Blue light induced reactive oxygen species from flavin mononucleotide and flavin adenine dinucleotide on lethality of HeLa cells

Accepted Manuscript Blue light induced reactive oxygen species from flavin mononucleotide and flavin adenine dinucleotide on lethality of HeLa cells

Ming-Yeh Yang, Chih-Jui Chang, Liang-Yü Chen PII: DOI: Reference:

S1011-1344(17)30119-7 doi: 10.1016/j.jphotobiol.2017.06.014 JPB 10875

To appear in:

Journal of Photochemistry & Photobiology, B: Biology

Received date: Revised date: Accepted date:

26 January 2017 7 June 2017 10 June 2017

Please cite this article as: Ming-Yeh Yang, Chih-Jui Chang, Liang-Yü Chen , Blue light induced reactive oxygen species from flavin mononucleotide and flavin adenine dinucleotide on lethality of HeLa cells, Journal of Photochemistry & Photobiology, B: Biology (2017), doi: 10.1016/j.jphotobiol.2017.06.014

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.

ACCEPTED MANUSCRIPT

Blue light induced reactive oxygen species from flavin mononucleotide and flavin adenine dinucleotide on

PT

lethality of HeLa cells

RI

Ming-Yeh Yang a,b, Chih-Jui Chang b, Liang-Yü Chen c,* a

SC

Institute of Medical Sciences, Tzu-Chi University, Hualien 970, Taiwan Department of Molecular Biology and Human Genetics, Tzu-Chi University, Hualien 970, Taiwan c Department of Biotechnology, Ming-Chuan University, Gui-Shan 333, Taiwan

MA

NU

b

2nd Revised Version

PT E

D

Submitted to “Photochemistry and Photobiology B: Biology” 2017/06/08

Corresponding Author

AC

*

CE

Running title: Cytocidal Effects of Riboflavin Photodegradation

Tel: (+886-3-3507001 est.3773) Fax: (+886-3-3593878) E-mail: [email protected]

1

ACCEPTED MANUSCRIPT Abstract Photodynamic therapy (PDT) is a safe and non-invasive treatment for cancers and microbial infections. Various photosensitizers and light sources have been developed for clinical cancer therapies. Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are the cofactor of enzymes and are used as photosensitizers in this study.

PT

Targeting hypoxia and light-triggering reactive oxygen species (ROS) are experimental

RI

strategies for poisoning tumor cells in vitro. HeLa cells are committed to apoptosis when

SC

treated with FMN or FAD and exposed to visible blue light (the maximum emitted wavelength of blue light is 462 nm). Under blue light irradiation at 3.744 J/cm2 (= 0.52

NU

mW/cm2 irradiated for 2 hours), the minimal lethal dose is 3.125 μM and the median lethal doses (LD50) for FMN and FAD are 6.5 μM and 7.2 μM, respectively. Individual

MA

exposure to visible blue light irradiation or riboflavin photosensitizers does not produce cytotoxicity and no side effects are observed in this study. The western blotting results

D

also show that an intrinsic apoptosis pathway is activated by the ROS during photolysis of

PT E

riboflavin analogues. Blue light triggers the cytotoxicity of riboflavins on HeLa cells in vitro. Based on these results, this is a feasible and efficient of PDT with an intrinsic

CE

photosensitizer for cancer research.

AC

Keywords: Hypoxia; Photodynamic therapy; Riboflavin photosensitizer; Apoptosis; Light emitting diode

2

ACCEPTED MANUSCRIPT

1. Introduction Photodynamic therapy (PDT) is a safe and non-invasive treatment for cancers and microbial infections [1]. PDT uses light irradiation and photosensitizers. Different

PT

wavelengths of light have different functions for various cell types [2, 3]. Light in the visible spectrum at wavelengths of about 380 to 740 nm is safer than ultraviolet (UV) and

RI

X-rays for medical or hygienic applications. Visible light with low intensity is used in

SC

clinical trials to promote the healing of wounds and to reduce inflammation [4]. Blue light

NU

irradiation is a potential approach in treatment of keloids, hypertrophic scars and fibrotic skin diseases [5]. Anterior cruciate ligament cells are damaged by 460 nm-light at 27

MA

J/cm2 [6]. Irradiation with light of 650 nm results in cytotoxicity in HeLa cells at 380

D

J/cm2 and induces an apoptosis pathway [7]. High intensity irradiation also induces cell

PT E

death through DNA damage [8].

Photosensitizers are transformed from the ground state to an excited state after

CE

exposure to light of a specific wavelength, which generates reactive oxygen species (ROS) and free radicals [9]. ROS has a high level of cytotoxicity and a short half-life [9]. Ideal

AC

photosensitizers accumulate in target cells and tissue. Photofrin was firstly used in PDT for cancer therapy in the 1970s [10]. However, it has many disadvantages, in that is non-specific, hydrophobic and cutaneously phototoxic [11]. The effects of ROS in a cell include lipid peroxidation, inactivate protein, cell necrosis and apoptosis [12]. Synthetic photosensitizes, such as porphyrin derivatives and 5-aminolevulinic acid (5-ALA), have been developed. The modification of porphyrin derivatives improves their 3

ACCEPTED MANUSCRIPT

solubility and increases the absorption wavelength to beyond the visible spectrum region [13] and decreases dark toxicity, such as core modification [14]. 5-ALA is used for the treatment of neck cancer [15], nodular basal cell carcinoma [16] and gastrointestinal

PT

cancers [11]. The photosensitizers conjugate with biomolecules to form a new generation of photosensitizers, such as dendrimer porphyrins, with 32 quaternary ammonium groups

RI

[17]. The design strategy is to decrease the target effects and to enhance pharmacokinetics.

SC

The major issues with photosensitizers in clinical applications are the specificity of targets,

NU

cutaneous phototoxicity, dark toxicity and photostability.

Flavin mononucleotide (riboflavin-5’-phosphate, FMN) and flavin adenine

MA

dinucleotide (FAD) are the cofactors of a reduction enzyme and riboflavin analogues

D

(shown in Fig. 1A). Riboflavin (RF) is a water-soluble molecule that is an essential

PT E

dietary supplement. It allows the body converts food or carbohydrates into energy. Excess RF is excreted through urine [18]. RF and its analogues (RFs) decompose after exposure

CE

to UV and visible light and this process generates ROS, such as superoxide anions and singlet oxygen [19].

AC

FMN/FAD is an electron transducer. Under hypoxic conditions, the level of the reduction form of FMNH2 and FADH2 is increased, which results in the production of O2•by auto-oxidation [20]. A previous study showed that RF and FMN are decomposed under irradiation by blue light, which generates hydroxyl radicals that de-activate microbes due to DNA damage [19]. RFs as dietary supplement products are ingested into the cell through riboflavin 4

ACCEPTED MANUSCRIPT

carrier protein (RCP) in the cell membrane. Previous studies have shown that the oxidation-reduction ratio of the FAD redox system in a cancerous cell is significantly greater than that for a normal cell [21]. The overexpression of RCP is an indicator of

PT

cancer in tissue such as the prostate [22], breast [23] or hepatocellular carcinoma [24]. As an anticancer drug, bioreductive prodrug is converted by reductase to a drug that inhibits

RI

the signal transduction pathway and DNA replication [25]. The characterization of RCP

SC

overexpression is used to track the metabolisms of cancer cells [26]. These features have

NU

been used in RF-aided photodynamic treatment for human breast adenocarcinoma cells therapy [27].

MA

Targeting the adaptive response to hypoxic cancer cells is a potential therapeutic

D

strategy [25]. Mehta et al. transferred the hypoxia-controlled expression genes of a tumor

PT E

suppressor protein P53 and a pro-apoptotic drug (Azurin) into a bacterial carrier [28]. This study proposes a versatile approach to overcome the diffusion barriers and the expression

CE

of cytocidal proteins in brain tumors. Cui et al. demonstrated that the inhibition of SOD2 activation and increased ROS generation results in an increase in apoptosis, inhibition of

AC

proliferation and enhanced sensitivity to chemotherapy for patients with epithelial ovarian cancer [29]. Increasing the ROS dose in tumor cells has also been proposed as a therapeutic target [26]. This study determines the effect of irradiation with visible light on HeLa cells and the effect of in vitro exposure to RF’s. Hypoxia is also a prognostic factor for cervical cancer [30-32] and increases proliferation of HeLa cells [33]. Targeting the hypoxic environment in cancer cells and generating light-triggering ROS generation has 5

ACCEPTED MANUSCRIPT

been used to achieve specific goals. A feasible and efficient PDT with an intrinsic photosensitizer is proposed is this paper. 2. Materials and methods

PT

2.1 Chemicals Dulbecco's modified eagle's medium (DMEM), fetal bovine serum (FBS), riboflavin,

RI

penicillin, streptomycin, FMN, FAD, 2’-thiobarbituric acid (TBA), trypsin-EDTA (0.05 %

SC

Trypsin, 0.53 mM EDTA), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NU

(MTT), trypan blue, dimethyl sulfoxide (DMSO), NaHCO3, KH2PO4, Na2HPO4 , Tris–HCl, glycerol, sodium dodecyl sulfate (SDS), 2-mercaptoethanol and Tween 20 were

MA

purchased from Sigma Chemical-Aldrich (USA). A chemi-luminescent substrate

D

(SuperSignal™ West Pico, 34080) was purchased from Thermo Fisher Scientific Inc.

PT E

(USA). NBT was purchased from Bio Basic Inc. (Canada). Ethyl acetate, methanol and pyridine were obtained from Merck (Germany).

CE

The antibody, anti-alpha tubulin (mouse mAb B512), was purchased from Sigma Chemical-Aldrich. The antibodies, Caspase-8 (mouse mAb 1C12) and Caspase-9 (rabbit

AC

pAB), were purchased from Cell Signaling Technology, Inc. (USA). The antibody, p53 (mouse mAb sc-126), was purchased from Santa Cruz Biotechnology Inc. (USA). All other chemicals were of analytical grade and used without further purification. The TBA (1% in 50 mM NaOH) and phosphate buffered solution (pH 7.8) were prepared before the experiment. The ultrapure water from a Milli-Q system (Millipore, USA) was used as a solvent in this study. 6

ACCEPTED MANUSCRIPT

2.2 Detection of superoxide anions by NBT reduction The NBT reduction method was modified using Beauchamp and Fridovichs’ method [34]. NBT reduction is used as an indicating scavenger and it is reduced by superoxide anions. Each chemical was fresh and was prepared before the experiment. The

PT

concentrations of RF (or FMN, or FAD) were 2.0 μM in 100 mM phosphate buffer with

RI

10 mM methionine and 0.16 mM NBT and the total volume of the reactant was 3 ml. The

SC

reactant was irradiated with blue light at 1.0 mW/cm2 for 10 min. The formazan was then

NU

formatted from the mixture solution after the photolysis reaction and was detected at 560 nm using a UV/Vis spectrometer (Lambda35, Perkin-Elmer).

MA

2.3 Setup of cell culture with the illumination unit

D

The photo-reaction system that is shown in Fig. 2A is composed of a cell incubator, a

PT E

culture plate and a light source with an independent power supply. The photosystem is home-made, with DC 12V 5050 light-emitting diode (LED) chips (vita LED Technologies

CE

Co., Taiwan) that provides a stable and well-defined irradiation source for the experimental period. A power supply (YP30-3-2, Chinatech Co., Taiwan) is used to

AC

maintain the luminous intensity at 520 μW/cm2, which is calibrated by a solar power meter (TM-207, Tenmars Electronics Co., Taiwan). The emission spectra for the lights were measured using an UV/visible/infrared Micro-spectrometer (VLS 1000, Rainbow Light Technology Co., Ltd., Taiwan) and the results are shown in Fig. 2B. The respective wavelengths of the emitted maxima for blue and green lights are 462 and 529 nm. 7

ACCEPTED MANUSCRIPT

2.4 Cell lines and culture HeLa cells were purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were incubated in DMEM that was supplemented with

PT

10% FBS and 1% penicillin-streptomycin. Cells were maintained and transfected at 37°C

2.5 Spectrometric analysis on FMN/FAD photolysis

RI

in a humidified atmosphere of 5% CO2, according to the instructions of the supplier.

SC

FMN/FAD solutions of 120 μM were irradiated using blue, green and red light for 10

NU

min. A fresh solution of FMN/FAD was prepared and stored in the dark for 10 min, as the control sample. The absorbance of illuminated FMN/FAD was measured at 200-600 nm

MA

using a UV/Vis spectrometer (Lambda35, Perkin-Elmer).

D

The light system was situated at the top of the CO2 incubator and the temperature of

PT E

the system was maintained at 37°C, to mimic the cell culture conditions. FMN/FAD solutions of 12.5 μM in a culture medium were irradiated using blue light for 0, 0.5, 1, 1.5

CE

and 2 h. The absorbance of illuminated FMN/FAD was measured at 350-750 nm using a UV-Vis spectrometer (NanoDrop™ 2000/2000c Spectrophotometers, Thermo Scientific).

AC

2.6 Cell viability

A total of 2000 cells/well were seeded onto a 96-well plate for 16 h. The cells were then treated and incubated at 37°C under different conditions, based on a two-variable experimental design. The photosensitizer concentrations were 0, 1.562, 3.125, 6.25 and 12.5 μM, for exposure to irradiation for 0, 2 and 4 h. The medium for the test groups (negative control, 12.5μM FMN/FAD, blue light and 8

ACCEPTED MANUSCRIPT

FMN/FAD that was exposed to irradiation) were removed and incubated for 6 h at 37°C with a culture medium containing 500 μg/ml of MTT. The supernatant was aspirated and 200 μL of DMSO was added to the wells to dissolve any precipitate. The optical density

PT

(OD) values were measured using an ELISA reader (Multiskan spectrum, Thermo Scientific) at a wavelength of 570 nm. The percentage of growth inhibition was

RI

determined using a MTT cell proliferation/viability assay. The relative survival rate was

SC

determined using the following equation:

NU

Cell viability (%) = [OD (treatment groups) / OD (negative control)] × 100%. The MTT assay was performed in triplicate. The mean and standard deviation for

MA

was calculated for each group. A blank control was first subtracted from the OD reading

D

for each group (background).

PT E

2.7 Intracellular ROS detection using FMN/FAD photolysis HeLa cells (5 × 105 cells/ml) were maintained in different conditions (negative

CE

control, 12.5 μM FMN/FAD, blue light and FMN/FAD that was exposed to irradiation) for 2 h and cellular ROS that was produced during the experiment assayed using a Total

AC

ROS detection kit (Enzo Life Sciences, USA, ENZ-51011) [35]. The solutions of HeLa cells were centrifuged for 5 min at 1,500 rpm. The supernatant was discarded and the cellular pellet was suspended with 1 × wash buffer (kit) and centrifuged for 5 min at 1,500 rpm. This cellular pellet was suspended with 0.5 ml ROS detection solution (1:5000) and incubated for 30 min at 37 °C in darkness. The ROS, including hydrogen peroxide, peroxynitrite and hydroxyl radicals, react with the reagent molecules that are locked in 9

ACCEPTED MANUSCRIPT

cell. The molecules are transformed to a fluorescent probe and the intracellular ROS levels were detected using a fluorescence microplate reader (Excitation filter: 485 nm, Emission filter: 538 nm; Fluoroskan Ascent FL, Thermo Labsystems). 2.8 Western blotting

PT

HeLa cells were maintained in four different conditions (negative control, 12.5μM

RI

FMN, blue light and FMN that was exposed to irradiation) and incubated for 2 h at 37°C.

SC

The FMN concentration and light irradiation were 12.5 μM and 2 h, respectively. Trypan

NU

blue was used to measure the number of cells and mixed the cells in a 5-fold sample buffer (60 mM Tris–HCl, pH 6.8, 25% glycerol, 2% SDS, 5% 2-mercaptoethanol). The

MA

cellular solutions were heated at 95°C for 20 min and then cooled and centrifuged for 10

D

min at 13,000 rpm and the supernatant was collected. Cellular extracts were separated on

PT E

10 % SDS-PAGE gels for protein analysis and proteins were transferred to PVDF transfer membranes (GE Healthcare) from gel and then blocked with 5 % nonfat milk in TBST (20

CE

mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.6) for 1 h. A PVDF membrane was incubated with anti-alpha tubulin (1:2000); Caspase-8 (1:1000); Caspase-9 antibody

AC

(1:1000); P53 (DO-1) (1:1000) at 4°C overnight, and a membrane was reacted with horseradish peroxidase-conjugated antibody for 1 h at room temperature. The signals were detected using a chemiluminescent substrate and alpha tubulin was used as a loading control.

10

ACCEPTED MANUSCRIPT

The levels of protein in the western blotting were quantified using ImageJ [36] and normalized with the corresponding tubulin band. The activation ratio for Caspase 9 was calculated using the following equation: [fragments Caspase 9] / [full length Caspase 9].

PT

2.9 Statistical analysis All data is presented as the mean with the standard deviation for at least three

RI

independent experiments. The results were analyzed using an unpaired, two-tailed

SC

Student’s t-test. A value of P <0.05 was designated as the level of significance.

NU

3. Results 3.1 ROS formation and photolysis factors

MA

Riboflavin and its analogues (FMN/FAD) are thermally stable compounds, but they

D

are sensitive to UV and visible light, even if illuminated for a very short period of time. In

PT E

the absence of external reductants, the isoalloxazine ring system undergoes intramolecular photoreduction. The superoxide anions are generated by intermediates during the

CE

photolysis of riboflavin analogues in an aqueous solution. NBT reduction was used as an indicating superoxide anions scavenger. As shown in Fig. 1B, the efficiency of NBT

AC

reduction is lower in riboflavin than in FMN/FAD for the same level of irradiation with blue light.

The configuration of the cell incubator with the light-based photo-reaction system that is used in this study is shown in Fig. 2. LED allows wavelength selection, intensity control and more stability than other light sources [6]. The emission spectra of the blue and green lights were compared and other physical parameters were maintained at 11

ACCEPTED MANUSCRIPT

relatively steady levels. Based on previous studies by the authors, the efficiency of FMN/FAD photolysis was mainly determined by the wavelength of the light used. The variation in the spectra of riboflavin analogues from photo-reactions by blue, green, and

PT

red light irradiation, with a dark control, were measured but the data is not shown here. Blue light illumination gives the most efficient photochemical reaction for FMN/FAD.

RI

The green light was found to be 3% less photochemically efficient than blue light so the

SC

green light was used as the non-activated light source, to attenuate the effects of

NU

illumination in the cell experiments.

3.2 Photolysis of FMN/FAD in cell culture medium

MA

HeLa cells were grown and maintained in a standard cell culture medium known as

D

DMEM, which was supplemented with FBS. The medium was buffered using NaHCO3

PT E

and CO2 and an incubator was used to maintain the pH between 7.2 and 7.4 (the red color of the medium is due to the presence of phenol red, which is a pH indicator).

CE

The photochemical reactions of FMN/FAD in cell culture medium were tested using spectrometry, as shown in Fig. 3. Two signals at 370 and 447 nm are seen for the spectra

AC

of the dark control. These are features of FMN/FAD. It is noticeable that the absorbance spectrum for FMN/FAD at 447 nm is significantly decreased when blue light is used for irradiation. A decrease in absorbance at 500-580 nm is also observed. These spectral changes are caused by the photolysis of FMN/FAD. 3.3 Cell toxicity induced by photolysis of FMN/FAD To quantify the cell viability, the cell number was normalized and counted using a 12

ACCEPTED MANUSCRIPT

trypan blue exclusion assay. Fortunately, a concentration of 12.5 μM photosensitizer that is irradiated with blue light for 2 h could has a cell death rate of 80% in the preliminary studies. The relationship between the threshold values and the dose-response was

PT

determined using two-variable experiments. The results are shown in the Fig. 4. The dose-cytocidal relationship is similar to the

RI

viability of HeLa by photolysis for FMN or FAD. However, the death of HeLa cells

SC

depends significantly on the concentration of the photosensitizer, rather than the

NU

irradiation dosage. The minimal lethal dose for FMN/FAD is 3.125 μM for irradiation with blue light at 0.52 mW/cm2 for 2 h. Increasing the exposure time to 4 h does not

MA

significantly increase the cell death rate for the same levels of photosensitizer and

D

irradiation intensity.

PT E

3.4 Cross validation of cell toxicity for the photosensitizers and the irradiation doses The concentration of the photosensitizer and the degree of irradiation are

CE

independent variables, but the cell toxicity is found to be a function of these two variables. The matrix effects for these two variables were carefully determined using the cytocidal

AC

ability as a dependent variable. The data in Fig. 5A shows that the concentrations of FMN/FAD used in this study represent a non-toxic dosage for HeLa cells that are cultured in darkness (without illumination). The data in Fig. 5B shows that the degree of irradiation with blue or green lights is also non-toxic to the viability of HeLa cells that are cultured without photosensitizers. The irradiation dose is the product of the light intensity (expressed as energy per unit surface area) and the exposure time. The equation for its 13

ACCEPTED MANUSCRIPT

calculation is as follows: Dose (mJ/cm2) = Intensity (mW/cm2) × Time (sec). The results in Fig. 5 show that the changes in cell viability are not significant and no side effects are observed for the use of the photosensitizers and the irradiation doses.

PT

3.5 Intracellular oxidative stress after photolysis of FMN/FAD To determine the intracellular ROS that is generated by FMN/FAD photoproducts,

RI

HeLa cells were kept under four different conditions: a negative control, blue light,

SC

FMN/FAD and FMN/FAD that is exposed to irradiation. The intracellular oxidative stress

NU

levels that were derived from these treatments for 2 h were measured. As shown in Fig. 6, the intracellular ROS levels are not changed significantly by an individual treatment with

MA

FMN, FAD or blue light irradiation. However, the FMN/FAD treatment with blue light

D

irradiation doubles ROS production in HeLa cells.

PT E

3.6 Apoptosis induced by the products of FMN photolysis To understand the cell death that is induced by riboflavin photoproducts, HeLa cells

CE

were maintained under four conditions: a negative control, blue light, FMN and blue light with FMN. P53 levels were significantly increased because of the irradiated

AC

riboflavin-induced cytotoxicity. The P53-mediated apoptosis that is induced by these conditions is shown in Fig. 7(a). P53 is a tumor suppressor protein and responds to cell stress, such as apoptosis, DNA repair and cell cycle arrest [37]. In order to determine the levels of apoptotic proteins that are caused by riboflavin photochemical treatment, the expression levels of the Caspase family, which plays a central role in the apoptotic process, were determined. Caspase-9 is an intrinsic apoptotic protein and Caspase-8 is an 14

ACCEPTED MANUSCRIPT

extrinsic apoptotic protein [38]. Apoptosis is always accompanied by downstream effector caspase-dependent cleavage of caspase-9 [39]. As shown in Fig. 7(b), full length and fragments of Caspase-9 were detected by western-blot analysis for different treatments.

PT

Under the same conditions, full length Caspase-8 was observed and not cleaved, as shown in Fig. 7(c). The activation ratio for Caspase-9 is increased significantly by the photolysis

RI

of FMN in Fig. 7(d). These results show that apoptosis is induced by the riboflavin

SC

photochemical reaction, probably through activation of an intrinsic pathway.

NU

4. Discussion

Most of the PDT studies show that the cellular toxicity depends on lethal

MA

concentrations of the toxic components. To induce biological activity, light must be

D

absorbed by certain molecules (photoacceptors), transforming them to an excited state

PT E

[40]. Blue-light irradiation can release bioactive nitric oxide (NO) from nitrosated proteins, which is known to initiate differentiation in skin cells for treating

CE

hyperproliferative skin conditions [41]. In this system, the ROS are the major toxic components and are induced by the photolysis of the photosensitizer. Therefore, the ROS

AC

formation is triggered by the photosensitizer concentration and the irradiation dose. The spectrometric data shows that blue light illumination increases photolysis of FMN/FAD, but green light has almost no impact. Blue light specifically triggers the photolysis of FMN/FAD and indirectly initiates the production of ROS in a cell culture medium. An increase in oxidative stress in HeLa cells is observed after blue light irradiation with FMN/FAD. ROS are also detected by the NBT assay after photolysis of 15

ACCEPTED MANUSCRIPT

FMN/FAD. The oxidative stress in HeLa cells might be a result of the riboflavin photochemical reaction. Excessive cellular ROS induces cell death. These results demonstrate that photodynamic cytotoxicity is enhanced by highly oxidative stress in

PT

HeLa cells and an increase in intracellular ROS level. However, blue light illumination is not hazardous to normal organisms if there are low levels of riboflavin, FMN and FAD, of

RI

less than 0.5 μM, in human plasma and erythrocytes [42].

SC

Hypoxia is a recognized feature in most cancer cells [43, 44]. Excess riboflavin is

NU

accumulated in cancer cells through RCP [45]. In PDT, irradiating the photosentizer releases ROS for nanoseconds and the action range is about 20 nm [46]. This

MA

photodynamic anticancer strategy decreases the side-effects of traditional anticancer

D

agents in normal cells.

PT E

This study focuses on understanding the intricate interplay between light quality (wavelength), irradiation dose and photosensitizer, which is used to derive the relationship

CE

between dose and response for the photo-induced inactivation of HeLa. Under blue light illumination with an irradiation dose of 3.744 J/cm2, the median lethal doses (LD50) for

AC

FMN and FAD are 6.5 μM and 7.2 μM, respectively. Cell death can occur through apoptosis, autophagy and necrosis. The different death mechanisms have been studied for keratocyte [47] and in HL-60 cells [48] after photolysis with riboflavin or riboflavin derivatives. Depending on the cellular context and the mechanisms that trigger death, these modes often cooperate and are used by cells in a complementary fashion to facilitate cell death [49]. A previous study [19] showed that 16

ACCEPTED MANUSCRIPT

cleavage of supercoiled plasmid DNA structure can be induced by ROS from exogenous riboflavin photolysis. The results indicate that photo-reactions that generate ROS may increase the level of DNA damage in the intracellular matrix.

PT

As shown in Fig. 4, the cytocidal percentage of HeLa cells increases as FMN/FAD concentration and blue light irradiance time increase. High concentrations of FMN/FAD

RI

reduce the survival rate of HeLa after 60 min light irradiation by blue light. However, if

SC

the irradiation time increases, as shown in Fig. 4, in the presence of low-concentration at

NU

30 µM FMN, a 96% de-activation rate is achieved. The effect of light irradiation on HeLa inactivation that is determined in this study is applicable as an easily accessible and safe

MA

photochemical treatment for in vivo requirements.

D

In order to determine the effect of riboflavin photochemical treatment on HeLa cells

PT E

that induce the process of the apoptotic pathway the protein levels in the apoptotic pathway, such as P53, Caspase-8 and Caspase-9, were assayed. The effect of ROS on

CE

cellular cytotoxicity causes the process of the apoptotic pathway. The intrinsic pathway for apoptosis is through mitochondria, cytochrome C, apoptotic protease-activating factor

AC

1 and Caspase 9. The extrinsic pathway for apoptosis is initiated by a death receptor and Caspase 8 [50]. After riboflavin-irradiation, the activated Caspase-9 is increased significantly. The cells commit suicide via an intrinsic apoptotic pathway by the photolysis of FMN. 5. Conclusion UV and blue light can be used to de-activate cancer cells. However, due to the highly 17

ACCEPTED MANUSCRIPT

characteristic photon energy, the use of shorter wavelength light can result in a high degree of damage to cells. Using an appropriate photosensitizer, illumination by blue light can also de-activate the cancer cell under some specific conditions. The energy dose,

PT

optimal illumination and FMN/FAD concentration are crucial. Individual exposure to visible light or RFs does not produce cytotoxicity. However, FMN/FAD photochemical

RI

treatment using blue light (420–500 nm) is shown to make HeLa cells toxic in this study.

SC

PDT using blue light and riboflavin photosensitizers is a simple and safe technique for

NU

hygienic process in practice and will be an effective strategy with biomedical

AC

CE

PT E

D

MA

applications.

18

ACCEPTED MANUSCRIPT References

PT

[1] S. Choudhary, K. Nouri, M.L. Elsaie, Photodynamic therapy in dermatology: a review, Lasers Med Sci, 24 (2009) 971-980. [2] D.S. Masson-Meyers, V.V. Bumah, C.S. Enwemeka, Blue light does not impair wound healing in vitro, J Photochem Photobiol B, 160 (2016) 53-60. [3] E.M. Vinck, B.J. Cagnie, M.J. Cornelissen, H.A. Declercq, D.C. Cambier, Green light emitting diode irradiation enhances fibroblast growth impaired by high glucose level, Photomed Laser Surg, 23 (2005) 167-171. [4] N. Adamskaya, P. Dungel, R. Mittermayr, J. Hartinger, G. Feichtinger, K. Wassermann, H. Redl, M. van Griensven, Light therapy by blue LED improves wound healing in an

SC

RI

excision model in rats, Injury, 42 (2011) 917-921. [5] C. Oplander, S. Hidding, F.B. Werners, M. Born, N. Pallua, C.V. Suschek, Effects of blue light irradiation on human dermal fibroblasts, J Photochem Photobiol B, 103 (2011) 118-125.

MA

NU

[6] Y.K. Seo, J.K. Park, C. Song, S.Y. Kwon, Comparison of light-emitting diode wavelength on activity and migration of rabbit ACL cells, Lasers Med Sci, 29 (2014) 245-255. [7] L. Zhang, Z. Xiong, Z. Li, B. Yao, D. Zhang, Effects of red light emitting diode on apoptosis of HeLa cells and suppression of implanted HeLa cells growth in mice, J Radiat Res, 50 (2009) 109-117.

AC

CE

PT E

D

[8] G.Y. Li, B. Fan, T.H. Ma, Visible light may directly induce nuclear DNA damage triggering the death pathway in RGC-5 cells, Mol Vis, 17 (2011) 3279-3289. [9] P. Fonda-Pascual, O.M. Moreno-Arrones, A. Alegre-Sanchez, D. Saceda-Corralo, D. Buendia-Castano, C. Pindado-Ortega, P. Fernandez-Gonzalez, K. Velazquez-Kennedy, M.I. Calvo-Sanchez, A. Harto-Castano, B. Perez-Garcia, L. Bagazgoitia, S. Vano-Galvan, J. Espada, P. Jaen-Olasolo, In situ production of ROS in the skin by photodynamic therapy as a powerful tool in clinical dermatology, Methods, 109 (2016) 190-202. [10] K. Moghissi, K. Dixon, M. Stringer, J.A. Thorpe, Photofrin PDT for early stage oesophageal cancer: long term results in 40 patients and literature review, Photodiagnosis Photodyn Ther, 6 (2009) 159-166. [11] H.W. Lim, Effects of porphyrins on skin, Ciba Found Symp, 146 (1989) 148-153; discussion 154-148. [12] T. Grune, L.O. Klotz, J. Gieche, M. Rudeck, H. Sies, Protein oxidation and proteolysis by the nonradical oxidants singlet oxygen or peroxynitrite, Free Radic Biol Med, 30 (2001) 1243-1253. [13] S. Weimin, Z. Gen, D. Guifu, Z. Yunxiao, Z. Jin, T. Jingchao, Synthesis and in vitro PDT activity of miscellaneous porphyrins with amino acid and uracil, Bioorg Med Chem, 16 (2008) 5665-5671. [14] Y.J. Tang, H.Y. Liu, J.Y. An, R. Han, Synthesis, characterization and photodynamic 19

ACCEPTED MANUSCRIPT

PT

activity of amino-substituted hypocrellin derivatives, Photochem Photobiol, 74 (2001) 201-205. [15] M.A. Biel, Photodynamic therapy of head and neck cancers, Methods Mol Biol, 635 (2010) 281-293. [16] D.I. Kuijpers, M.R. Thissen, C.A. Thissen, M.H. Neumann, Similar effectiveness of methyl aminolevulinate and 5-aminolevulinate in topical photodynamic therapy for nodular basal cell carcinoma, J Drugs Dermatol, 5 (2006) 642-645. [17] N. Nishiyama, H.R. Stapert, G.D. Zhang, D. Takasu, D.L. Jiang, T. Nagano, T. Aida, K. Kataoka, Light-harvesting ionic dendrimer porphyrins as new photosensitizers for photodynamic therapy, Bioconjug Chem, 14 (2003) 58-66.

SC

RI

[18] R.R.E. Y. Lin, W.O. Landen, Riboflavin. Vitamin Analysis for the Health and Food Sciences, (second ed.)CRC Press, New York, (2008) pp. 329-360 (Chapter 327). [19] J.Y. Liang, J.M. Yuann, C.W. Cheng, H.L. Jian, C.C. Lin, L.Y. Chen, Blue light induced free radicals from riboflavin on E. coli DNA damage, J Photochem Photobiol B, 119 (2013)

MA

NU

60-64. [20] M.A. Kertesz, Riding the sulfur cycle--metabolism of sulfonates and sulfate esters in gram-negative bacteria, FEMS Microbiol Rev, 24 (2000) 135-175. [21] J.H. Ostrander, C.M. McMahon, S. Lem, S.R. Millon, J.Q. Brown, V.L. Seewaldt, N. Ramanujam, Optical redox ratio differentiates breast cancer cell lines based on estrogen

AC

CE

PT E

D

receptor status, Cancer Res, 70 (2010) 4759-4766. [22] T. Johnson, A. Ouhtit, R. Gaur, A. Fernando, P. Schwarzenberger, J. Su, M.F. Ismail, H.I. El-Sayyad, A. Karande, Z.A. Elmageed, P. Rao, M. Raj, Biochemical characterization of riboflavin carrier protein (RCP) in prostate cancer, Front Biosci (Landmark Ed), 14 (2009) 3634-3640. [23] A.A. Karande, L. Sridhar, K.S. Gopinath, P.R. Adiga, Riboflavin carrier protein: a serum and tissue marker for breast carcinoma, Int J Cancer, 95 (2001) 277-281. [24] P.N. Rao, J. Crippin, E. Levine, J. Hunt, S. Baliga, L. Balart, L. Anthony, M. Mulekar, M.H. Raj, Elevation of serum riboflavin carrier protein in hepatocellular carcinoma, Hepatol Res, 35 (2006) 83-87. [25] W.R. Wilson, M.P. Hay, Targeting hypoxia in cancer therapy, Nat Rev Cancer, 11 (2011) 393-410. [26] N. Beztsinna, Y. Tsvetkova, M. Bartneck, T. Lammers, F. Kiessling, I. Bestel, Amphiphilic Phospholipid-Based Riboflavin Derivatives for Tumor Targeting Nanomedicines, Bioconjug Chem, 27 (2016) 2048-2061. [27] E.V. Khaydukov, K.E. Mironova, V.A. Semchishen, A.N. Generalova, A.V. Nechaev, D.A. Khochenkov, E.V. Stepanova, O.I. Lebedev, A.V. Zvyagin, S.M. Deyev, V.Y. Panchenko, Riboflavin photoactivation by upconversion nanoparticles for cancer treatment, Sci Rep, 6 (2016) 35103. 20

ACCEPTED MANUSCRIPT

PT

[28] N. Mehta, J.G. Lyon, K. Patil, N. Mokarram, C. Kim, R.V. Bellamkonda, Bacterial carriers for glioblastoma therapy, Molecular Therapy: Oncolytics, (2017). [29] Y. Cui, K. She, D. Tian, P. Zhang, X. Xin, miR-146a Inhibits Proliferation and Enhances Chemosensitivity in Epithelial Ovarian Cancer via Reduction of SOD2, Oncol Res, 23 (2016) 275-282. [30] M. Hockel, K. Schlenger, B. Aral, M. Mitze, U. Schaffer, P. Vaupel, Association between tumor hypoxia and malignant progression in advanced cancer of the uterine cervix, Cancer Res, 56 (1996) 4509-4515. [31] J. Markowska, J.P. Grabowski, K. Tomaszewska, Z. Kojs, J. Pudelek, M. Skrzypczak, J. Sobotkowski, J. Emerich, A. Olejek, V. Filas, Significance of hypoxia in uterine cervical

SC

RI

cancer. Multicentre study, Eur J Gynaecol Oncol, 28 (2007) 386-388. [32] K. Dellas, M. Bache, S.U. Pigorsch, H. Taubert, M. Kappler, D. Holzapfel, E. Zorn, H.J. Holzhausen, G. Haensgen, Prognostic impact of HIF-1alpha expression in patients with definitive radiotherapy for cervical cancer, Strahlenther Onkol, 184 (2008) 169-174.

MA

NU

[33] B.A. Setty, N. Pillay Smiley, C.M. Pool, Y. Jin, Y. Liu, L.D. Nelin, Hypoxia-induced proliferation of HeLa cells depends on epidermal growth factor receptor-mediated arginase II induction, Physiological reports, 5 (2017). [34] C. Beauchamp, I. Fridovich, Superoxide dismutase: improved assays and an assay applicable to acrylamide gels, Analytical biochemistry, 44 (1971) 276-287.

AC

CE

PT E

D

[35] H. Lu, X. Li, Y. Lu, S. Qiu, Z. Fan, ASCT2 (SLC1A5) is an EGFR-associated protein that can be co-targeted by cetuximab to sensitize cancer cells to ROS-induced apoptosis, Cancer Letters, 381 (2016) 23-30. [36] T.J. Collins, ImageJ for microscopy, Biotechniques, 43 (2007) 25-30. [37] J.P. Kruse, W. Gu, Modes of p53 regulation, Cell, 137 (2009) 609-622. [38] K.H. Khan, M. Blanco-Codesido, L.R. Molife, Cancer therapeutics: Targeting the apoptotic pathway, Critical reviews in oncology/hematology, 90 (2014) 200-219. [39] J.B. Denault, B.P. Eckelman, H. Shin, C. Pop, G.S. Salvesen, Caspase 3 attenuates XIAP (X-linked inhibitor of apoptosis protein)-mediated inhibition of caspase 9, Biochem J, 405 (2007) 11-19. [40] T.I. Karu, L.V. Piatibrat, G.S. Kalenko, Photosensitized inactivation of HeLa tumor cells by phthalocyanines, Radiobiologiia, 29 (1989) 353-358. [41] J. Liebmann, M. Born, V. Kolb-Bachofen, Blue-light irradiation regulates proliferation and differentiation in human skin cells, J Invest Dermatol, 130 (2010) 259-269. [42] S. Hustad, M.C. McKinley, H. McNulty, J. Schneede, J.J. Strain, J.M. Scott, P.M. Ueland, Riboflavin, flavin mononucleotide, and flavin adenine dinucleotide in human plasma and erythrocytes at baseline and after low-dose riboflavin supplementation, Clin Chem, 48 (2002) 1571-1577. [43] K.L. Eales, K.E. Hollinshead, D.A. Tennant, Hypoxia and metabolic adaptation of 21

ACCEPTED MANUSCRIPT

PT

cancer cells, Oncogenesis, 5 (2016) e190. [44] Z. Yun, Q. Lin, Hypoxia and regulation of cancer cell stemness, Adv Exp Med Biol, 772 (2014) 41-53. [45] C.T. Lee, M.K. Boss, M.W. Dewhirst, Imaging tumor hypoxia to advance radiation oncology, Antioxid Redox Signal, 21 (2014) 313-337. [46] J. Moan, K. Berg, The photodegradation of porphyrins in cells can be used to estimate the lifetime of singlet oxygen, Photochem Photobiol, 53 (1991) 549-553. [47] S. Esquenazi, J. He, N. Li, H.E. Bazan, Immunofluorescence of rabbit corneas after collagen cross-linking treatment with riboflavin and ultraviolet A, Cornea, 29 (2010) 412-417.

SC

RI

[48] M.A. Munoz, A. Pacheco, M.I. Becker, E. Silva, R. Ebensperger, A.M. Garcia, A.E. De Ioannes, A.M. Edwards, Different cell death mechanisms are induced by a hydrophobic flavin in human tumor cells after visible light irradiation, J Photochem Photobiol B, 103 (2011) 57-67.

AC

CE

PT E

D

MA

NU

[49] V. Nikoletopoulou, M. Markaki, K. Palikaras, N. Tavernarakis, Crosstalk between apoptosis, necrosis and autophagy, Biochim Biophys Acta, 1833 (2013) 3448-3459. [50] V. Massey, Activation of molecular oxygen by flavins and flavoproteins, J Biol Chem, 269 (1994) 22459-22462.

22

ACCEPTED MANUSCRIPT Figure Captions Fig. 1 (A) The chemical structures of the photosensitizers that are used in this study. The isoalloxazine moiety (in red brackets) in the FMN or FAD molecule is a redox agent and is sensitive to light. (B) NBT reduction to determine the reactive oxygen species by photolysis of riboflavin analogues (2.0 μM) using irradiation with 1.0

PT

mW/cm2 blue LEDs for 10 min. RF is the abbreviation for riboflavin and the data

RI

is presented as mean ± standard deviation, where n = 3.

SC

Fig. 2 (A) Schematic representation of the cell incubator with an LED-based photo-reaction system and (B) the emission spectra of blue and green LEDs that

NU

are used in this study.

Fig. 3 The effects of blue light irradiation on the photolysis reactions. 12.5 μM of (A)

MA

FMN or (B) FAD was solubilized in DMEM and irradiated by blue light at 0.52 mW/cm2 for the indicated time. The absorption spectra were recorded using a

D

UV/Vis spectrometer, after irradiation.

PT E

Fig. 4 The dose-cytocidal relationship for sensitizer photolysis on the viability of HeLa. The cells were treated with different levels of (A) FMN or (B) FAD and irradiated

CE

using blue light for a specified period. The (G) represents the samples that were irradiated by green light. The data is shown as mean ± standard deviation, where n

AC

= 5.

Fig. 5 Matrix effect for photolysis factors on the viability of HeLa. (A) The effect of the dose of the photosensitizer on cell viability in the dark for 2 h. (B) The effect of 0.5 mW/cm2 light irradiation, without photosensitizer, on cell viability. The data is presented as mean ± standard deviation, where n = 5. Fig. 6 Intracellular ROS levels were measured in HeLa cells that underwent different treatments. Irradiation used blue light at 0.52 mW/cm2 for 2 h. The data is presented as mean ± standard deviation, where n = 3. 23

ACCEPTED MANUSCRIPT

Fig. 7 The effect of FMN-irradiation treated with HeLa cell on the apoptosis protein level: (a) P53, (b) Caspase-9 and (c) Caspase-8 level in HeLa cells with 12.5 μM FMN and 0.5 mW/cm2 blue light, dependent exposure and independent, for 2 h. The

PT

expression of protein, as determined by immunoblotting, using extracts obtained from HeLa cells that were maintained in different conditions. Tubulin was used as

RI

the internal control. (d) The quantitative analysis of apoptosis proteins expression

AC

CE

PT E

D

MA

NU

SC

used ImageJ.

24

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

Figure 1A

25

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

Figure 1B

26

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

Figure 2A

27

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

Figure 2B

28

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

Figure 3A

29

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

Figure 3B

30

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

Figure 4A

31

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

Figure 4B

32

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

Figure 5A

33

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

Figure 5B

34

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

Figure 6

35

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

Figure 7A

36

CE

AC

Figure 7B

PT E

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

37

Graphical abstract

AC

CE

PT E

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

38

ACCEPTED MANUSCRIPT

Highlights: Targeting hypoxia is a potential strategy of photodynamic therapy in cancer

2.

Excess riboflavins were accumulated in cancer cell under hypoxia condition

3.

Blue light triggers the cytotoxicity of riboflavins on HeLa cells in vitro

4.

Individual of blue light irradiation and riboflavin analogues is non-hazard on

PT

1.

AC

CE

PT E

D

MA

NU

SC

RI

cells

39