TiO2 nanoparticles enhance the chemotherapeutic effects of 5-fluorouracil in human AGS gastric cancer cells via autophagy blockade

Journal Pre-proof TiO2 nanoparticles enhance the chemotherapeutic effects of 5-fluorouracil in human AGS gastric cancer cells via autophagy blockade

Shiva Azimee, Marveh Rahmati, Hossein Fahimi, Mohammad Amin Moosavi PII:

S0024-3205(20)30214-9

DOI:

https://doi.org/10.1016/j.lfs.2020.117466

Reference:

LFS 117466

To appear in:

Life Sciences

Received date:

23 November 2019

Revised date:

15 February 2020

Accepted date:

21 February 2020

Please cite this article as: S. Azimee, M. Rahmati, H. Fahimi, et al., TiO2 nanoparticles enhance the chemotherapeutic effects of 5-fluorouracil in human AGS gastric cancer cells via autophagy blockade, Life Sciences(2020), https://doi.org/10.1016/j.lfs.2020.117466

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© 2020 Published by Elsevier.

Journal Pre-proof

TiO2 nanoparticles enhance the chemotherapeutic effects of 5-fluorouracil in human AGS gastric cancer cells via autophagy blockade

Shiva Azimee1,2, Marveh Rahmati3, Hossein Fahimi1, Mohammad Amin Moosavi2* 1

Department of Genetics, Faculty of Advanced Science and Technology, Tehran Medical

Sciences, Islamic Azad University, Tehran, Iran. Department of Molecular Medicine, Institute of Medical Biotechnology, National Institute

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of Genetic Engineering and Biotechnology, P.O. Box: 14965/161, Tehran, Iran. Cancer Biology Research Center, Cancer Institute, Tehran University of Medical Sciences,

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Tehran, Iran.

*Corresponding author:

Assistant Professor,

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Moosavi MA,

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National Institute of Genetic Engineering and Biotechnology, Shahrak-e Pajoohesh, km 15, Tehran –Karaj Highway, P.O. Box: 14965/161, Tehran, Iran. Tel/Fax:(+98)2144787335

Email: [email protected]

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Journal Pre-proof Abstract Aims: Nanoparticles (NPs)-based drugs have been recently introduced to improve the efficacy of current therapeutic strategies for the treatment of cancer; however, the molecular mechanisms by which a NP interacts with cellular systems still need to be delineated. Here, we utilize the autophagic potential of TiO2 NPs for improving chemotherapeutic effects of 5fluorouracil (5-FU) in human AGS gastric cells.

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Materials and Methods: Cell growth and viability were determined by trypan blue exclusion test and MTT assay, respectively. Vesicular organelles formation was evaluated by acridine

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orange staining of cells. Cell cycle and apoptosis were monitored by flow cytometry.

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Reactive oxygen species (ROS) level were measured by DCHF-DA staining. Autophagy was

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interaction with autophagic proteins.

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examined by q-PCR and western blotting. Molecular docking was used for studying NP

Key findings: TiO2 NPs increase ROS production, impair lysosomal function and

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subsequently block autophagy flux in AGS cells. In addition, the autophagy blockade induced by non-toxic concentrations of TiO2 NPs (1 µg/ml) can promote cytotoxic and apoptotic

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effects of 5-FU in AGS cells.

Significance: These results confirm the beneficial effects of TiO2 NPs in combination with chemotherapy in in vitro model of gastric cancer, which may pave the way to develop a possible solution to circumvent chemoresistance in cancer.

Keywords: 5-fluorouracil, autophagy, chemotherapy, gastric cancer, nanoparticle, titanium dioxide.

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Journal Pre-proof Introduction Tumor formation is the consequence of genetic and epigenetic alterations that trigger inactivation or overactivation of specific genes, leading to uncontrolled proliferation, poor differentiation, and inhibition of programmed cell death pathways in cancerous cells [1, 2]. In addition, recent findings revealed that most tumors display dysregulation of autophagy, an essential quality control process which maintain cells homeostasis under stressful conditions such as nutrient starvation and hypoxia [2-4]. In context of cancer, autophagy acts as a

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double-edged sword; whereas autophagy level diminishes at early phases of cancer

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formation, its level is overactivated during tumor progression and cancer cell survival at

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advanced stages [5, 6]. Therefore, autophagy manipulation (ie, both its inhibition and

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induction) is promising for designing new anti-cancer therapies [6-8]. Gastric cancer is started from the lining of the stomach, and according to

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GLOBOCAN2018, this malignancy is the 5th most commonly diagnosed cancer and 3rd

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leading cause of cancer mortality in the world [9]. Current therapeutic protocols for the treatment of human gastric adenocarcinoma include surgery (gastrectomy), radiotherapy,

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chemotherapy and adjuvant chemoradiotherapy [10]. 5-fuorouracil (5-FU), either alone or in combination with other drugs, is typically used as the main chemotherapeutic protocol for the treatment of human gastric cancer [11, 12]. This pyrimidine analog inhibits thymidylate synthase enzyme and provokes DNA and RNA strand breaks, leading to the induction of cell death [11, 13]. However, the efficacy of chemotherapy with 5-FU is limited in gastric cancer patients, mainly due to rapid drug clearance and the development of drug resistance [14, 15]. Therefore, novel adjuvant therapeutic strategies are desired, such as those derived from nanomedicine [16]. Nanoparticles (NPs) have received considerable attention for cancer therapy due to their unique physicochemical properties at nanoscale size (1-100 nm), which allow them to

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Journal Pre-proof penetrate cell membrane and localize into tumor sites higher than routine drugs [17]. Various NPs have provided the convenient tools for combination therapy, which is mechanistically mediated through enhancing the cytotoxic mechanism of chemotherapy [18] and/or combating cancer chemoresistance [19]. Recent evidence suggests that NPs are potent activators of autophagy, and this autophagic potential may enable them to overcome the limitations of the current cancer treatments, including the low therapeutic efficacy of chemotherapy and drug resistance [20-22]. Among NPs, titanium dioxide (TiO2) is typically

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considered as an inert and nontoxic particle [23]. This NP has three crystallographic

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structures (anatase, rutile, and brookite), and among them, anatase TiO2 exerts highest

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cytotoxic effects [24]. TiO2 NPs showed antitumor activities in various in vitro and in vivo

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models of cancers, which is mechanistically mediated through the induction of oxidative stress response and DNA damage, leading to the activation of apoptotic and/or necrotic cell

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death pathways [23, 25-27]. Like most other NPS, TiO2 NPs can also modulate autophagy

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pathways in different cancerous cells [28-30]. However, the underlying mechanisms of anticancer effects of TiO2 NP, and particularly, the possibility of its therapeutic potentials in

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gastric cancer have yet to be addressed. In this study we investigate the autophagic and cytotoxic potentials of anatase TiO2 NP as well as its potential for the improvement of chemotherapy in AGS gastric cancer cells. Our results demonstrate that non-toxic concentrations of TiO2 NPs can enhance 5-FU-induced cell death in AGS cells, which is supposed to be due to the blockade of autophagy flux. Materials and methods Materials All culture flask and plates were purchased from SPL Life Science (South Korea). RPMI1640 and trypsin/EDTA were prepared from BIO-IDEA Co. (Tehran, Iran). Fetal bovine serum (FBS) was purchased from Gibco (MD, USA). The penicillin and streptomycin were

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Journal Pre-proof prepared from Cinnagen Co. (Tehran, Iran). Bafilomycin A1 (Baf A1), Propidium iodide (PI), dichlorodihydrofluorescein diacetate (DCFH-DA), trypan blue, 3-(4, 5-dimethythiazol2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), tris-HCl, acridine orange (AO), tween-20, sodium dodecyl sulfate (SDS), dimethyl sulfoxide (DMSO), ethylene diamine tetra acetic acid (EDTA) and polyvinylidene difluoride (PVDF) were obtained from Sigma-Aldrich (Germany). Primary antibodies against GAPDH (sc-32233) and p62/SQSTM1 (sc-28359) as

LC3B antibody was purchased from Abcam (ab51520).

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Characterization of TiO2 NPs

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well as secondary antibodies were purchased from Santa Cruz Biotech (USA). The anti-

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Anatase TiO2 NP (99% anatase; 10-25 nm) was purchased from US Research Nanomaterial,

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Inc (Houston, TX, USA). The crystal structure and phase purity of NP was studied by X-ray powder diffraction (XRD) method using the Siemens X-ray diffractometer D5000 (Siemens

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AG, Germany). The morphology and size of NPs were determined by transmission electron

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microscopy (TEM) (Philips, Germany). The hydrodynamic size of NPs was measured by dynamic light scattering (DLS) (Nanotrac Wave model, Microtrac Inc., USA). All

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measurements were performed at room temperature. Cell culture and treatment

Human gastric adenocarcinoma AGS cell line was purchased from Pasture Institute Cell Bank (Tehran, Iran). The cells were cultured in RPMI 1640 medium plus FBS (10%, v/v) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) at 37 ºC and humidified condition with 5% CO2. The stock solution of 5-FU (1 mM) was prepared in DMSO. TiO2 NP suspensions (1 mg/ml) were prepared in culture medium. NPs were dispersed in a sonicator bath (JAC ultrasonic, Jeio Tech Co, Republic of Korea) for 10 min at 50 W and stabilized by adding 10% FBS [31]. The NP stock suspensions were further diluted in culture media and sonicated (5 min)

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Journal Pre-proof before exposure to the cells. For combination therapy, the cells were simultaneously treated with TiO2 NPs and different doses of 5-FU for 24 h. For autophagy inhibition experiments, the cells were pretreated with Baf A1 (10 nM dissolved in DMSO) for 60 min and then NPs and/or drugs were added to the cell culture wells. Cell growth and cytotoxicity Cell growth was determined by trypan blue exclusion test. Briefly, cells were stained with 0.4 mg/ml trypan blue, and then the number of live and dead (blue-stained) cells were counted

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using a hemocytometer under an inverted light microscope (Olympus, Japan). In parallel, cell

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viability or cytotoxicity were evaluated by colorimetric MTT assay as previously reported

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[32]. Briefly, 20 μl MTT (5 mg/ml) was added to the control and treated cells and allowed to

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be incubated for 4 h at cell culture condition. The tetrazolium dye MTT can be reduced to purple-blue formazan crystals in the viable cells. The absorbance of dissolved formazan (570

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nm) in DMSO was measured using an ELISA Reader (Lab systems Multiskan MS, Finland).

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Lysosome acidity and acidic vesicular organelles (AVOs) formation The acidity of lysosomes and the accumulation of AVOs were evaluated by acridine orange

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(AO) staining of control and treated cells. AO is a cell-permeable green lysotropic fluorophore that can emit read fluorescent upon accumulation in acidic compartments, such as lysosomes. [31]. Briefly, the cells were collected, washed and resuspended in PBS, and then stained with 1 μl AO (10 μg/ml) for 10 min in the dark. The microscopic photographs were taken by a fluorescent microscope (Carl Zeiss, Germany) with 20X magnification. Cell cycle flow cytometry Cell cycle pattern of cells were determined by flow cytometry (FACS Calibur, BD bioscience, USA). Cells were harvested, washed in PBS and fixed in cold 70% ethanol (v/v) for 30 min at 4 °C. After two times washing in PBS, the cells were treated with 50 μl DNasefree RNase (100 μg/ml) and stained with 450 μl PI solution (50 μg/ml) in the dark. The DNA

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Journal Pre-proof content of the cells and population of sub-G1 (apoptosis), G0/G1, S and G2/M phases were analyzed by FlowJo 7.6.1 software. Gene expression evaluation The gene expression levels were quantitatively evaluated by real-time PCR. Total RNAs were extracted using RNX-Plus kit (Cinaclone Co, Tehran, Iran) and used for cDNA synthesis (PrimeScriptTM RT reagent Kit, Takara Bio Inc, Japan) according to the manufacturer’s protocol. The real-time PCR was performed using SYBR green and PCR

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master mix (Premix Ex Taq II, Takara Bio Inc, Japan) by an Applied Bio molecular system

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(Australia). β-actin was used as housekeeping gene and the relative quantification of gene

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expression was determined by the 2-ΔΔCt method. The specific sequences of primers have

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been shown in Table 1. Western blot analysis

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Total proteins were extracted by ice-cold RIPA buffer containing fresh protease

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inhibitors cocktail (CMG Co., Esfahan, Iran). Eual amount of proteins (35 µg) were separated with SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membranes. The

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membranes were blocked with 5% skimmed milk in TBST (Tris-buffered saline with 0.05% Tween-20) for 90 min at room temperature and then incubated with the primary antibodies, including anti-GAPDH (1:2000), anti-p62/SQSTM1 (1:1000) and anti-LC3B (1:1000) overnight at 4 °C. After three times washing in TBST (0.2%), the corresponding HRPconjugated secondary antibodies (1:10000) were added to the membranes and allowed to be incubated for 2 h at room temperature. The protein bands were visualized by an enhanced chemiluminescence kit (Amersham Life Sciences, UK) on X-ray films. ROS level assay To evaluate the intracellular level of ROS, control and treated cells were stained by DCHFDA (25 mg/ml) for 15 min in the dark, then mean fluorescent intensity was measured at 488

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Journal Pre-proof nm (excitation) and 530 nm (emission) by a fluorimeter microplate reader device (Bio Tek Instrument, USA) as mentioned elsewhere [31]. Molecular docking Crystallographic structure of anatase TiO2 (TiO6 octahedra) was used for docking study. The crystallographic structures structures of ZZ domain of p62/SQSTM1 (PDB ID: 5YP7) and LC3-II (PDB ID: 2ZJD) were obtained from the Protein Data Bank (http://www.rcsb.org). The molecular docking study was carried out by HEX 6.3 software. The CHIMERA

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(www.cgl.ucsf.edu/chimera) and free Molegro Virtual Docker software were applied as

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graphical tools for the visualization of the docked site and interaction among the residues.

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Statistical analysis

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All experiments are from at least three independent experiments each performed in triplicates. The results were presented as the mean ± standard deviation (SD) and statistically

Results

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(version 6.07).

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analyzed using two-tailed Student's t-test by Microsoft Excel 2013 and GraphPad Prism

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TiO2 NP potentiates the anti-cancer effects of 5-FU in AGS cells First, we confirmed the physicochemical characterization and size of prepared TiO2 NPs. As can be observed in Fig. 1A, the XRD peaks of TiO2 NPs were corresponded to the (101), (004), (200), (105), (211), (204), (220) and (215), which represent good crystallography and anatase form of NP. The TEM images of TiO2 powder confirmed that NP size was mostly ranged between 20-30 nm (Fig. 1B). The DLS analysis was also in agreement with this result and a slightly larger hydrodynamic size (46.2 ± 15.6) was recorded (Fig. 1C). To test effects of TiO2 NP (1-100 μg/ml) and 5-FU (0.01-100 μM) on the growth and viability of AGS cells, we used trypan blue exclusion and MTT assays, respectively. The results showed that 1, 10 and 100 μg/ml concentrations of TiO2 NPs could inhibit growth by

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Journal Pre-proof 4±3%, 13±4 % and 51±7% after 24 h, respectively (Fig. 2A). Similarly, viability was also decreased by 1%, 7±5%, and 33±5% (Fig. 2A). In addition, 5-FU at concentrations between 0.01-100 μM could inhibit growth and viability between 8-73% and 1-56%, after 24 h, respectively (Fig. 2B). We then studied the effects of combination of nontoxic concentration of TiO2 NP (1 μg/ml) with different doses of 5-FU (0.01-10 μM) after 24 h (Fig.1C, 2D). The results showed that combination of TiO2 NP could potentiate the anti-cancer effects of low doses of 5-FU (0.01-1 μM). For instance, TiO2 NPs (1 μg/ml) promoted both growth

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inhibitory and cytotoxic effects of 1 μM 5-FU up to 1.9-fold (from 39.27% to 74.68%) and

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2.7-fold (from 21.31% to 57.5%), respectively (Fig. 2C and D). In addition, combination of

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TiO2 NP (1 μg/ml) with 5-FU (1 μM) led to the observation of higher portion of cells with

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morphological signs of death (detached cells with membrane shrinkage) compared with chemotherapy alone after 24 h (Fig 2E, block arrows).

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TiO2 NP enhances the occurrence of apoptosis in 5-FU-treted AGS cells

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To further confirm the effectiveness of TiO2 NPs in sensitizing AGS cells to chemotherapy with 5-FU, we evaluated the rate of apoptosis by flow cytometry (sub-G1 cells) and q-PCR

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(Bax/Bcl2 ratio). No statistically significant changes in apoptosis rate (from 3.8% in control to 7% in treated cells) was observed when the cells exposed to NP (1 μg/ml) for 24 h (Fig. 3A). However, an increase in sub-G1 peak (apoptotic cells) accompanied with a decrease in G0/G1 and G2./M cells were recorded after exposing the cells to 5-FU (1 μM) for 24 h (Fig. 3A), and this was significantly augmented (from 22.7% to 42.9%) when AGS cells were cotreated with NP and 5-FU (Fig. 3A and B). To further investigate apoptosis, we evaluated the gene expressions levels of Bax (pro-apoptotic protein) and Bcl-2 (anti-apoptotic protein), two typical markers of mitochondrial pathway of apoptosis [33]. The gene expression ratio of Bax/Bcl-2 was significantly increased (2.53 ± 0.43-fold) in 5-FU-treated cells compared with control cells (Fig. 3C). Interestingly, the combination of TiO2 and 5-FU could further enhance

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Journal Pre-proof the Bax/Bcl-2 ratio (8.6-fold compared with control and 3.4-fold compared to 5-FU treated cells), indicating that TiO2 NP can sensitize AGS cell to low doses of chemotherapy through eliciting mitochondrial pathway of apoptosis. However, TiO2 NP (1 μg/ml) treatment not only did not increase but also decreased the ratio of Bax/Bcl-2 gene expression in AGS cells, suggest that low doses of NP alone may inhibit apoptosis and promote viability in AGS cells (Fig. 3C). TiO2 NP blocks autophagy flux in control and 5-FU-treated cells

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To seek how nontoxic TiO2 NPs can promote the anti-cancer (cytotoxic and apoptotic) effects

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of 5-FU, we examined the involvement of autophagy that may have both pro-survival and

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pro-death roles in cancer, depending on the condition [3, 5]. First, we probed for the protein

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levels of two typical autophagy markers p62/SQSTM1 and microtubule associated protein light chain 3 (LC3) proteins (Fig. 4A). The LC3 proteins is cleaved by ATG4B to form LC3-

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I, then LC3-I is lipidated to form LC3-II, which is a key factor during autophagosome

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membrane expansion and fusion [3, 5]. Our results revealed that 5-FU, and with higher extent TiO2 NP, increased the formation of LC3B-II compared with control cells (Fig. 4A).

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Interestingly, TiO2 (1 μg/ml) + 5-FU (1 μM) treatment was more effective in inducing LC3BII accumulation than each monotherapy alone (Fig. 4A). The changes at the protein levels of LC3B-II may be related to the activation of autophagy flux or alternatively to the blockade of autophagy flux [34]. To get a better picture of autophagy in our system, we evaluated the protein level of p62/SQSTM1 (p62), which is degraded during autophagy flux. A decrease at p62 level is a hallmark of autophagy flux while an increase at the level of this autophagy receptor may be an indicator of autophagy blockade [34]. The protein levels of p62 was moderately downregulated in the cells treated with 5-FU (1 μM) alone, pointing to the occurrence of an autophagy flux response (Fig. 4A). By contrast, in TiO2 NP (1 μg/ml) treated cells, p62 levels substantially increased compared with control group, suggesting the

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Journal Pre-proof possibility of the autophagy blockade in this condition (Fig. 4A). More importantly, p62 accumulation was further increased in combination therapy group compared with 5-FUtreated cells, meaning that TiO2 NPs can also block autophagy flux induced by chemotherapy in AGS cells (Fig 4A). To get a more mechanistical insight, the mRNA expression levels of autophagy-related genes ATG5, BECN1 (ATG6) and ATG7, which play key roles during formation and elongation of autophagosome, were also evaluated by q-PCR [3, 5]. The results showed that

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both ATG5 and ATG7 expression levels were significantly increased in the cell treated with

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TiO2 NP or 5-FU, and this trend was enhanced when the cells exposed to the combination of

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TiO2 NP and 5-FU (Fig. 4B). For example, when compared with control cells, the relative

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ratio of ATG5 gene expression levels were 3.4 ± 0.3-fold in TiO2, 2.5 ± 0.18-fold in 5-FU, and 6 ± 0.05-fold in TiO2+5-FU treated AGS cells. Similarly, the ATG7 fold changes were

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2.1 ± 0.39-fold in TiO2, 0.27 ± 0.06-fold in 5-FU, and 11.8 ± 0.45-fold in TiO2+5-FU group

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(Fig. 4B). However, exposure to TiO2 NP or 5-FU did not result in any significant change at the gene expression levels of BECN1 (ATG6) in AGS cells (Fig. 4B), leads us to propose the

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involvement of BECN1-independent (non-canonical) autophagy pathway. Autophagy inhibitor of Baf A1 potentiates the anti-cancer effects of TiO2 NPs and/or 5FU in AGS cells

To better understand functional importance of autophagy in our experiments, we pretreated the cells with the late autophagy inhibitor Baf A1. Similar to TiO2 NPs effects, the blocking autophagy with Baf A1 could enhance cytotoxic effects of 5-FU and sensitize AGS cell to chemotherapy (Fig. 5). Interestingly, pretreatment with nontoxic dose of Baf A1 (10 nM) led to a profound increase in cytotoxic effects of TiO2 + 5-FU (from 49.9% to 89%), suggests that autophagy blockade is important for the observed synergistic effects of NPs on chemotherapy in our experiments.

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Journal Pre-proof TiO2 NP enhances ROS generation, impairs lysosome function and may interact with autophagic proteins NPs can modulate autophagy through direct interaction with autophagic proteins, the regulation of expression of autophagic genes, lysosome dysfunction, the production of ROS, and the induction of ER stress [20-22, 35]. First we evaluated the oxidative potential of these NPs. The results showed that TiO2 NP (1 mg/ml) significantly increased ROS level in AGS cells up to 1.8-fold compared with control after 24 h (Fig. 6A). The increase at the ROS

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levels may damage lysosomes, which in turn may block autophagy. To test this hypothesis,

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we evaluated the integrity and acidity of lysosomes by AO staining, a lysosomotropic probe

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for evaluating gross abnormalities in the lysosomal pH [36]. It has been well documented that

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the autophagosome-lysosome fusion depends on the optimal acidic condition of AVOs so that any significant change in the pH of lysosomes can block autophagic flux. Fluorescent

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microscopic images revealed the appearance of orange/red dots in AGS cells exposed to TiO2

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NPs after 24 h (Fig. 6B), suggesting an increase in the lysosomal acidity that may lead to the impairment of lysosome function. Finally, we used bioinformatics tools to see if a direct

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interaction of NPs with autophagic proteins may be another possible scenario for the observed autophagy blockade in AGS cells. Our in silico-based results by molecular docking method showed that anatase TiO2 has ability to interact with p62 and LC3-II proteins (Fig. 6C-F). The results from docked complex revealed that the hydrogen bond between anatase TiO2 and ZZ domain of p62 and the closest interacting amino acids are Ile-127, Asp-129, Pro146, Asp-147, Asn-132 residues in binding pocket (Fig. 6C and D). The hydrogen bond between anatase TiO2 and closest interacting amino acids of LC3-II in binding pocket include Gln-43, Gln-77, Asn-76, Ala-78, Gln-116 and Glu-117 residues (Fig. 6E and F). However, further experiments are needed to experimentally confirm these findings.

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Journal Pre-proof Discussion Despite improvements in the treatment of cancer, the acquisition of drug resistance and therapeutic management of disease still remain as major challenges [15]. Currently, the combination of hydroxychloroquine, a non-specific autophagy inhibitor, with chemotherapy could promote therapeutic outcomes in some cancer patients, but for clinical translation, we still need to develop more specific and potent autophagic modulators [37]. In this study, we demonstrated the therapeutic potential of TiO2 NPs in sensitizing human AGS gastric cells to

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chemotherapy that seems to be mediated through the blockade of autophagy flux.

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The autophagic response of cells to NPs is complex and can be either autophagy flux or

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autophagy dysfunction (block), depending on the dose and physicochemical properties (e.g.,

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size, charge, dispersity). For instance, TiO2 NPs with the same physicochemical characteristics was found to evoke the autophagic flux and/or autophagy blockade responses,

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depending on the dose and treatment time [21, 38, 39]. It has been recently reported that

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constant concentrations of TiO2 NPs activate an autophagic flux response at short times (24 h), which is followed with the occurrence of an autophagic blockade response after longer

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times [40]. Here, we demonstrated that anatase TiO2 NPs at low concentration (1 mg/ml) are potent blocker of autophagy flux in AGS cells (Fig. 3). The autophagic potential of NPs may play paradoxical roles (ie., cytoprotective or cytotoxic roles) in different cells. For example, autophagy induced by AgNPs is cytoprotective in HeLa cells [41], while this response is associated with cell death on other cells [35, 42]. In addition, autophagy flux induced by photodynamic N-TiO2 NPs are associated with apoptosis in leukemia K562 cells [31]. In our current study, however, autophagy blocked by TiO2 NP (1 mg/ml) was not cytotoxic (Fig. 2A) and even could decrease Bax/Bcl2 ratio in AGS cells (Fig. 3B). The combination of nontoxic concentration of TiO2 NP and Baf A1 led to the induction of a cell death response in NP-exposed cells (Fig. 5), suggesting prosurvival role of

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Journal Pre-proof autophagy in TiO2 NP-exposed cells. Therefore, it seems that consequence of treatment with TiO2 NPs is mostly cytoprotective accompanied with an increase in overall survival in AGS gastric cells [43], and also other cell types such as HaCaT skin keratinocytes [44]. However, Nasr and coworkers reported that TiO2 NPs promoted apoptosis and invasion in MKN-45 gastric cancer cells [45]. Recent findings suggest that ROS has an essential role in controlling membrane integrity and proper function of lysosomes [46]. Our results confirm the enhancement of ROS

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(DCFH fluorescent) and the alteration in lysosomal activity (AO attained cells) in AGS cells

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exposed to TiO2 NPs alone (Fig. 6). It is possible that ROS produced by TiO2 NPs and the

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subsequent oxidative stress is responsible for the impairment of the proper function of

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lysosomes and subsequently autophagy blockade [47]. However, we cannot rule out the possibility of other scenarios, such as a direct binding ability between NPs and autophagy

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proteins as previously evidenced by other groups [48]. In this line, we showed by means of

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bioinformatics tools that TiO2 NPs can interact with LC3-II and ZZ domain of p62 in AGS cells (Fig. 6). The amino acids involved in these interactions have mostly positive charges

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and are bonded to the negative surface of TiO2 NPs. Similarly, bioactive silica NPs showed strong and direct interaction with the autophagy proteins of LC3-II and p62, which is associated with autophagy dysfunction and osteoblast differentiation [48]. Autophagy may contribute to the cytotoxic mechanisms of drugs or, alternatively, it may serve as a drug-resistance mechanism in cancerous cells [15]. Recently, it has been shown that autophagy inhibition promoted the effect of chemotherapy with 5-FU in different cancers, such as colon cancer and human skin squamous cell carcinoma [49, 50]. Autophagy disruption by means of either 3-MA treatment or BECN1 depletion potentiated the cytotoxic effects of 5-FU in human gastric BGC 823 cells [51]. In line with these reports, we confirm that autophagy blockade by means of Baf A1 and/or low doses of TiO2 NPs enhance the

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Journal Pre-proof effectiveness of chemotherapy with 5-FU in AGS cells. The potential of these autophagy blockers in promoting the cytotoxic effects of 5-FU suggests that autophagy blockade may be a new strategy for enhancing the effectiveness of chemotherapy in in AGS cells. Similarly, titania-coated gold nano-bipyramids synergistically potentiated effects of the protease inhibitor bortezomib via autophagy blockade in glioblastoma cancer [52]. In addition, combination of graphene-oxide silver NPs with cisplatin enhanced apoptotic and autophagic responses to chemotherapy in human cervical HeLa cancer cells [53]. Hopefully, these NP-

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based combination therapy approaches are promising to be a part of new anti-cancer therapies

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[39].

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Conclusion

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We demonstrated that combination of TiO2 NPs with 5-FU promoted the cytotoxic and apoptotic effects of chemotherapy via autophagy blockade in AGS gastric cancer cells. The

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autophagy modulating effects of TiO2 NPs is mediated through the increase in ROS level, the

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impairment of proper function of lysosomes and possibly direct interaction with key autophagic proteins. The property of autophagic TiO2 NP in sensitizing AGS gastric cancer

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cells to low doses of 5-FU highlights may alleviate the potential dose limiting toxicity and extend the therapeutic options in gastric cancers, warranting further experiments.

Acknowledgment

This work was supported by National Institute of Genetic Engineering and Biotechnology (grant number 980301-I-728). AM also appreciates Islamic Azad University and NIMAD grants. MR is financially supported by NIMAD and Tehran University of Medical Sciences grants.

Conflict of interest: The authors report no conflicts of interest in this work.

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Journal Pre-proof References

10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

27.

of

9.

ro

8.

-p

7.

re

6.

lP

5.

na

3. 4.

Sell, S., Cancer stem cells and differentiation therapy. Tumor Biology, 2006. 27(2): p. 59-70. Chaabane, W., et al., Autophagy, apoptosis, mitoptosis and necrosis: interdependence between those pathways and effects on cancer. Archivum immunologiae et therapiae experimentalis, 2013. 61(1): p. 43-58. Mizushima, N., Autophagy: process and function. Genes & development, 2007. 21(22): p. 2861-2873. Yang, Z. and D.J. Klionsky, An overview of the molecular mechanism of autophagy, in Autophagy in infection and immunity. 2009, Springer. p. 1-32. Kroemer, G., G. Mariño, and B. Levine, Autophagy and the integrated stress response. Molecular cell, 2010. 40(2): p. 280-293. Galluzzi, L., et al., Autophagy in malignant transformation and cancer progression. The EMBO journal, 2015. 34(7): p. 856-880. Levine, B., M. Packer, and P. Codogno, Development of autophagy inducers in clinical medicine. The Journal of clinical investigation, 2015. 125(1): p. 14-24. Levy, J.M.M., C.G. Towers, and A. Thorburn, Targeting autophagy in cancer. Nature Reviews Cancer, 2017. 17(9): p. 528. Bray, F., et al., Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a cancer journal for clinicians, 2018. 68(6): p. 394-424. Benbrahim, Z., et al., Gastric Cancer: An Epidemiological Overview. Epidemiology (Sunnyvale), 2017. 7(304): p. 2161-1165.1000304. Longley, D.B., D.P. Harkin, and P.G. Johnston, 5-fluorouracil: mechanisms of action and clinical strategies. Nature reviews cancer, 2003. 3(5): p. 330. Park, S.C. and H.J. Chun, Chemotherapy for advanced gastric cancer: review and update of current practices. Gut and liver, 2013. 7(4): p. 385. Chu, E., et al., Thymidylate synthase inhibitors as anticancer agents: from bench to bedside. Cancer chemotherapy and pharmacology, 2003. 52(1): p. 80-89. Chang, H.J., et al., Molecular mechanism of chemoresistance and restoration in human gastric cancer cells. 2019, American Society of Clinical Oncology. Holohan, C., et al., Cancer drug resistance: an evolving paradigm. Nature Reviews Cancer, 2013. 13(10): p. 714-726. Bogdan, J., J. Pławińska-Czarnak, and J. Zarzyńska, Nanoparticles of titanium and zinc oxides as novel agents in tumor treatment: a review. Nanoscale research letters, 2017. 12(1): p. 225. Vinardell, M.P. and M. Mitjans, Antitumor activities of metal oxide nanoparticles. Nanomaterials, 2015. 5(2): p. 1004-1021. Zhao, C.-Y., et al., Nanotechnology for cancer therapy based on chemotherapy. Molecules, 2018. 23(4): p. 826. Hu, C.-M.J. and L. Zhang, Therapeutic nanoparticles to combat cancer drug resistance. Current drug metabolism, 2009. 10(8): p. 836-841. Peynshaert, K., et al., Exploiting intrinsic nanoparticle toxicity: the pros and cons of nanoparticleinduced autophagy in biomedical research. Chemical reviews, 2014. 114(15): p. 7581-7609. Popp, L. and L. Segatori, Differential autophagic responses to nano-sized materials. Current opinion in biotechnology, 2015. 36: p. 129-136. Soenen, S.J., et al., Turning a frown upside down: Exploiting nanoparticle toxicity for anticancer therapy. Nano Today, 2013. 8(2): p. 121-125. McNamara, K. and S.A. Tofail, Nanoparticles in biomedical applications. Advances in Physics: X, 2017. 2(1): p. 54-88. Gupta, S.M. and M. Tripathi, A review of TiO 2 nanoparticles. Chinese Science Bulletin, 2011. 56(16): p. 1639. Xue, C., et al., Nano titanium dioxide induces the generation of ROS and potential damage in HaCaT cells under UVA irradiation. Journal of nanoscience and nanotechnology, 2010. 10(12): p. 8500-8507. Petković, J., et al., DNA damage and alterations in expression of DNA damage responsive genes induced by TiO2 nanoparticles in human hepatoma HepG2 cells. Nanotoxicology, 2011. 5(3): p. 341353. Zhu, Y., J.W. Eaton, and C. Li, Titanium dioxide (TiO2) nanoparticles preferentially induce cell death in transformed cells in a Bak/Bax-independent fashion. PloS one, 2012. 7(11): p. e50607.

Jo ur

1. 2.

16

Journal Pre-proof

35. 36. 37. 38.

39. 40. 41. 42. 43. 44. 45. 46. 47. 48.

49. 50. 51. 52. 53.

of

34.

ro

33.

-p

32.

re

31.

lP

30.

na

29.

Dai, X., et al., Titanium dioxide nanoparticles induce in vitro autophagy. Human & experimental toxicology, 2018: p. 0960327118777849. Zhao, Y., et al., Exposure to titanium dioxide nanoparticles induces autophagy in primary human keratinocytes. Small, 2013. 9(3): p. 387-392. Yu, K.-N., et al., Inhalation of titanium dioxide induces endoplasmic reticulum stress-mediated autophagy and inflammation in mice. Food and Chemical Toxicology, 2015. 85: p. 106-113. Moosavi, M.A., et al., Photodynamic N-TiO 2 nanoparticle treatment induces controlled Ros-mediated autophagy and terminal differentiation of leukemia cells. Scientific reports, 2016. 6: p. 34413. Seyed-Gogani, N., et al., Nucleostemin depletion induces post-g1 arrest apoptosis in chronic myelogenous leukemia k562 cells. Advanced pharmaceutical bulletin, 2014. 4(1): p. 55. Oltval, Z.N., C.L. Milliman, and S.J. Korsmeyer, Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programed cell death. cell, 1993. 74(4): p. 609-619. Klionsky, D.J., et al., Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy, 2016. 12(1): p. 1-222. Zielinska, E., et al., Silver nanoparticles of different sizes induce a mixed type of programmed cell death in human pancreatic ductal adenocarcinoma. Oncotarget, 2018. 9(4): p. 4675. Thomé, M.P., et al., Ratiometric analysis of acridine orange staining in the study of acidic organelles and autophagy. J Cell Sci, 2016: p. jcs. 195057. Chude, C. and R. Amaravadi, Targeting autophagy in cancer: update on clinical trials and novel inhibitors. International journal of molecular sciences, 2017. 18(6): p. 1279. Mohammadalipour, Z., et al., Different Concentrations of Titanium Dioxide Nanoparticles Induce Autophagy Followed by Growth Inhibition or Cell Death in A375 Melanoma Cells. Journal of Skin and Stem Cell, 2017. 4(2). Mohammadinejad, R., et al., Necrotic, apoptotic and autophagic cell fates triggered by nanoparticles. Autophagy, 2019. 15(1): p. 4-33. Popp, L., et al., Autophagic response to cellular exposure to titanium dioxide nanoparticles. Acta biomaterialia, 2018. 79: p. 354-363. Lin, J., et al., Inhibition of autophagy enhances the anticancer activity of silver nanoparticles. Autophagy, 2014. 10(11): p. 2006-2020. Lee, Y.-H., et al., Cytotoxicity, oxidative stress, apoptosis and the autophagic effects of silver nanoparticles in mouse embryonic fibroblasts. Biomaterials, 2014. 35(16): p. 4706-4715. Botelho, M.C., et al., Effects of titanium dioxide nanoparticles in human gastric epithelial cells in vitro. Biomedicine & Pharmacotherapy, 2014. 68(1): p. 59-64. Lopes, V.R., et al., Dose-dependent autophagic effect of titanium dioxide nanoparticles in human HaCaT cells at non-cytotoxic levels. Journal of nanobiotechnology, 2016. 14(1): p. 22. Nasr, R., et al., Induction of apoptosis and inhibition of invasion in gastric cancer cells by titanium dioxide nanoparticles. Oman medical journal, 2018. 33(2): p. 111. Zhang, X., et al., MCOLN1 is a ROS sensor in lysosomes that regulates autophagy. Nature communications, 2016. 7: p. 12109. Cai, R., et al., Induction of cytotoxicity by photoexcited TiO2 particles. Cancer research, 1992. 52(8): p. 2346-2348. Ha, S.-W., M.N. Weitzmann, and G.R. Beck Jr, Bioactive silica nanoparticles promote osteoblast differentiation through stimulation of autophagy and direct association with LC3 and p62. ACS nano, 2014. 8(6): p. 5898-5910. Coker-Gurkan, A., et al., Inhibition of autophagy by 3-MA potentiates purvalanol-induced apoptosis in Bax deficient HCT 116 colon cancer cells. Experimental cell research, 2014. 328(1): p. 87-98. Zhang, L., et al., Autophagy in human skin squamous cell carcinoma: Inhibition by 3-MA enhances the effect of 5-FU-induced chemotherapy sensitivity. Oncology reports, 2015. 34(6): p. 3147-3155. He, X.X., C.K. Huang, and B.S. Xie, Autophagy inhibition enhanced 5‑FU‑induced cell death in human gastric carcinoma BGC‑823 cells. Molecular medicine reports, 2018. 17(5): p. 6768-6776. Wan, H.Y., et al., Titania‐Coated Gold Nano‐Bipyramids for Blocking Autophagy Flux and Sensitizing Cancer Cells to Proteasome Inhibitor‐Induced Death. Advanced Science, 2018. 5(3): p. 1700585. Yuan, Y.-G. and S. Gurunathan, Combination of graphene oxide–silver nanoparticle nanocomposites and cisplatin enhances apoptosis and autophagy in human cervical cancer cells. International journal of nanomedicine, 2017. 12: p. 6537.

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Table legends: Table 1. The specific sequences of oligonucleotide primers for real-time RT-PCR. Forward primer (5′-3′)

Forward primer (5′-3′)

β-actin

5′-CCTGGCCATGGAGTCCTGT-3′

5′-ATCTCCTTCTGCATCCTGTCG-3′

153bp

ATG5

5′-ACTGGGCTGGTCTTACTTTGC-3

5′-CCTTCAGTGGTCCGGTAAGTC-3′

99bp

BECN1

5′-AGGAACTCACAGCTCCATTAC-3

5′-AATGGCTCCTCTCCTGAGTT-3′

88bp

ATG7

5′-GTTTTGCTATCCTGCCCTC-3′

5′-GCTGTGACTCCTTCTGTTTG-3′

144bp

Bax

5'-CTGACATGTTTTCTGACGGCAA-3′

5'-GAAGTCCAATGTCCAGCCCA-3'

140bp

Bcl2

5‘-TGAGTTCGGTGGGGTCATGTGT-3′

5'-AAAGGCATCCCAGCCTCCGTTA-3′

142bp

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Journal Pre-proof Figure legends: Fig. 1. Characterization of TiO2 NPs. The XRD (A) and TEM (B) and DLS (C) were used to determine crystalline phase, average size and the hydrodynamic size of NP powder, respectively, as described in the Materials and Methods. Scale bar in B = 50 nm. Fig. 2. Effect of 5-FU and/or TiO2 NPs on the growth, viability and morphology of AGS cells. A and B) the cells were exposed to various concentrations of TiO2 NPs (1-100 μg/ml), or 5-FU (0.01-100 μM) for 24 h, then growth and viability were studied using trypan blue

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exclusion test and MTT assay, respectively. The results are from duplicates/triplicated of

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three independent experiments and represented as means ± SD. Statistically different results

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are indicated with *( p< 0.05) and ** (p < 0.01) compared to untreated cells. C and D) the

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combination of TiO2 NPs and 5-FU in AGS cells. The cells were cotreated with TiO2 (1 μg/ml) with different doses of 5-FU (0.01-10 μM) for 24 h, then growth (C) viability (D) and

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morphological changes (E) were studied by trypan blue test, MTT assay and an invert light

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microscope (magnification 32X), respectively. The result are means ± SD with *p< 0.05 or **p < 0.01 compared to 5-FU-treated alone.

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Fig. 3. Effect of TiO2 NPs and/or 5-FU on apoptosis in AGS cells. A and B) cells were treated with TiO2 NPs (1g μ/ml), 5-FU (1 μM) or a combination TiO2 NPs (1 μg/ml) with 5FU (1 μM) for 24 h, then collected and stained with PI for flow cytometric analyses of cell cycle. The percentage of cells in each phase of cell cycle as well as sub-G1 fraction were determined by FlowJet software. C) the gene expression levels of Bax, Bcl-2 and GAPDH genes were evaluated in the cells exposed to TiO2 NPs and/or 5-FU by real-time RT-PCR. The Bax/Bcl2 ratio was calculated and normalized to GAPDH, and the results were represented as fold change. Results are from three independent experiments each performed in duplicates and expressed as means ± SD. *p < 0.05 and** p < 0.01 compared with corresponding control.

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Journal Pre-proof Fig. 4. Autophagic effects of TiO2 NPs and/or 5-FU in AGS cells. Cells were exposed to TiO2 NPs (1g μ/ml), 5-FU (1 μM) or a combination TiO2 NPs (1 μg/ml) with 5-FU (1 μM) for 24 h, then protein (A) and gene (B) expression levels of autophagic genes were determined by western blotting and real-time PCR, respectively. A) The representative western blot image corresponding to the protein levels LC3-II and p62 in the control and treated AGS cells. The protein expressions were normalized to GAPDH as a loading control and results were quantified by the Image J software. The results were expressed as protein fold changes

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(means ± SD). B) the gene expression level of ATG5, BECN1 (ATG6) and ATG7 genes in the

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control and treated AGS cells. The results were normalized to β-actin housekeeping gene

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Fig. 5. Effects of autophagy inhibitor Baf A1 on cytotoxicity of TiO2 NPs and/or 5-FU in in AGS cells. Cells were pretreated with or without Baf A1 (10 nM) for 60 min, then exposed

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to TiO2 NPs (1 μg/ml), 5-FU (1 μM) or their combinations for 24 h. The cytotoxicity was

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Fig. 6. Mechanism of autophagy blockade induce by TiO2 NPs in AGS cells. A) cells were exposed to TiO2 NPs (1 μg/ml) for 24 h, then collected and stained with DCHF for evaluating ROS levels as mentioned in materials and methods. B) the acidity and integration of lysosomes were determined after AO staining of control and NP-exposed cells. The images were captured by a florescent microscope (magnification 20X). C-F) molecular docking studies between anatase TiO2 NPs and p62 or LC3-II proteins. The interaction between crystallographic structure of anatase TiO2 with highlighted TiO6 polyhedra and LC3-II (C) or p62 (E) proteins. Blue and red atoms represent titanium and oxygen, respectively. The hydrogen bond of interacting residues LC3-II (D) or p62 (F) with anatase TiO2.

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