RESEARCH ARTICLE

Nitric oxide regulates the expression of heme
carrier protein-1 via hypoxia inducible factor1α stabilization
Hiromi Kurokawa1, Hiromu Ito2, Masahiko Terasaki3, Daisuke Matano3, Atsushi Taninaka4,
Hidemi Shigekawa4, Hirofumi Matsui1*
1 Faculty of Medicine, University of Tsukuba, Ibaraki, Japan, 2 Graduate School of Medical and Dental
Sciences, Kagoshima University, Kagoshima, Japan, 3 Graduate School of Comprehensive Human
Sciences, University of Tsukuba, Ibaraki, Japan, 4 Faculty of Pure and Applied Sciences, University ofTsukuba, Ibaraki, Japan* hmatsui@md.tsukuba.ac.jp

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OPEN ACCESS
Citation: Kurokawa H, Ito H, Terasaki M, Matano D,
Taninaka A, Shigekawa H, et al. (2019) Nitric oxide
regulates the expression of heme carrier protein-1
via hypoxia inducible factor-1α stabilization. PLoS
ONE 14(9): e0222074. https://doi.org/10.1371/
journal.pone.0222074
Editor: Surinder K. Batra, University of Nebraska
Medical Center, UNITED STATES
Received: December 8, 2018
Accepted: August 21, 2019
Published: September 12, 2019
Copyright: © 2019 Kurokawa et al. This is an open
access article distributed under the terms of theCreative Commons Attribution License, which
permits unrestricted use, distribution, andreproduction in any medium, provided the original
author and source are credited.

Abstract
Photodynamic therapy (PDT) is a cancer therapy that capitalizes on cancer-specific porphyrin accumulation. We have investigated this phenomenon to propose the following threeconclusions: 1) the mechanism underlying this phenomenon is closely related to both nitricoxide (NO) and heme carrier protein-1 (HCP-1), 2) NO inactivates ferrochelatase, and thus,
the intracellular porphyrin levels in the cells are increased by the administration of an NO
donor after 5-aminolevulinic acid treatment, 3) HCP-1 transports not only heme but also
other porphyrins. Since NO stabilizes hypoxia-inducible factor (HIF)-1α, resulting in the
upregulation of heme biosynthesis, HCP-1 expression can be increased by HIF-1α stabilization. In this study, we determined whether NO regulates HCP-1 expression by stabilizing
HIF-1α expression. For this purpose, rat gastric cancer cell line RGK36 was treated with Larginine or N6-(1-iminoethyl)-L-lysine (L-NIL). L-arginine treatment increased the intracellular NO concentration, and both HCP-1 and HIF-1α expression, while L-NIL treatment
decreased them. Cytotoxicity of PDT was enhanced by L-arginine, following intracellular
hemato-porphyrin dihydrochloride (HpD) accumulation. Both Cytotoxicity of PDT and HpD
accumulation were decreased by L-NIL. The HCP-1 and HIF-1α expression, intracellular
HpD accumulation and PDT cytotoxicity were decreased by 2-methoxyestradiol, which is a
HIF-1α inhibitor. Moreover, these phenomena were not increased by a combination of bothL-arginine and 2-Me. Thus, HCP-1 can be a downstream target of HIF-1α. These effects
were also induced in the human gastric cancer cell line MKN45. Taken together, we conclude that HCP-1 expression is regulated by NO via HIF-1α stabilization.

Data Availability Statement: All relevant data arewithin the manuscript and its Supporting
Information files.
Funding: The authors received no specific funding
for this work.
Competing interests: The authors have declaredthat no competing interests exist.

Introduction
Porphyrins are substrates used for photodynamic therapy (PDT), a cancer treatment using the
combination of a photosensitizer and laser irradiation [1]. The advantages of this therapy
include reduced treatment time, minimal normal tissue toxicity, no long-term systemic

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Nitric oxide regulates the expression of heme carrier protein-1 via hypoxia inducible factor-1α stabilization

toxicity and lack of drug resistance mechanisms [2, 3]. PDT is used the capability of combination with chemotherapy make it more commonly used in many fields of medicine [3]. Developed countries are evolving into super-aged socieites, i.e. socieites with a larger porportion of
population in the 65 and older age group such as Japan, German, and Italy [4]. PDT is desired
as a less invasive treatment because it has no requirement for anesthesia and a lower risk of
bleeding than with surgery [2]. In these conditions, the usefulness of PDT has been re-recognized [5]. The fundamentals of this therapy involve the cancer-specific accumulation of porphyrins [6]. Lee et al. reported that porphyrin accumulation and PDT effect were different
between normal and cancer skin sell line [7] Jedrych et al. reported that cell viability after PDT
treatment in normal mouse embryo was higher than that in human lung carcinoma [8]. However, the mechanism of this accumulation remains unknown, and many clinicians have disputed the ability of cancer to accumulate porphyrins specifically. Thus, PDT has not become amajor form of cancer therapy.
Heme carrier protein-1 (HCP-1) is a proton-coupled folate transporter (SLC46A1) thattransports heme into cells [9, 10]. We have reported that cancer-specific accumulation of porphyrins is involved in this heme transport because the structure of porphyrin is virtually thesame as that of heme [11, 12]. We confirmed that HCP-1 expression is cancer-specific and that
hematoporphyrin fluorescence is higher in HCP-1-overexpressing HeLa cells than in wildtype cells after treatment with hematoporphyrin [11]. Cell viability after PDT is significantly
lower in HCP-1-overexpressing HeLa cells compared to control cells. In contrast, when HCP1 was silenced with siRNA, the intracellular fluorescence intensity of hematoporphyrin was
decreased. From these results, we concluded that HCP-1 is a cancer-specific porphyrin transporter. Moreover, HCP-1 expression in normal cells was lower than that in cancer cells, thus
we concluded that the reason of cancer specific porphyrin accumulation is HCP-1 expression[12].
Nitric oxide (NO), which is a free radical gas, acts as a second messenger and has both physiological and pathological effects. The biological roles of NO and ROS are equally important[13]. Many studies have reported that NO has functions in cancer tissue related angiogenesis[14], vascular permeability [15], DNA damage [16], and apoptosis [17]. Moreover, NO regulates intracellular porphyrin accumulation in cancer cells. Indeed, we had previously reported
that the fluorescence intensity of the porphyrins in the MKN45 cells increased with the administration of an NO donor after 5-aminolevulinic acid (ALA) treatment [18]. Moreover, transfection of the inducible nitric oxide synthase (iNOS) gene into the normal mouse kidney cellline HEK293T decreased the ferrochelatase activity and increased the PDT effect [19]. However, it is unclear the relationship between NO and HCP-1.
Hypoxia inducible factor (HIF)-1α is stabilized by NO and upregulates vascular endothelial
growth factor (VEGF) and heme oxygenase 1, which control the vascular system [20, 21]. Both
VEGF and heme oxygenase 1 stimulate angiogenesis and improve the impaired post-ischemic
blood flow [22]. The HIF-1α is also related to heme biosynthesis as it upregulates the erythropoietin receptor and ferrochelatase [23]. Hence, it may also regulate HCP-1 expression to
obtain heme.
In this study, we determined whether the increase in NO production upregulated theexpression of HCP-1 to enhance the PDT effect by stabilizing the expression of HIF-1α.

Materials and methods
Materials
L-arginine, 2-methoxyestradiol (2-Me), deoxycholic acid, hydrochloric acid, and NaOH
(Wako Pure Chemicals, Osaka, Japan); N6-(1-iminoethyl)-L-lysine (L-NIL) (Cayman,

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Nitric oxide regulates the expression of heme carrier protein-1 via hypoxia inducible factor-1α stabilization

Michigan, USA); cell counting kit-8 (Dojindo, Tokyo, Japan); DAF-2DA (Daiichi Pure Chemicals, Tokyo, Japan); hematoporphyrin dihydrochloride (HpD), NaCl, sodium dodecyl sulfate(SDS), Trizma base, Triton X-100, and Tween 20 (Sigma-Aldrich Japan K.K., Tokyo, Japan);
NuPAGE Novex 12% Bis-Tris gels (Life Technologies Japan, Tokyo, Japan); polyvinylidene
difluoride (PVDF) Blocking Reagent for Can Get Signal, Can Get Signal Immunoreaction
Enhancer Solution 1, and Can Get Signal Immunoreaction Enhancer Solution 2 (Toyobo,
Osaka, Japan); HIF-1α, β-actin, and horseradish peroxidase (HRP)-linked anti-rabbit IgG
antibodies (Cell Signaling Technology Japan, K.K., Tokyo, Japan); HCP-1 antibody (Abcam,
Cambridge, U.K.); iNOS antibody (BiossAntibodies, Massachusetts, U.S.A.) and Lumina Fortewestern HRP substrate (EMD Millipore, Billerica, MA, USA) were purchased and used without further purification or modification.

Cell culture
The rat cancerous gastric mucosa cell line RGK36 (S1 File), which is established in our laboratory [24], was cultured in DMEM/F12 without L-glutamine (Sigma-Aldrich Japan K.K.). The
human gastric cancer cell line MKN45 was cultured in RPMI1640 (Wako Pure Chemicals).
The culture media were supplemented with 10% inactivated fetal bovine serum (FBS) (Biowest
LLC, Kansas City, MO) and 1% penicillin/streptomycin (Life Technologies Japan Ltd.). Cells
were cultured in 5% CO2 cell culture incubator at 37˚C.

NO detection with DAF-2DA
The RGK36 cells were cultured in 96-well plates at 3×103 cells/well and incubated overnight.
Then, the cells were incubated with 10 mM L-arginine or 3 μM L-NIL, a potent and selective
inhibitor of iNOS, for 24 h. The supernatant was aspirated, and the cells were incubated with
10 μM DAF-2DA for 1 h. The fluorescence intensity of DAF-2DA was measured using a Synergy H1 (BioTek Instruments Inc., Winooski, VT, USA). The excitation and emission wavelengths were 480 and 520 nm, respectively.

Western blotting analyses
The RGK36 cells were incubated in medium containing 10 mM L-arginine, 3 μM L-NIL, or
1 μM 2-Me for 24 h. After incubation, the whole-cell lysates were prepared by rinsing thecells three times with PBS; NuPAGE LDS sample buffer was added on ice and then heated at
70˚C for 10 min. For SDS-polyacrylamide gel electrophoresis, the cell lysates were added
into the wells of the NuPAGE Novex 4–12% Bis-Tris gels. The gels were electrophoresed at
100 V for 70 min, and the proteins were transferred onto a PVDF membrane by electrophoresis at 1.2 mA/cm2 for 60 min. The membrane was blocked for 60 min with PVDF blocking
reagent for the Can Get Signal kit, and probed with primary and secondary antibodies.
Anti-rabbit HCP-1 (ab25134), HIF-1α (#14179), or iNOS (bs-2072R) antibodies (1:1000)
were added to Can Get Signal Immunoreaction Enhancer Solution 1. The membrane was
exposed to the solution overnight. After the primary antibody solution was aspirated, themembrane was washed three times with 1x Tris-buffered saline containing Tween 20. The
secondary HRP-linked anti-rabbit IgG antibody (#7074) was added to the Can Get Signal
Immunoreaction Enhancer Solution 2. The solution was exposed to the membrane for 60
min. Chemiluminescence was used to develop the membrane. Images of the blots were captured by ImageQuant LAS4000 (GE Health Care Japan, Tokyo, Japan). β-Actin (#4967) wasused as the control for protein loading.

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Nitric oxide regulates the expression of heme carrier protein-1 via hypoxia inducible factor-1α stabilization

Cellular uptake of HpD
The RGK36 and MKN45 cells were incubated overnight in 12-well plates at 5×104 cells/well,each. Cells were then incubated with 10 mM L-arginine, 3 μM L-NIL, or 1 μM 2-Me for 24 h.
After incubation, 20 μM HpD was added to the cells and incubated for 6 h. The cells wererinsed with PBS and lysed in 100 μL RIPA buffer containing 25 mM Tris-HCl (pH 7.6), 150
mM NaCl, 1% (v/v) Triton X-100, 0.1% (w/v) SDS, and 0.2% (w/v) deoxycholic acid. Cell
homogenates were transferred to a 96-well plate. The fluorescence intensity of HpD was measured using a Varioskan microplate reader. The excitation and emission wavelengths were 415
and 625 nm, respectively.

Cell viability assay after PDT
The RGK36 and MKN45 cells were incubated overnight in 96-well plates at 1×103 cells/well,each, and then incubated with 10 mM L-arginine, 3 μM L-NIL, or 1 μM 2-Me for 24 h. Afterthe treatments, the cells were incubated with 20 μM HpD for 24 h and rinsed with PBS. Freshmedium without phenol red was added. Cells were irradiated by excimer dye laser light (630
nm, 0.5 J/cm2) for PDT by using PDT EDL-1 (Hamamatsu Photonics K.K., Hamamatsu,
Japan) and incubated for 24 h. The medium was replaced with fresh medium containing 10%
Cell Counting Kit-8, and further incubated. The absorbance at 450 nm was measured on a
DTX880 multi-mode microplate reader.

Statistical analysis
Data are expressed as the mean ± SD and were assessed by analysis of variance (ANOVA).
Individual groups were compared by Student’s t-test or Tukey’s test, with p<0.05 considered
statistically significant.

Results
Intracellular NO concentration was regulated by L-arginine and/ or L-NIL
The intracellular NO concentration was determined using DAF-2DA. L-arginine was used as aprecursor for NO production, whereas L-NIL was used as iNOS inhibitor. The fluorescenceintensity of the dye significantly increased in 10 mM L-arginine-treated cells, while it significantly decreased in 3 μM L-NIL-treated cells compared to control cells (Fig 1A and 1B). Moreover, L-NIL treatment induced the downregulation of iNOS expression (Fig 1C and 1D).
These results indicate that the intracellular NO concentration was increased by L-arginine anddecreased by L-NIL.

HIF-1α and HCP-1 protein expression increased by L-arginine treatment
and decreased by L-NIL or 2-Me
The RGK36 cells were treated with L-arginine, L-NIL, or 1 μM 2-Me for 24 h and then analyzed for the expression of HIF-1α and HCP-1 by western blotting. Both HIF-1α and HCP-1
expression in cells treated with L-arginine was significantly higher than that in cells treated
without L-arginine (Fig 2A–2C). The expression of both the proteins in L-NIL- treated cells,
where the production of NO was inhibited, was significantly decreased (Fig 2D–2F). 2-Me is
known as a HIF-1α inhibitor. The HIF-1α expression in 2-Me-treated cells was significantly
decreased (Fig 2G and 2H). Moreover, the HCP-1 expression also decreased by 2-Me treatment (Fig 2G and 2I).

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Nitric oxide regulates the expression of heme carrier protein-1 via hypoxia inducible factor-1α stabilization

Fig 1. NO concentration and iNOS expression in RGK36 cells. Intracellular NO concentration increased with Larginine (A) while decreased with L-NIL (B). Data are expressed as the mean ± SD (n = 4). � p < 0.05, �� p < 0.01. iNOS
expression decreased with L-NIL (C, D). Data are expressed as the mean ± SD (n = 3). � p < 0.05.
https://doi.org/10.1371/journal.pone.0222074.g001

The fluorescence intensity of HpD increased in L-arginine treatment cells
whereas decreased in L-NIL or 2-Me
The RGK36 cells and the MKN45 cells were treated with L-arginine, L-NIL, or 2-Me for 24 h,and then the cells were treated with 20 μM HpD for 6 h. The intracellular fluorescence intensity of HpD was measured with a microplate reader. In both RGK36 and MKN45 cells, theintracellular fluorescence intensity of HpD was higher after the L-arginine treatment, while
treatment with L-NIL and 2-Me significantly decreased the intensity compared to the control
cells (Fig 3A–3F).

Cytotoxicity of PDT was enhanced by L-arginine while suppressed by
L-NIL or 2-Me
The RGK36 cells and the MKN45 cells were treated with L-arginine, L-NIL, or 2-Me for 24 h;then the cells were treated with 20 μM HpD for 24 h. The cells were irradiated with 630 nm
laser. Cellular injuries after the PDT effect was evaluated with the WST assay. In case of
RGK36, the cytotoxicity of PDT was accelerated in the L-arginine-treated cells, compared to
the non-treated cells (Fig 4A). However, the PDT effect for cells treated with L-NIL or 2-Me
was suppressed (Fig 4B and 4C). The same phenomenon was also induced in MKN 45 for theeffect of L-arginine, L-NIL or 2-Me treatments.

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Nitric oxide regulates the expression of heme carrier protein-1 via hypoxia inducible factor-1α stabilization

Fig 2. Expression of HCP-1 and HIF-1α in RGK36 cells. HCP-1, HIF-1α, and β-actin were detected by westernblot. Both HCP-1 and HIF-1α expression was increased with L-arginine (A-C), while it decreased with L-NIL (D-F) or
2-Me (G-I). Data are expressed as the mean ± SD (n = 3). � p < 0.05, �� p < 0.01.
https://doi.org/10.1371/journal.pone.0222074.g002

Porphyrin accumulation was inhibited by 2-Me even though the cells weretreated with L-arginine
Cells were treated with L-arginine, 2-Me, or both for 24 h and then analyzed for HIF-1α and
HCP-1 expression by western blotting. L-arginine treatment increased the HCP-1 expression.
However, the combination of L-arginine and 2-Me could not increase the expression. Similarly, L-arginine treatment increased HIF-1α expression, while the combination treatment
could not increase (Fig 5A–5C). The intracellular fluorescence intensity of HpD in L-argininetreated cells became higher in comparison to the control cells. However, the fluorescenceintensity in the combination treated-cells became lower in comparison to the control cells (Fig
5D).

Discussion
In this study, we demonstrated for the first time that NO increased the intracellular porphyrinaccumulation by upregulation of HCP-1 expression via HIF-1α stabilization. It is reported that
HCP-1 transports not only heme but also porphyrins [11, 12]. NO regulates the blood flow to
obtain O2; thus, we hypothesized that NO also regulates HCP-1 expression to increase theintracellular heme concentration. NO is produced through the conversion of L-arginine to L-

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Nitric oxide regulates the expression of heme carrier protein-1 via hypoxia inducible factor-1α stabilization

Fig 3. Intracellular HpD accumulation in RGK36 and MKN45 cells. Cells were treated with 10 mM L-arginine (A, D), 3 μM L-NIL (B, E), or 1 μM 2-Me (C,F) for 24 h. Then, the cells were exposed to culture medium containing 20 μM HpD for 6 h. Data are expressed as the mean ± SD (n = 4). � p < 0.05, �� p < 0.01.
https://doi.org/10.1371/journal.pone.0222074.g003

citrulline by the enzyme nitric oxide synthase (NOS) [25]. In this study, we used L-arginine toenhance the NO production, whereas L-NIL was used to inhibit the NO production. At a concentration of 0.4 to 3.3 μM, L-NIL works as a selective iNOS inhibitor [26]. Therefore, theintracellular NO concentration increased by L-arginine and decreased by L-NIL (Fig 1A–1D).
We concluded that HCP-1 expression could be regulated by L-arginine or L-NIL. L-arginine,
the NO producer, upregulated HCP-1 expression, while L-NIL, the NO inhibitor, downregulated HCP-1 expression (Fig 2A, 2B, 2D and 2E). Taken together, we concluded that regulationof intracellular NO influenced HCP-1 expression.
We also investigated the relationship between NO and HIF-1α. In normoxia, HIF-1α ishydroxylated by prolyl hydroxylase (PHD) and binds to the von Hippel Lindau protein. Under
this condition, HIF-1α is ubiquitinated and degraded by the 26S proteasome [27]. Asparagine803 of HIF-1α is also hydroxylated by an oxygen-dependent asparagine hydroxylase, referred
to as factor-inhibiting HIF-1 (FIH). The recruitment of CBP/p300 is prevented by the hydroxylated asparagine residue, thereby inhibiting transactivation by the stabilized HIF-1α. In hypoxia, HIF-1α is stabilized as a result of inhibition of prolyl hydroxylase. Even in normoxia,
HIF-1α can be stabilized by NO, which inhibits the PHD activity through the nitrosylation ofcysteine residues [28]. The FIH also appears to be inhibited by NO, and the mechanism islikely similar to that of PHD. HIF-1α expression was increased by L-arginine and decreased by
L-NIL, similarly to HCP-1 expression (Fig 2A, 2C, 2D and 2F). Regulation of NO concentration by L-arginine or L-NIL affected the expression of HIF-1α, which when stable can

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Nitric oxide regulates the expression of heme carrier protein-1 via hypoxia inducible factor-1α stabilization

Fig 4. The PDT effect was examined after exposure to HpD in RGK36 and MKN45 cells. Cells were treated with 10 mM L-arginine (A, D), 3 μM
L-NIL (B, E), or 1 μM 2-Me (C, F) for 24 h. Cell viability was evaluated after HpD exposure and laser irradiation. Data are expressed as the mean ± SD
(n = 4). � p < 0.05, �� p < 0.01.
https://doi.org/10.1371/journal.pone.0222074.g004

accelerate HCP-1 expression. 2-Me is a derivative of estradiol and downregulates HIF-1α at
the post-transcriptional level [29, 30]. After the 2-Me treatment, both HIF-1α and HCP-1
expression decreased in RGK36 cells (Fig 2G–2I). These results also indicate that HIF-1α regulates HCP-1 expression.
We investigated the relationship between porphyrin accumulation and the PDT effect. The
HCP-1 expression was increased in the L-arginine-treated cells, while L-NIL or 2-Me treatment decreased the expression. We proposed that the intracellular porphyrin accumulation
can be controlled with L-arginine, L-NIL, and 2-Me. Fluorescence intensity of porphyrins was
significantly higher in L-arginine-treated cells than control cells (Fig 3A). However, the fluorescence intensity decreased in L-NIL or 2-Me-treated cells (Fig 3B and 3C). In other words,
intracellular porphyrin accumulation increased by L-arginine and decreased by L-NIL or
2-Me. Moreover, cytotoxicity of PDT increased in L-arginine treatment while it decreased in
L-NIL or 2-Me (Fig 4A–4C). Since inhibition of HIF-1α with 2-Me decreased porphyrin accumulation and PDT effect, we proposed that HCP-1 expression was regulated by NO via HIF1α stabilization. HCP-1 expression, upon treatment with the combination of L-arginine and2-Me, was lower than that upon treatment with L-arginine alone. The HIF-1α expression was
decreased by L-arginine and 2-Me, similar to the HCP-1 expression. However, there was no
significant difference between each group (Fig 5A–5C). On the other hand, intracellular porphyrin accumulation was inhibited by L-arginine and 2-Me (Fig 5D). Based on these results,

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Nitric oxide regulates the expression of heme carrier protein-1 via hypoxia inducible factor-1α stabilization

Fig 5. The activity of L-arginine was inhibited by 2-Me. Expression of HCP-1 and HIF-1α in RGK36 cells was inhibited by 1 μM 2-Me (A-B). Data are expressed as themean ± SD (n = 3). Intracellular HpD accumulation was also suppressed by 1 μM 2-Me (D). Data are expressed as the mean ± SD (n = 4). � p < 0.05 and �� p < 0.01 vscontrol. ##p < 0.01 vs L-arginine. $p < 0.05 vs 2-Me.
https://doi.org/10.1371/journal.pone.0222074.g005

we considered that the HCP-1 expression was regulated by NO via HIF-1α stabilization andassumed that the strong PDT effect by L- arginine may attenuate in combination with 2-Me.
We previously reported that iNOS overexpression decreased the ferrochelatase activity of
HEK293T cells [19]. Moreover, PDT-induced cell death in iNOS-expressing HEK293T cells
was greater than that in mock transfected cells. Since NO participates in porphyrin accumulation via ferrochelatase inactivation, we assumed that NO would be involved in the HCP-1
expression. In this study, we showed that HCP-1 expression increased after treatment of
RGK36 cells with L-arginine, whereas it decreased after treatment with L-NIL or 2-Me. HIF1α upregulate the ferrochelatase gene expression [31]. However 2-Me treatment decreased
intracellular porphyrin accumulation from in this study. We showed the level and activity offerrochelatase were decreased by the expression of iNOS, however the degree of the decreaseof the enzyme activity was somewhat different from that of the enzyme level [18]. Thus weregarded that upregulation of ferrochelates does not necessarily directly increase porphyrinaccumulation. These results suggest that the accumulation of HpD in response to NO involved
not only ferrochelatase inhibition but also HCP-1 over-expression. This phenomenon also
accelerated the PDT effect.

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Nitric oxide regulates the expression of heme carrier protein-1 via hypoxia inducible factor-1α stabilization

Fig 6. The mechanism of regulation of HCP-1 expression. HCP-1 expression increase by L-arginine, while itdecrease by L-NIL or 2-Me.
https://doi.org/10.1371/journal.pone.0222074.g006

In this study, we used both rat and human gastric cancer cells, RGK36 and MKN45. In
MKN45 cells, the fluorescence intensity of porphyrin increased by L-arginine and decreasedby L-NIL or 2-Me. Thus, the intracellular porphyrin accumulation was regulated by thesetreatments (Fig 3D–3F). The cytotoxicity of PDT was also enhanced by L-arginine anddecreased by L-NIL or 2-Me in the RGK36 cells (Fig 4D–4F). Thereby PDT cytotoxicity can beenhanced by increasing the NO production in clinical cases.
In conclusion, NO stabilizes the HIF-1α to accelerate HCP-1 expression, resulting inenhanced intracellular porphyrin accumulation that improves the PDT effect (Fig 6).

Supporting information
S1 File. Cell lines.
(DOCX)

Author Contributions
Conceptualization: Hiromi Kurokawa, Hiromu Ito, Hirofumi Matsui.
Data curation: Hiromi Kurokawa, Masahiko Terasaki, Daisuke Matano, Atsushi Taninaka.
Methodology: Hiromi Kurokawa.
Supervision: Hidemi Shigekawa.
Writing – original draft: Hiromi Kurokawa.
Writing – review & editing: Hiromu Ito, Masahiko Terasaki, Hirofumi Matsui.

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