nude mice bearing MCF-7 xenograft were constructed as animal models for in vivo study. The mice were provided by the Experimental Animal Center in Zhangjiang Campus, Fudan University (Shanghai, China), and were cared and handled under the standard protocols approved and supervised by Fudan University Experimental Ethics Committee. Twenty female nude mice were injected with MCF-7 cells subcutaneously to yield tumors. After about 2 weeks, when the tumor volume reached around 100 mm3, the mice were randomly divided into 4 treatment groups (n=5) and each mouse was intratumorally injected with blank PBS (control group and CQ group) or IONP solutions (25 µL,0.8 mg Fe/mL; IONP group and IONP/CQ group). The mice in groups with CQ addition were injected with CQ (10 mg/kg, i.p.) every other day since ACS Paragon Plus Environment


day 0 to day 10. In all four groups, mice were anesthetized two hours after the initial treatment and their tumor region were illuminated with laser (808 nm, 2.5 W/cm2) for 4 min. During the light treatment, temperatures of tumor areas were recorded using a NIR camera. Mice tumor volume and body weight were measured and recorded every other day. On day 12, all mice were humanly euthanized and their tumor tissues were dissected and weighted before. Another in vivo photothermal anti-tumor evaluation at a lower laser power of 2 W/cm2 were conducted on fifteen female tumor bearing mice. The mice were randomly divided into three groups (n=5): control group, IONP group and IONP/CQ group and were subjected to the similar treatment regimen as the previous animal experiment with the 808 nm laser exposure at 2 W/cm2 for 4 min. 2.14 Histology assessment After the mice were sacrificed 12 days after initial treatment, their main organs including heart, liver, spleen, lung and kidney as well as tumor were obtained and fixed in Formalin solution. Then, these tissues were embedded, sliced and placed onto glass slides. For immunohistology and apoptosis assay, sample slides were stained with an LC3 antibody and TUNEL reagent (as in section 2.12), respectively. The slides were observed and the images were acquired by a microscope. 2.15 Statistic analysis Statistical analysis was carried out using unpaired two-tailed student t-test. n.s. is short for not significant (p ≥ 0.05) and p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***) were considered different statistically and were marked in each figure.

3. Results and discussion 3.1 Synthesis and characterization of IONP Hydrophobic OA-IONP were synthesized via hydrothermal reaction following a well-documented procedure with slight modifications and DHCA was used to transfer the as-synthesized hydrophobic nanoparticle into aqueous solutions in order to render OA-IONP with good dispersibility in water and accessibility for biomedical applications.24-27 ACS Paragon Plus Environment

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Figure 2. (A) TEM image of as-synthesized IONP. (B) HRTEM image of as-synthesized IONP. (C) Representative DLS data of hydrodynamic diameter of IONP suspension at room temperature. (D) Photothermal heating curves as a function of laser exposure time for as-prepared IONP suspensions at different Fe concentrations under an 808 nm laser at a power density of 3 W/cm2 for 480 s. Water was used as a control.

In Figure 2A, TEM image reveals that the final products IONP are monodispersed, well-shaped spheroids with uniformed sizes (average diameter in TEM is 13.2 ± 0.7 nm). In HRTEM image (Figure 2B), well-defined crystal lattice of IONP could be clearly observed indicating their magnetite crystalline nature. This result is in line with previous publications confirming the successful synthesis of crystalized photo-responsive IONP.22, 24 DLS was employed to measure IONP’s size, zeta potential and to monitor its stability in water solutions. Results show that the as-prepared photothermal agent has an average surface charge of -29.9 ± 1.9 mV and an average hydrodynamic diameter of 32.1 ± 1.3 nm with the PDI of 0.141 (Figure 2C). And the resultant IONP could maintain well-dispersed and stable in both water and complete cell culture medium for days (Supporting Information Figure S1).



Before further cell and animal study, IONP’s photothermal property was first validated by monitoring the temperature increment of IONP dispersions under laser irradiation. As shown in Figure 2D, IONP exhibit iron concentration-dependent photothermal effect. That is, after 480s of continuous laser illumination (808 nm, 3W/cm2), IONP can efficiently raise the solution temperature from ~24 oC to 41.8 oC (50 µg Fe/mL) and 54 oC (100 µg Fe/mL) while water alone fail to reach a final temperature of 30 oC under the same experimental condition.

3.2 Autophagy induced by IONP’s photothermal effect Before investigating how the photothermal effect of IONP would influence tumor cells autophagy level, we carried out a study to see if IONP alone would have any impact on cellular autophagic flux. Results from Cyto-ID staining, GFP-LC3/MCF-7 cells assessment as well as western blotting assay of LC3-I/LC3-II (data not shown here) all agree that MCF-7 cells incubated with up to 50 µg Fe/mL IONP would not exhibit detectable variation in terms of autophagy level. We are aware that in some previous studies, iron-related nanomaterials showed different influence in autophagy process which are inconsistent with our finding. This is probably due to the relatively low nanoparticle concentration we are working with and the different cell lines adopted among reports.28-30 Also, particle size, shape and surface chemistry of iron-based nanomaterials may also influence the overall results since these factors play important roles in particle-cell interactions.31-33


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Figure 3. Autophagy evaluation in MCF-7 cells. (A) Confocal fluorescence microscope images of MCF-7 cells stained with Cyto-ID (green) and hoechst 33342 (blue). Cells were treated with blank medium (Ctrl), IONP at an Fe concentration of 50µg/mL and equivalent IONP with 808 nm laser irradiation for 3 min and 5 min, respectively. (B) Fluorescence microscope images of GFP-LC3/MCF-7 cells treated with blank medium (Ctrl), an 808 nm laser treatment of 5 min at 3 W/cm2, 20 µM CQ, 50µg Fe/mL IONP and IONP with laser treatment of 3 min and 5 min, respectively. In both two figures, shorter laser exposure time is represented by L+ while longer laser duration time is represented by L++.

After confirming their little impact on MCF-7 cellular autophagy flux, a series of experiments were conducted to test our hypothesis that IONP’s photothermal effect might lead to the induction of cell autophagy. Cyto-ID is a green fluorescence probe that selectively accumulates in the autophagic vacuoles including autolysosomes, autophagosomes and phagosomes.6, 14 After incubated with IONP, MCF-7 cells were treated with or without NIR laser illumination and were observed after staining with Cyto-ID autophagic detection kit. As shown in Figure 3A, cells with only IONP incubation do not show obvious green fluorescence. This result again demonstrates that IONP have no detectable influence on MCF-7 autophagy level at testing concentration. And IONP incubated cells with laser treatments apparently display ACS Paragon Plus Environment


more green fluorescent punctate compared with the cells with no laser irradiation. Moreover, the cells with longer laser exposure time show stronger fluorescence compared with the ones with less laser intervention. To further confirm the autophagic induction of IONP’s photothermal effect, a more specific assay was carried out using GFP-LC3/MCF-7 cells. GFP-LC3/MCF-7 is a cell line with stable expression of GFP-LC3, a fusion protein of microtubule-associated light chain (LC3) and the optical probe green fluorescent protein (GFP), which distributes evenly in the cytoplasm at basal level while accumulates on the membrane of autophagosomes to show scattered green fluorescence upon autophagy. In consistent with the Cyto-ID staining results, GFP-LC3/MCF-7 cells with IONP’s photothermal treatment show more punctate green fluorescent dots compared with the cells with either laser irradiation or IONP incubation alone (Figure 3B and Supporting Information Table S1), demonstrating that the IONP’s photothermal effect can cause cellular autophagosomes accumulation while IONP or laser alone have little impact on the autophagic process. And the longer the laser illumination time, the more green dots with higher fluorescence intensity can be seen within the cells. Besides, CQ treated cells also present an enhanced fluorescent signal since CQ inhibits the degradation of the autophagosomes leading to their accumulation within the cytoplasm. To afford additional concrete proof of this photothermal-induced autophagy and to discriminate whether the autophagosomes accumulation is caused by the induction of autophagic flux or the interdiction of autophagosomes degradation, the cellular level of autophagic protein LC3 and p62 were determined via western blot analysis. During autophagy induction, LC3-I dispersed throughout the cell is recruited to the membrane of autophagosomes and lapidated into LC3-II which marks the maturation of the autophagosomes, so the formation of autophagosomes and the induction of autophagy can be monitored by the LC3-I to LC3-II transition process.1, 34 And p62 is a protein that degrades specifically relied on autophagy, so the expression level of p62 is also an indicator of autophagic flux.7, 9 As shown in Figure 4A-C, IONP-incubated MCF-7 cells illuminated with laser result in a significant increase of cellular LC3-II ACS Paragon Plus Environment

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protein level and a significant decrease of p62 compared to the cells without laser intervention, meaning that IONP’s photothermal effect enhances the accumulation of autophagosomes by the activation of autophagic flux instead of blocking it. This result is also confirmed by the increased LC3-I/II conversion upon the addition of Bafilomycin A1, another autophagy regulator inhibiting the fusion process of autophagosomes and lysosomes, with the same nanoparticles photothermal treatment (Supporting Information Figure S3).

Figure 4. (A) The cellular level of LC3-I, LC3-II and p62 in MCF-7 cells treated with PBS and IONP (50 µg Fe/mL) at different laser exposure time of 3, 5 and 7 min, respectively. GAPDH was used as a loading control. The relative level of LC3-II/GAPDH (B) and p62/GAPDH (C) in MCF-7 cells treated as described in (A) are listed in the up-right panel. * indicates p < 0.05 and ** indicates p

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