Yamada-oka, Suita-shi, Osaka 565-0871, Japan, Tel: 81 6 ......2017/02/21 · mitochondrial oxidative phosphorylation (OXPHOS) and were relatively resistant to various drugs including

1

Calcium-dependent enhancement by extracellular acidity of the cytotoxicity of

mitochondrial inhibitors against melanoma

Fumihito Noguchi1,2,7, Shigeki Inui1, Clare Fedele2, Mark Shackleton2,3,4,5,6, Satoshi Itami1,6

1Department of Regenerative Dermatology, Graduate School of Medicine, Osaka University, 2-2,

Yamada-oka, Suita-shi, Osaka 565-0871, Japan, Tel: 81 6 6879 3032; Fax: 81 6 6879 3039,

E-mail: [email protected]

2Cancer Development and Treatment Laboratory and 3Division of Cancer Medicine, Peter

MacCallum Cancer Centre, 305 Grattan Street, Melbourne VIC 3000, Australia, Tel: 61 3 8559

5000; Fax: 61 3 8559 7379

4Sir Peter MacCallum Department of Oncology and 5Department of Pathology, University of

Melbourne, Parkville VIC 3052, Australia

6Equal contribution

7Correspondance: [email protected]; Tel: 61 3 8559 5000; Fax: 61 3 8559

7379

on August 26, 2021. © 2017 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 21, 2017; DOI: 10.1158/1535-7163.MCT-15-0235

http://mct.aacrjournals.org/

2

Keywords

acidic pH; OXPHOS; calcium; alkalizer therapy; ROS

Running title

OXPHOS-inhibition, calcium and pHe in melanoma

Abbreviations

pHe, extracellular pH; OXPHOS, oxidative phosphorylation; TIS, therapy-induced senescence;

ROS, reactive oxygen species; PGC1alpha, peroxisome proliferator-activated receptor gamma

coactivator 1-alpha; JARID1B, Jumonji/ARID Domain-Containing Protein 1B; OCR, oxygen

consumption rate; FCCP, trifluoromethoxy carbonyl cyanide phenylhydrazine; AIF,

apoptosis-inducing factor; ATA, aurintricarboxylic Acid; MFI, median fluorescence intensity

Disclosure of potential conflict of interest

There are no conflicts of interest.

Financial Support

F. Noguchi was supported by Uehara Memorial Foundation. M. Shackleton was supported by




3

Pfizer Australia, the Victorian Cancer Agency, the Australian National Health and Medical

Research Council (NHMRC) and veski.

Abstract

Extracellular acidity is a hallmark of cancers and is independent of hypoxia. Since acidity

potentiates malignant phenotypes, therapeutic strategies that enhance the targeting of oncogenic

mechanisms in an acidic microenvironment should be effective. We report here that drugs which

abrogate mitochondrial respiration show enhanced cytotoxicity against melanoma cells in a

normoxic but acidic extracellular pH, independent from P53 mutations, BRAF (V600E)

mutations and/or resistance against BRAF inhibitors. Conversely, the cytotoxicity against

melanoma cells of mitochondrial inhibitors is impaired by a neutral or alkaline extracellular pH

and in vivo systemic alkalinization with NaHCO3 enhanced subcutaneous tumor growth and

lung metastasis of B16F10 cells in mice treated with the mitochondrial inhibitor phenformin.

Intracellular calcium (Ca2+) was significantly increased in melanoma cells treated with

mitochondrial inhibitors at an acidic extracellular pH and an intracellular Ca2+ chelator,

BAPTA/AM, inhibited cytoplasmic Ca2+ as well as melanoma cell death. Surprisingly, ROS

scavengers synergized with increased apoptosis in cells treated with mitochondrial inhibitors,

suggesting that ROS contributes to cell survival in this context. Notably, the cytotoxic

enhancement of mitochondrial inhibitors by acidity was distinct from PGC1alpha-driven




4

mitochondrial addiction, from therapy-induced senescence and from slow,

JARID1B-high-associated cell cycling, all of which have been shown to promote vulnerability

to mitochondrial inhibition. These data indicate that extracellular pH profoundly modulates the

cytotoxicity of mitochondrial inhibitors against cancer cells.

Introduction

Melanoma, the most aggressive form of skin cancer, typically responds poorly to conventional

cancer therapies, including chemotherapy and radiation therapy, after it progresses and

subsequently metastasizes (1). Although new therapies such as BRAF inhibitors (e.g.

vemurafenib) and immunotherapies prolong the survival of many patients, relapses occur in

most cases (2,3). Accordingly, identifying new therapies and ascertaining ways in which to

combine and potentiate existing therapies are high research priorities in this disease.

Recently, a subpopulation was identified of proliferative but slow-cycling melanoma cells that

express the histone3 K4 demethylase JARID1B (4,5). These cells showed enhanced

mitochondrial oxidative phosphorylation (OXPHOS) and were relatively resistant to various

drugs including vemurafenib, thus playing a critical role in melanoma cell survival. Furthermore,

it was reported that the mitochondrial master regulator gene PGC1alpha is regulated in

melanoma cells through microphthalmia-associated transcription factor (MITF), the central




5

melanocytic lineage regulator, resulting in mitochondrial addiction, tolerance to vemurafenib (6)

and oxidative stress (7). As PGC1alpha-driven mitochondria-addicted melanoma cells (6) as

well as JARID1B-high slow-cycling cells (5,8) are sensitive to drugs that inhibit OXPHOS

(OXPHOSi), OXPHOSi have therapeutic potential against melanoma (9-11).

Interest in OXPHOSi is also driven by links between mitochondrial activity and

therapy-induced senescence (TIS) (12). Therapies such as radiation and chemotherapy can drive

either apoptosis or senescence in cancer cells, with senescent cells appearing in tumor remnants

that remain after therapy. These cells are important as they may possess a slow-cycling,

drug-resistant phenotype that serves as a reservoir for recurrent disease (12). Importantly,

senescent cancer cells also exhibit activation of OXPHOS and sensitivity to OXPHOSi (12,13).

The extracellular pH (pHe) is acidic in most cancers due to hypoxia, insufficient perfusion and

aerobic glycolysis (the so-called “Warburg effect”), although hypoxia and extracellular acidity

often lack spatial correlation (14). The pHe is reportedly 6.2–6.9 in tumor tissues, while the pHe

is 7.3-7.4 in normal tissues (15,16). Extracellular acidity, independently from hypoxia,

facilitates malignant behaviors of cancer cells including migration, invasion,

epithelial-mesenchymal transition, metastasis, lymphangiogenesis, autophagy, mutagenesis,

chemoresistance, immune evasion and dedifferentiation (17-29). Indeed, high intratumoral

acidity is linked to poor patient prognosis (30) and alkalizer therapy that manipulates the acidic




6

pHe in a tumor towards neutral was reported to inhibit tumor growth and metastasis in several

types of cancers (20,28,31,32). These observations highlight the importance of considering pHe

in cancer treatment. In this study, we assessed the effects of an acidic pHe on melanoma cells

and on modulating the anti-melanoma effects of OXPHOSi.

Materials and Methods

Cell lines and cell culture

B16F10 (ATCC Number CRL-6475) mouse melanoma cells were purchased from the American

Type Culture Collection (ATCC) (Manassas, VA, USA) in April 2009. Human melanoma cell

lines MEWO (ATCC Number HTB-65, in January 2012), SK-MEL-28 (ATCC Number

HTB-72, in October 2014) and A375 (ATCC Number CRL-1619, in November 2014) were

purchased from ATCC. Normal human dermal fibroblasts were derived from the scalp skin of a

14-year-old female patient in Jun 2003. All cell lines were stored in liquid nitrogen and

authenticated for viability a month before use. Cells were grown in Dulbecco's Modified Eagle

Medium (DMEM) (MediaTech, Herndon, VA, USA) supplemented with 4.5 g/L glucose, 2 mM

L-glutamine, 5% penicillin-streptomycin, 2.5 μg/ml amphotericin B and 10% fetal bovine serum

(FBS) at pH 7.4. All cell lines were cultured in a humidified 5% CO2 atmosphere at 37°C. The

pH of media was adjusted with hydrochloric acid, sodium hydroxide or bicarbonate to 6.2, 6.7




7

and 8.0 for extracellular pH-manipulation in vitro. The medium was changed every day to

normal/acidic medium with/without oligomycin (1404-19-9, Wako Pure Chemical Industries,

Ltd, Osaka, Japan), phenformin (P7045, Sigma-Aldrich, St. Louis, MO, USA), rotenone (R8875,

Sigma-Aldrich), 2-thenoyltrifluoroacetone (TTFA) (T27006, Sigma-Aldrich), cisplatin

(15663-27-1, Wako Pure Chemical Industries, Ltd.), vemurafenib (PLX4032, RG7204) (S1267,

Selleckchem.com, Houston, TX, USA), Z-VAD-FMK (sc-3067, Santa Cruz Biotechnology,

Santa Cruz, CA, USA), Z-DEVD-FMK (FMK004, R&D Systems, Minneapolis, MN, USA),

Trolox (sc-200810, Santa Cruz Biotechnology), sodium pyruvate (sc-208397, Santa Cruz

Biotechnology), (aurintricarboxylic Acid (ATA) (sc-3525, Santa Cruz Biotechnology), E64

(sc-201276, Santa Cruz Biotechnology), antipain (sc-291907, Santa Cruz Biotechnology) and/or

BAPTA/AM (1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid

tetrakis(acetoxymethyl ester) (sc-202488, Santa Cruz Biotechnology). To derive

vemurafenib-resistant sublines, SK-MEL-28 and A375 cells were seeded at low density, treated

with vemurafenib at 10 µM for 6 weeks and clonal colonies isolated and maintained. All in vitro

experiments were performed with cells at 70-80% confluency in normoxic conditions.

Cell viability assay

The viability of drug-treated cells at each pH was calculated using a Cell Counting Kit-8

(CCK8) (Dojindo Laboratories, Kumamoto, Japan) by the following formula: relative viability




8

= value of drug-treated cells / value of cells with no treatment.

Western Blotting

Immunoblotting was performed as previously described (33). Antibodies used were: mouse

monoclonal anti-ATP synthase beta IgG (1:10000) (612518, BD Biosciences), rabbit polyclonal

anti-PGC-1 IgG (1:200) (sc-13067, Santa Cruz Biotechnology), rabbit polyclonal anti-cleaved

caspase-3 p11 IgG (1:200) (sc-22171-R, Santa Cruz Biotechnology), rabbit polyclonal

anti-cleaved caspase-6 (Asp162) IgG (1:1000) (#9761, Cell Signaling) and anti-β-actin

(1:20000) (A5441, Sigma-Aldrich).

Detection of mitochondrial membrane potential

For detection of mitochondrial membrane potential, MitoRed (PromoKine, PK-CA707-70055,

Heidelberg, Germany) was used following the manufacturer’s protocol.

Oxygen Consumption

The oxygen consumption rate was measured using a Seahorse Bioscience XF96 Extracellular

Flux Analyzer. Briefly, cells (2 × 104 /well) were cultured in XF96 tissue culture plates at pH 7.4

or pH 6.7 for 72 h and then media was changed into unbuffered XF Base Medium 1 h before the

measurement. Maximal oxygen consumption rate was measured under a mitochondrial

uncoupler trifluoromethoxy carbonyl cyanide phenylhydrazine (FCCP) (0.25 µM for

SK-MEL-28 and 0.1 µM for MEWO, respectively). The number of cells was normalized using




9

CCK8.

Immunocytochemistry

Cells were fixed in 4% paraformaldehyde for 10 min at room temperature, and then

permeabilized with 0.5% Triton X-100 for 5 min. The expression of P53 and P21 was detected

by incubating cells with rabbit polyclonal anti-p53 IgG (1:100) (sc-6243, Santa Cruz) or rabbit

polyclonal anti-p21 IgG (1:100) (sc-756, Santa Cruz) for 1 h at room temperature followed by

incubation with Alexa fluor 488 conjugated goat anti-rabbit IgG. DAPI was added to label cell

nuclei. The expression of senescence associated β-galactosidase was detected using Cellular

Senescence Kit (GX0003, OZ Biosciences) following the manufacturer’s protocol. Cells were

observed using an All-in-one Fluorescence Microscope BZ-9000 (Keyence, Osaka, Japan).

Immunohistochemistry

For TUNEL staining, a TACS2 TdT in situ Apoptosis Detection Kit (Trevigen, Gaithersburg,

MD, 20877) and a ChemMate ENVISION/ HRP kit (Dako, Glostrup, Denmark) were used. In

brief, 3 μm thick tumor sections on the slides were covered with Proteinase K Solution for 15

min and were then immersed in Quenching Solution for 5 min. After washing with PBS, the

slides were immersed in 1×TdT Labeling Buffer for 5 min and were then covered with Labeling

Reaction Mix for 60 min at 37°C, followed by covering with Stop Buffer for 5 min. The slides

were then washed 2 times in deionized H2O for 5 min each, followed by treatment with alkaline




10

phosphatase-conjugated streptavidin (K1018, Dako) for 15 min. Reaction products were

visualized using the Fuchsin+ Substrate-Chromogen System (K0625, Dako). 1% Methyl Green

was used as a counterstain.

Ki67 staining was performed as previously described (34). Briefly, sections were autoclaved

and stained with rabbit polyclonal anti-Ki67 IgG (1:500; Leica Microsystems, Buffalo Grove,

IL), followed by incubation and visualization with a ChemMate ENVISION/ HRP kit (Dako).

FACS analysis

Cellular ROS was labeled with CellROX Deep Red Reagent (Ex/Em = 644/665 nm) (C10422,

Life Technologies, Frederick, MD, USA) and measured using a BD FACS Canto II (BD

Biosciences). Briefly, cells were treated with the reagent (5 μM) with/without the drug(s) at pH

7.4 or 6.7 and were then incubated at 37°C for 30 min and collected. Menadione (100 μM,

M5625, Sigma-Aldrich) was used for a positive control. After distinguishing dead cells, median

fluorescence intensities (MFIs) of cells were determined (Ex/Em = 650/660 nm). MFIs of cells

in each condition were normalized to cells at pH 7.4 with no treatment.

For apoptosis assays, cells were cultured with/without the drug(s) at pH 7.4 or 6.7 for the

indicated periods and then apoptotic cells were detected using FITC Annexin V (640905,

BioLegend, San Diego, CA, USA) according to the manufacturer’s protocol using a BD FACS

Canto II.




11

For intracellular calcium measurement, cells were cultured with/without the drug(s) at pH 7.4 or

6.7 for the indicated periods and then loaded with Fluo3/AM (sc-202612, Santa Cruz

Biotechnology) (5 μM) in PBS at 37°C for 1 h. After 2 washes, cells were cultured in PBS at

37°C for 30 min (de-esterification) and then analyzed on a BD FACS Canto II with excitation

wavelength of 488 mm. For cell sorting, cells stained by Fluo3/AM were analyzed on a BD

FACS Aria and sorted by Fluo3/AM fluorescence.

For cell cycle analysis, 1×105 cells were fixed with ice cold 70% EtOH for 4 h and then

suspended in DNA staining solution including 2.5 μg/ml propidium iodide and 0.5 mg/ml

RNase A in PBS. They were then analyzed using a BD FACS Canto II.

For analysis of JARID1B expression after incubation with/without the drug(s) at pH 7.4 or 6.7

for the indicated periods, 1×106 cells were fixed in 70% ethanol for 10 min on ice and then

washed with 2% bovine serum albumin (BSA) in PBS. Cells were then incubated with rabbit

polyclonal anti-JARID1B antibody (1:1000) (22260002, Novus Biologicals, Littleton, CO,

USA) for 30 min on ice. After washing, cells were incubated with a secondary fluorescein

isothiocyanate conjugated anti-rabbit antibody for 20 min on ice and analyzed using a BD FACS

Canto II.

Measurement of lactate

Measurement of lactate was performed using a Lactate Colorimetric Assay Kit II (#K627,




12

BioVision, Milpitas, CA, USA). Briefly, cells were seeded (5×103/well) in 96-well plates and

were incubated at pH 7.4 or 6.7 for the indicated periods. The culture medium of each well was

then recovered and mixed with Reaction Mix. After incubation for 30 min at room temperature,

OD 450 nm was measured using a microplate reader. Values were normalized by the number of

viable cells evaluated using the CCK8 kit. The values of cells in acidic conditions were

normalized to cells at pH 7.4.

Measurement of ATP

Cellular ATP was measured using “Cellno” ATP ASSAY reagent (CA50, Toyo Inc, Tokyo,

Japan). Briefly, cells were seeded (5 × 103/well) in 96-well plates and incubated at pH 7.4 or 6.7

with/without the drug(s) for the indicated periods. The reagent was added to each well and the

plates were stirred for 1 min. Luminescence was measured using a CentroXS3 LB960 plate

reader (Berthold Technologies, Bad Wildbad, Germany). Values were normalized as described

in the section of Lactate measurement.

Animals

All animal studies were performed according to protocols approved by the Institutional Animal

Care and Use Committee at Osaka University. C57BL/6J mice (7-week-old, female) were

purchased from Nippon Charles River (Tokyo, Japan). Mice were maintained under light/dark

(12 h/12 h) cycles with free access to standard chow and water. For mice treated with oral




13

administration of NaHCO3, water was replaced with 200 mM NaHCO3.

Subcutaneous tumor growth

For studies of subcutaneous tumor growth, mice (n = 6 per group) were inoculated with 2.5×105

B16F10 cells in their flanks. Mice were randomly divided into four groups: a) no treatment, b)

oral administration of 200 mM NaHCO3 ad libitum, c) daily intraperitoneal injection (i.p.) of

100 mg/kg phenformin, and d) both NaHCO3 and phenformin. Treatments started from the day

of inoculation and were continued for the duration of the experiment. Tumor diameters were

measured at the indicated time points. Tumor volumes were estimated by V = (a2×b)/2, where ‘a’

is the short axis and ‘b’ is the long axis of the tumor.

Metastasis assay

Mice were injected intravenously with 5 × 104 B16F10 cells in 0.1 ml PBS via the tail vein and

were treated daily with phenformin (100 mg/kg, i.p.) and/or NaHCO3 (200 mM, oral

administration ad libitum) from the day of injection, which continued for the duration of the

experiment (n = 5 per group). After 21 days, mice were sacrificed and their lungs resected and

photographed. The numbers of metastases on the surfaces of the lungs were counted

macroscopically.

Statistics

Data are expressed as means ± SE. To compare two mean values, an unpaired Welch’s t test was




14

used. For comparisons of multiple mean values, a Tukey-Kramer test following a single-factor

ANOVA was used. For subcutaneous tumor growth analysis, an unpaired Welch’s t test was

used. For metastasis analysis, a nonparametric Mann-Whitney U test was used. Differences of

*P <0.05 were considered statistically significant.

Results

Extracellular pH modulates the cytotoxicity of OXPHOSi on melanoma cells in vitro

To elucidate whether cytotoxicity of mitochondrial inhibitors on melanoma cells is affected by

pHe in vitro, we examined human melanoma cells at pH 7.4 or 6.7 treated with oligomycin

(0.01 μg/ml), an inhibitor of mitochondrial ATP synthase, or phenformin (1 mM), an

anti-diabetic drug from the biguanide class known to inhibit mitochondrial complex I. Increased

numbers of apoptotic cells were detected when A375, SK-MEL-28 or MEWO cells were

cultured with oligomycin or phenformin at an acidic pHe, while there were only modest

numbers of apoptotic cells when cells were cultured without the drugs at an acidic pHe or with

the drugs at pHe 7.4 (Fig. 1A to 1D, S1A to S1C). A similar enhancement of apoptosis was

observed in cells treated with the mitochondrial complex II inhibitor TTFA (0.1 mM) at an

acidic pHe (Fig. S1D, S1E). An increase in apoptosis was also seen in A375 cells cultured with

oligomycin at pH 6.2 rather than at pH 6.7, and even in A375 cells cultured at pH 6.2 without




15

the drug (Fig. S1F, S1G). The number of apoptotic A375 cells cultured at pH 8.0 was

comparable to that at pH 7.4 (Fig. S1F, S1G). These data indicate that extracellular acidity

enhances the cytotoxic effect of OXPHOSi on melanoma cells in vitro. Interestingly,

patient-derived normal human dermal fibroblasts were also sensitive to phenformin (1 mM) at

an acidic pHe (Fig. S1J to S1L), indicating that this phenomenon occurs in normal as well as

malignant cells.

Since cancer cells in an acidic microenvironment generally acquire multidrug-resistance (15,28),

we tested the cytotoxicity of cisplatin (20 μM) on melanoma cells at varying pHe. In contrast to

the effects of OXPHOSi, A375 and SK-MEL-28 cells displayed less apoptosis following

cisplatin exposure at pH 6.7 compared to pH 7.4, although comparable levels of apoptosis were

detected across pHs after treatment of MEWO cells with cisplatin (Fig. 1E to 1G, S1H, S1I).

Since a low pHe augments the cytotoxicity of weak acids and as cisplatin is a weak acid (35),

the cytotoxicity enhancement of OXPHOSi by acidity must not merely be because cells are

damaged by acidity. Rather, OXPHOS-inhibition by these drugs may specifically cooperate with

an acidic pHe to augment cytotoxicity.

Systemic buffering attenuates the anti-cancer effect of phenformin on B16F10 cells in vivo

Given that an acidic pHe enhanced cytotoxicity of OXPHOSi (including phenformin) against




16

melanoma cells in vitro (Fig. 1), we hypothesized that systemic alkali buffering with NaHCO3

would reduce anti-tumor effects of phenformin in vivo, which have been shown for cancers such

as melanoma (8,36). Since it was reported that extracellular acidity promotes immune evasion

of cancer cells (26) and that host immune responses critically affect tumor development (37),

we utilized B16F10 cells and a syngeneic allograft model using C57BL/6 mice to test this.

In preparatory in vitro work, phenformin decreased the viability of B16F10 cells at pHe 7.4 as

well as pHe 6.7 (Fig. S2A, S2B). However, far more apoptosis was induced in B16F10 cells at

the acidic pHe compared to the normal pHe (Fig. 2A, 2B). Consistent with previous studies

(8,36), in vivo tumor growth was significantly inhibited in mice treated with phenformin (148

mm3 ± 28 SE) compared to untreated mice (1504 mm3 ± 106 SE) (Fig. 2C, 2D). As

hypothesized, a significant increase in tumor growth was observed in phenformin +

NaHCO3-treated mice compared to phenformin only treated mice (303 mm3 ± 39 SE on day 16

post-injection) (Fig. 2C, 2D). Correspondingly, immunohistochemical analysis suggested that

apoptotic cells were decreased while proliferating cells were increased in tumors from

phenformin + NaHCO3-treated mice (Fig. 2E, 2F). Interestingly, tumor growth was also

inhibited in mice treated with oral bicarbonate (452 mm3 ± 81 SE on day 16 post-injection)

compared to mice without treatment (Fig. 2C, 2D), regardless of phenformin administration.




17

We also evaluated experimental metastasis. Twenty-one days after injection, the number of lung

atmetastases was reduced in mice treated with daily phenformin, compared to mice with no

treatment (Fig. 2G, 2H). Noticeably, metastasis was increased in mice with daily phenformin +

NaHCO3 treatment compared to phenformin only treated mice (Fig. 2G, 2H). Collectively, these

data are consistent with the possibility that OXPHOS inhibition preferentially targets melanoma

cells in the acidic in vivo tumor microenvironment that otherwise facilitates malignant

progression.

OXPHOS-inhibition causes the activation of caspase-dependent apoptosis at an acidic pHe

We next interrogated mechanisms of action of OXPHOSi targeting in an acidic

microenvironment. Cleavage of caspase-3 as well as caspase-6 was increased in A375 cells after

treatment with oligomycin at an acidic pHe (Fig. 3A, 3B). Although the substrate-specific

caspase inhibitor Z-DEVD-FMK failed to suppress apoptosis in A375 cells after treatment with

oligomycin at pH 6.7 (Fig. S3A), the pan-caspase inhibitor Z-VAD-FMK suppressed apoptosis

in these cells (Fig. 3C, 3D). In contrast, treatment with the apoptosis-inducing factor (AIF)

specific inhibitor ATA (38) and E64 and antipain both of which inhibit cathepsin-B (39) failed to

suppress apoptosis in A375 cells treated with oligomycin at pH 6.7 (Figs. S3B and S3C). These

data indicate caspase-dependent apoptosis in the cytotoxicity of OXPHOSi elicited by acidity.




18

Inhibition of both caspase-3 and caspase-6 by Z-VAD-FMK, but not caspase-3-specific

inhibition by Z-DEVD-FMK, may be necessary to suppress apoptosis in A375 cells treated with

oligomycin at an acidic pHe.

Intracellular Ca2+ level is increased in melanoma cells after treatment with OXPHOSi at

an acidic pHe

Since oxidative stress increases the concentration of intracellular Ca2+ (40), leading to

intracellular Ca2+ overload and apoptosis (41), we next tested if intracellular Ca2+ is altered in

melanoma cells after treatment with OXPHOSi at an acidic pHe. When A375 cells were treated

with the OXPHOSi at pH 6.7, large fractions of surviving cells showed high cytoplasmic Ca2+.

This was not seen following OXPHOSi at pH 7.4 or at pH 6.7 without OXPHOSi (Fig. 4A, 4B).

Similar results were obtained with SK-MEL-28 cells and MEWO cells (Fig. S4A to S4D). The

viable Ca2+-high cell population induced by OXPHOSi at pH 6.7 was impaired, as A375 cells

sorted from the propidium iodide-excluding Ca2+-high gate showed markedly impaired in vitro

proliferation (Fig. 4C to 4E). The importance of intracellular Ca2+ in this context was further

revealed by treatment with BAPTA/AM, an intracellular calcium chelator, which decreased

Ca2+-high cells as well as death in A375 cells treated with oligomycin at pH 6.7 (Fig. 4F to 4I).




19

These results indicate that intracellular Ca2+ overload contributes substantially to the enhanced

cytotoxicity of OXPHOSi in an acidic pH.

ROS scavenging synergizes with OXPHOSi against melanoma cells in an acidic pHe

Cytotoxicity of OXPHOSi against cancer cells may be due to deleterious effects of ROS

production (12). Since extracellular acidosis enhances ROS in cancer cells (42,43), we next

studied whether increased ROS production in melanoma cells at acidic pHe modulates the

cytotoxic effects of OXPHOSi. As expected, ROS production in A375 and MEWO cells was

significantly increased after OXPHOS-inhibition at pH 6.7 compared to pH 7.4 and Trolox and

Sodium pyruvate, both ROS scavengers, suppressed ROS production at pH 6.7 (Fig. 5A, S5A).

However, when ROS production was suppressed after treatment with OXPHOSi at pH 6.7,

more rather than less apoptosis was unexpectedly seen in both A375 (Fig. 5B to 5E) and

MEWO cells (Fig. S5B to S5E). As a moderate increase of ROS correlates with aggressive

cancer cell phenotypes (44), these data raise the possibility that OXPHOSi in an acidic pH

increases ROS to a threshold that provides a level of adaptive cytoprotection that may be

exploited therapeutically by concurrent ROS scavengers (44).

Enhanced cytotoxicity in melanoma cells of OXPHOSi in acid pHe is independent of




20

PGC1alpha

PGC1alpha is up-regulated in some melanoma cells, resulting in mitochondrial addiction,

tolerance to oxidative stress (7) and vulnerability against OXPHOSi (6). As expected (7), we

found that MEWO cells robustly expressed PGC1alpha (Fig. 6A). Interestingly, expression of

PGC1alpha and ATP synthase were increased by acidification in MEWO cells, indicating the

activation of OXPHOS (Fig. 6A). Consistent with this, mitochondrial membrane potential was

activated (Fig. 6B) and oxygen consumption rate was increased (Fig. 6D) in acid pHe,

suggesting that acidification potentiates mitochondrial activity in MEWO cells. However,

although SK-MEL-28 and A375 cells were also sensitive to OXPHOSi at acidic pHe, they

expressed very low levels of PGC1alpha (Fig. 6A) and had much lower mitochondrial

membrane potentials and oxygen consumption rates after acidification (Fig. 6B, 6C). This

argues against a consistent PGC1alpha-dependency of the cytotoxicity elicited by OXPHOSi in

acidic pHe (6,7).

Melanoma cells at an acidic pHe acquire senescent phenotypes that do not mediate the

cytotoxicity of OXPHOSi

To examine further potential mechanisms of OXPHOSi cytotoxicity in acidic pHe and as cells

undergoing senescence have increased sensitivity to OXPHOSi (12,13), we tested whether




21

melanoma cells that survive an acidic milieu are senescent (12). Some cell lines showed some

but not all features of senescence in acidic pHe. Consistent with previous data (17), A375 cells

were slow-cycling and had increased G1 phase cells (Fig. S6A to S6C). SK-MEL-28 cells, but

not A375 or MEWO cells, showed increased expression of senescence-associated

β-galactosidase (Fig. S6G) (45). In metabolism studies, both lactate and ATP production were

increased by acidification in A375, SK-MEL-28 and MEWO cells (Fig. S6H, S6I), consistent

with a hyperactive hybrid metabolism, characterized by increased glycolysis as well as

mitochondrial respiration, that is linked to the senescent state (12,13). However, A375 and

SK-MEL-28 cells did not display increased mitochondrial membrane potential after

acidification (Fig. 6B, 6C), which would be expected of senescent cells (12). As P53 and P21

upregulation characterize the senescent state, we also tested expression of these proteins in

acidic pHe in A375, SK-MEL-28 and MEWO cells, with contrasting results. Although P53 and

P21 were increased after acidification in A375 cells (which harbor wild-type p53 (46)) (Fig.

S6D), neither protein was increased in MEWO cells (which carry p53 mutations (47)) (Fig.

S6F). In SK-MEL-28 cells, which are also p53 mutant (48), P53 but not P21 was increased after

acidification (Fig. S6E). Thus, P53 and P21 expression was not consistently linked to sensitivity

to OXPHOSi. Altogether, these data indicate that although some melanoma cells at an acidic




22

pHe show some features of senescence, this phenotype is not unequivocally and universally

linked to the cytotoxicity conferred by OXPHOSi in an acidic pHe.

The JARID1B-high population is not increased in melanoma cells by an acidic pHe

Treatment with vemurafenib has been shown to enrich JARID1B-high slow-cycling melanoma

cells, which are sensitive to OXPHOSi (5). Since melanoma cells exhibited enhanced sensitivity

to OXPHOSi after acidification, we also tested whether JARID1B-high cells are increased by

acidification. SK-MEL-28-R and A375-R cells, resistant to vemurafenib, were generated from

SK-MEL-28 and A375 cells, which harbor a homozygous BRAF (V600E) mutation (Fig. 6E,

S6J). Apoptotic SK-MEL-28-R and in A375-R cells were significantly increased by treatment

with oligomycin at pH 6.7, compared to oligomycin at pH 7.4 or untreated cells at pH 6.7 (Fig.

6F, 6G, S6K, S6L). However, this was not associated with an increase in JARID1B-high cells at

pH 6.7 (Fig. 6H), although JARID1B-high SK-MEL-28-R cells were decreased after treatment

with oligomycin or rotenone at pH 6.7 (Fig. 6I). JARID1B-high A375-R cells were also

decreased after treatment with oligomycin or rotenone at acidic pHe (Fig. S6M, 6I). Thus, the

augmented sensitivity of melanoma cells to OXPHOSi at an acidic pHe cannot be attributed to

increased JARID1B-high cells in an acidic milieu. Despite this, the vulnerability of

JARID1B-high cells to OXPHOSi is preserved after acidification.




23

Discussion

We demonstrate that the cytotoxicity of OXPHOSi against melanoma is strongly mediated by

pHe. Since not only melanoma cells but also normal human dermal fibroblasts are relatively

protected against phenformin by a neutral pH (Fig. S1J to S1L), the neutral pHe in normal

tissues (15,16) should ameliorate potential side-effects in patients of OXPHOSi, while

preserving cytotoxic effects that will be more specific for acidic tumor microenvironments. On

the other hand, when tissue physiological oxygenation is disturbed in patients with pathological

conditions like severe diabetes, coronary heart disease or stroke (49), acidosis can occur in

involved organs and tissues. It is thus possible that OXPHOSi could exert untoward toxicities

on non-malignant tissues that are unusually acidotic as a result of non-malignant pathological

conditions.

As an acidic pHe promotes aggressive phenotypes in cancers (15), manipulation of intratumoral

pHe has been considered as a potential cancer therapy (32,50,51). Systemic buffering by oral

administration of NaHCO3 increased intratumoral pHe and inhibited local invasion and tumor

growth in breast and in colon cancer cells (20). Moreover, treatment with oral bicarbonate

increased the average tumor pHe from 7.0 to 7.4 and inhibited the spontaneous metastasis of

metastatic breast and prostate cancer cells in immunodeficient mice (31). Our data show that the




24

acidic microenvironment that promotes tumor progression also renders cancer cells susceptible

to therapies that target mitochondrial metabolism, and that systemic alkalinization ameliorates

this susceptibility.

It is notable that the vulnerability of melanoma cells to OXPHOSi in acidic pHe was

independent of P53 mutation status, BRAF (V600E) mutation status and resistance against

vemurafenib. This indicates potential application of OXPHOSi across a range of clinical

contexts, such as a second-line treatment against drug-resistant melanomas or combined with

first-line therapies (12) to reduce primary resistance. Regardless, identifying biomarkers of

acidic pHe, perhaps by tumor sampling (52,53), will be important to identify subgroups of

patients likely to respond to OXPHOSi, as recent clinical trials in unselected patients failed to

show improved survival in cancer patients treated with OXPHOSi (54,55). Clearly, concurrent

use of alkalizer therapy and OXPHOSi should not be applied to melanoma patients.

In examining mechanisms of action of the increased susceptibility of melanoma cells to

OXPHOSi in acidic pHe, we unexpectedly found that this vulnerability is not consistently

dependent on PGC1alpha (6,7), induction of senescence (12) or JARID1B expression (5,8).

Rather, we found that caspase- and pHe dependent apoptotic effects of OXPHOSi were closely

linked to increased intracellular calcium levels. The role of calcium in regulating cell

metabolism in some pathophysiological states is well understood. For example, myocardial cells




25

undergo intracellular Ca2+ overload upon myocardial ischemia-reperfusion injury (56). In this

context, a decrease in extracellular as well as intracellular pH during ischemia leads to Na+/H+

exchange and Na+ overload in myocardial cells. Subsequently, Ca2+ uptake occurs through

reverse-mode Na+/Ca2+ exchange, resulting in intracellular Ca2+ overload. Concurrently

impaired ATP production in mitochondria inhibits Ca2+ extrusion by plasmalemmal Ca2+-ATPase,

compounding the intracellular Ca2+ increase, which results in mitochondrial-driven myocardial

cell apoptosis (40,41,56,57).

We speculate that the acidification of melanoma cells mimics this by inducing compensatory ion

fluxes that increase intracellular calcium to synergize with reduced ATP production conferred by

OXPHOSi. On the other hand, in A375 and SK-MEL-28 cells mitochondrial activities are

already decreased at acidic pHe without OXPHOSi (Fig. 6B, 6C). Therefore, it is possible that

inhibition of OXPHOS at acidic pH might induce intracellular Ca2+ increase and activation of

cell death signaling pathway in a manner independent from a decrease in ATP production

(58,59), although further studies are needed to address this mechanism. Our demonstration of

intracellular Ca2+ overload and activation of apoptosis in acidic melanoma cells after treatment

with OXPHOSi (Fig. 3, Fig. 4, Fig. S3, Fig. S4) suggests that the combination of OXPHOSi and

drugs that increase cytoplasmic Ca2+ level via manipulating Ca2+ channels, pumps or storage

(57) might have synergistic cytotoxicity against melanoma cells. This highlights the importance




26

of understanding complex interactions between cell metabolism, tumoral pHe and ionic

homeostasis in developing novel therapy combinations that target specific susceptibilities in

cancer cells.

Acknowledgements

We appreciate Professor Seiji Takashima and Dr. Yasunori Shintani, Department of Medical

Biochemistry, Osaka University Graduate School of Medicine, for provision of convenience in

measuring oxygen consumption rates. We also greatly appreciate Ms. Ayako Sato, Ms. Yuko

Yoshikawa and Ms. Miyuki Nakamura for their technical assistance.

References

1. Miller AJ, Mihm MC, Jr. Melanoma. N Engl J Med 2006;355(1):51-65.

2. Sosman JA, Kim KB, Schuchter L, Gonzalez R, Pavlick AC, Weber JS, et al. Survival

in BRAF V600-mutant advanced melanoma treated with vemurafenib. N Engl J

Med 2012;366(8):707-14.

3. Zaretsky JM, Garcia-Diaz A, Shin DS, Escuin-Ordinas H, Hugo W, Hu-Lieskovan S,

et al. Mutations Associated with Acquired Resistance to PD-1 Blockade in Melanoma.

N Engl J Med 2016;375(9):819-29 doi 10.1056/NEJMoa1604958.

4. Roesch A, Fukunaga-Kalabis M, Schmidt EC, Zabierowski SE, Brafford PA, Vultur A,

et al. A temporarily distinct subpopulation of slow-cycling melanoma cells is

required for continuous tumor growth. Cell 2010;141(4):583-94.

5. Roesch A, Vultur A, Bogeski I, Wang H, Zimmermann KM, Speicher D, et al.

Overcoming intrinsic multidrug resistance in melanoma by blocking the

mitochondrial respiratory chain of slow-cycling JARID1B(high) cells. Cancer Cell

2013;23(6):811-25.

6. Haq R, Shoag J, Andreu-Perez P, Yokoyama S, Edelman H, Rowe GC, et al.

Oncogenic BRAF regulates oxidative metabolism via PGC1alpha and MITF. Cancer




27

Cell 2013;23(3):302-15.

7. Vazquez F, Lim JH, Chim H, Bhalla K, Girnun G, Pierce K, et al. PGC1alpha

expression defines a subset of human melanoma tumors with increased

mitochondrial capacity and resistance to oxidative stress. Cancer Cell

2013;23(3):287-301.

8. Yuan P, Ito K, Perez-Lorenzo R, Del Guzzo C, Lee JH, Shen CH, et al. Phenformin

enhances the therapeutic benefit of BRAF(V600E) inhibition in melanoma. Proc

Natl Acad Sci U S A 2013;110(45):18226-31.

9. Miskimins WK, Ahn HJ, Kim JY, Ryu S, Jung YS, Choi JY. Synergistic anti-cancer

effect of phenformin and oxamate. PLoS One 2014;9(1):e85576.

10. Corazao-Rozas P, Guerreschi P, Andre F, Gabert PE, Lancel S, Dekiouk S, et al.

Mitochondrial oxidative phosphorylation controls cancer cell's life and death

decisions upon exposure to MAPK inhibitors. Oncotarget 2016;7(26):39473-85 doi

10.18632/oncotarget.7790.

11. Petrachi T, Romagnani A, Albini A, Longo C, Argenziano G, Grisendi G, et al.

Therapeutic potential of the metabolic modulator phenformin in targeting the stem

cell compartment in melanoma. Oncotarget 2016 doi 10.18632/oncotarget.14321.

12. Wolf DA. Is Reliance on Mitochondrial Respiration a "Chink in the Armor" of

Therapy-Resistant Cancer? Cancer Cell 2014;26(6):788-95.

13. Dorr JR, Yu Y, Milanovic M, Beuster G, Zasada C, Dabritz JH, et al. Synthetic lethal

metabolic targeting of cellular senescence in cancer therapy. Nature

2013;501(7467):421-5.

14. Taddei ML, Giannoni E, Comito G, Chiarugi P. Microenvironment and tumor cell

plasticity: an easy way out. Cancer Lett 2013;341(1):80-96.

15. Calorini L, Peppicelli S, Bianchini F. Extracellular acidity as favouring factor of

tumor progression and metastatic dissemination. Exp Oncol 2012;34(2):79-84.

16. Gerweck LE, Seetharaman K. Cellular pH gradient in tumor versus normal tissue:

potential exploitation for the treatment of cancer. Cancer research

1996;56(6):1194-8.

17. Marino ML, Pellegrini P, Di Lernia G, Djavaheri-Mergny M, Brnjic S, Zhang X, et al.

Autophagy is a protective mechanism for human melanoma cells under acidic stress.

J Biol Chem 2012;287(36):30664-76.

18. Hjelmeland AB, Wu Q, Heddleston JM, Choudhary GS, MacSwords J, Lathia JD, et

al. Acidic stress promotes a glioma stem cell phenotype. Cell Death Differ

2011;18(5):829-40.

19. Riemann A, Schneider B, Gundel D, Stock C, Thews O, Gekle M. Acidic priming




28

enhances metastatic potential of cancer cells. Pflugers Arch 2014;466(11):2127-38.

20. Estrella V, Chen T, Lloyd M, Wojtkowiak J, Cornnell HH, Ibrahim-Hashim A, et al.

Acidity generated by the tumor microenvironment drives local invasion. Cancer

research 2013;73(5):1524-35.

21. Moellering RE, Black KC, Krishnamurty C, Baggett BK, Stafford P, Rain M, et al.

Acid treatment of melanoma cells selects for invasive phenotypes. Clin Exp

Metastasis 2008;25(4):411-25.

22. Rofstad EK, Mathiesen B, Kindem K, Galappathi K. Acidic extracellular pH

promotes experimental metastasis of human melanoma cells in athymic nude mice.

Cancer research 2006;66(13):6699-707.

23. Peppicelli S, Bianchini F, Contena C, Tombaccini D, Calorini L. Acidic pH via

NF-kappaB favours VEGF-C expression in human melanoma cells. Clin Exp

Metastasis 2013;30(8):957-67.

24. Peppicelli S, Bianchini F, Torre E, Calorini L. Contribution of acidic melanoma cells

undergoing epithelial-to-mesenchymal transition to aggressiveness of non-acidic

melanoma cells. Clin Exp Metastasis 2014;31:423-33.

25. Kato Y, Lambert CA, Colige AC, Mineur P, Noel A, Frankenne F, et al. Acidic

extracellular pH induces matrix metalloproteinase-9 expression in mouse metastatic

melanoma cells through the phospholipase D-mitogen-activated protein kinase

signaling. J Biol Chem 2005;280(12):10938-44.

26. Fischer K, Hoffmann P, Voelkl S, Meidenbauer N, Ammer J, Edinger M, et al.

Inhibitory effect of tumor cell-derived lactic acid on human T cells. Blood

2007;109(9):3812-9.

27. Raghunand N, Mahoney B, van Sluis R, Baggett B, Gillies RJ. Acute metabolic

alkalosis enhances response of C3H mouse mammary tumors to the weak base

mitoxantrone. Neoplasia 2001;3(3):227-35.

28. Raghunand N, He X, van Sluis R, Mahoney B, Baggett B, Taylor CW, et al.

Enhancement of chemotherapy by manipulation of tumour pH. Br J Cancer

1999;80(7):1005-11.

29. Morita T, Nagaki T, Fukuda I, Okumura K. Clastogenicity of low pH to various

cultured mammalian cells. Mutat Res 1992;268(2):297-305.

30. Walenta S, Wetterling M, Lehrke M, Schwickert G, Sundfor K, Rofstad EK, et al.

High lactate levels predict likelihood of metastases, tumor recurrence, and restricted

patient survival in human cervical cancers. Cancer research 2000;60(4):916-21.

31. Robey IF, Baggett BK, Kirkpatrick ND, Roe DJ, Dosescu J, Sloane BF, et al.

Bicarbonate increases tumor pH and inhibits spontaneous metastases. Cancer




29

research 2009;69(6):2260-8.

32. Silva AS, Yunes JA, Gillies RJ, Gatenby RA. The potential role of systemic buffers in

reducing intratumoral extracellular pH and acid-mediated invasion. Cancer

research 2009;69(6):2677-84.

33. Noguchi F, Inui S, Nakajima T, Itami S. Hic-5 affects proliferation, migration and

invasion of B16 murine melanoma cells. Pigment Cell Melanoma Res

2012;25(6):773-82.

34. Noguchi F, Nakajima T, Inui S, Reddy JK, Itami S. Alteration of skin wound healing

in keratinocyte-specific mediator complex subunit 1 null mice. PLoS One

2014;9(8):e102271 doi 10.1371/journal.pone.0102271.

35. Herman TS, Teicher BA, Collins LS. Effect of hypoxia and acidosis on the

cytotoxicity of four platinum complexes at normal and hyperthermic temperatures.

Cancer research 1988;48(9):2342-7.

36. Shackelford DB, Abt E, Gerken L, Vasquez DS, Seki A, Leblanc M, et al. LKB1

inactivation dictates therapeutic response of non-small cell lung cancer to the

metabolism drug phenformin. Cancer Cell 2013;23(2):143-58.

37. Landsberg J, Kohlmeyer J, Renn M, Bald T, Rogava M, Cron M, et al. Melanomas

resist T-cell therapy through inflammation-induced reversible dedifferentiation.

Nature 2012;490(7420):412-6.

38. Chan LL, Pineda M, Heeres JT, Hergenrother PJ, Cunningham BT. A general

method for discovering inhibitors of protein-DNA interactions using photonic crystal

biosensors. ACS chemical biology 2008;3(7):437-48 doi 10.1021/cb800057j.

39. Erdel M, Trefz G, Spiess E, Habermaas S, Spring H, Lah T, et al. Localization of

cathepsin B in two human lung cancer cell lines. The journal of histochemistry and

cytochemistry : official journal of the Histochemistry Society 1990;38(9):1313-21.

40. Pinton P, Giorgi C, Siviero R, Zecchini E, Rizzuto R. Calcium and apoptosis:

ER-mitochondria Ca2+ transfer in the control of apoptosis. Oncogene

2008;27(50):6407-18 doi 10.1038/onc.2008.308.

41. Vervliet T, Parys JB, Bultynck G. Bcl-2 proteins and calcium signaling: complexity

beneath the surface. Oncogene 2016;35(39):5079-92 doi 10.1038/onc.2016.31.

42. Riemann A, Schneider B, Ihling A, Nowak M, Sauvant C, Thews O, et al. Acidic

environment leads to ROS-induced MAPK signaling in cancer cells. PLoS One

2011;6(7):e22445.

43. Gupta SC, Singh R, Pochampally R, Watabe K, Mo YY. Acidosis promotes

invasiveness of breast cancer cells through ROS-AKT-NF-kappaB pathway.

Oncotarget 2014;5(23):12070-82.




30

44. Trachootham D, Alexandre J, Huang P. Targeting cancer cells by ROS-mediated

mechanisms: a radical therapeutic approach? Nat Rev Drug Discov

2009;8(7):579-91.

45. Ewald JA, Desotelle JA, Wilding G, Jarrard DF. Therapy-induced senescence in

cancer. J Natl Cancer Inst 2010;102(20):1536-46.

46. Kroumpouzos G, Eberle J, Garbe C, Orfanos CE. P53 mutation and c-fos

overexpression are associated with detection of the antigen VLA-2 in human

melanoma cell lines. Pigment Cell Res 1994;7(5):348-53.

47. Ho CK, Li G. Mutant p53 melanoma cell lines respond differently to

CP-31398-induced apoptosis. Br J Dermatol 2005;153(5):900-10.

48. Soengas MS, Capodieci P, Polsky D, Mora J, Esteller M, Opitz-Araya X, et al.

Inactivation of the apoptosis effector Apaf-1 in malignant melanoma. Nature

2001;409(6817):207-11.

49. Carreau A, El Hafny-Rahbi B, Matejuk A, Grillon C, Kieda C. Why is the partial

oxygen pressure of human tissues a crucial parameter? Small molecules and hypoxia.

Journal of cellular and molecular medicine 2011;15(6):1239-53 doi

10.1111/j.1582-4934.2011.01258.x.

50. McCarty MF, Whitaker J. Manipulating tumor acidification as a cancer treatment

strategy. Altern Med Rev 2010;15(3):264-72.

51. Neri D, Supuran CT. Interfering with pH regulation in tumours as a therapeutic

strategy. Nat Rev Drug Discov 2011;10(10):767-77.

52. Lindner D, Raghavan D. Intra-tumoural extra-cellular pH: a useful parameter of

response to chemotherapy in syngeneic tumour lines. Br J Cancer

2009;100(8):1287-91.

53. Tapmeier TT, Moshnikova A, Beech J, Allen D, Kinchesh P, Smart S, et al. The pH

low insertion peptide pHLIP Variant 3 as a novel marker of acidic malignant lesions.

Proc Natl Acad Sci U S A 2015;112(31):9710-5 doi 10.1073/pnas.1509488112.

54. O'Day SJ, Eggermont AM, Chiarion-Sileni V, Kefford R, Grob JJ, Mortier L, et al.

Final results of phase III SYMMETRY study: randomized, double-blind trial of

elesclomol plus paclitaxel versus paclitaxel alone as treatment for

chemotherapy-naive patients with advanced melanoma. J Clin Oncol

2013;31(9):1211-8.

55. Azvolinsky A. Repurposing to fight cancer: the metformin-prostate cancer

connection. J Natl Cancer Inst 2014;106(2):dju030.

56. Brookes PS, Yoon Y, Robotham JL, Anders MW, Sheu SS. Calcium, ATP, and ROS: a

mitochondrial love-hate triangle. Am J Physiol Cell Physiol 2004;287(4):C817-33 doi




31

10.1152/ajpcell.00139.2004.

57. Monteith GR, McAndrew D, Faddy HM, Roberts-Thomson SJ. Calcium and cancer:

targeting Ca2+ transport. Nat Rev Cancer 2007;7(7):519-30 doi 10.1038/nrc2171.

58. Vier J, Gerhard M, Wagner H, Hacker G. Enhancement of death-receptor induced

caspase-8-activation in the death-inducing signalling complex by uncoupling of

oxidative phosphorylation. Molecular immunology 2004;40(10):661-70.

59. Linsinger G, Wilhelm S, Wagner H, Hacker G. Uncouplers of oxidative

phosphorylation can enhance a Fas death signal. Molecular and cellular biology

1999;19(5):3299-311.

Figure Legends

Figure 1. pHe is a threshold that modulates the cytotoxicity of OXPHOSi on melanoma

cells in vitro

A, Representative microscopic images of A375 and of MEWO cells treated with/without

oligomycin (0.01 µg/ml) or phenformin (1 mM) at pHs of 7.4 and 6.7 for 48 h (A375) or 72 h

(MEWO) (magnification × 100). Bar, 100 µm.

B, C, D, Apoptosis (late apoptotic + early apoptotic) detected in A375 and in MEWO cells after

treatment with/without oligomycin (0.01 µg/ml) or phenformin (1 mM) for 48 h (A375) or 72 h

(MEWO). (n = 3).

E, Representative microscopic images of A375 cells treated with/without cisplatin (20 µM) for

48 h at pHs of 7.4 and 6.7 (magnification × 100). Bar, 100 µm.

F, G, Apoptosis (late apoptotic + early apoptotic) detected in A375 cells after treatment

with/without cisplatin (20 µM) at pHs of 7.4 and 6.7 for 72 h (n = 3).




32

Images are representative of 3 independent experiments. Bars = means ± SE. **P<0.01, N.S.,

not significant.

Figure 2. Systemic buffering attenuates the anti-cancer effect of phenformin on B16F10

cells in vivo

A, B, Apoptosis (late apoptotic + early apoptotic) detected in B16F10 cells after treatment

with/without phenformin (1 mM) at pH 7.4 or 6.7 for 48 h (n = 3).

C, Representative images of tumors in each group of mice 15 days after the subcutaneous

injection of B16F10 cells; dashed lines indicate areas of tumors.

D, Tumor volumes after the subcutaneous injection of B16F10 cells in mice with the indicated

treatments (n = 6). Bars = means ± SE. *P <0.05, **P<0.01.

E, F, TUNEL and Ki67 staining of tumors 16 days after treatment with phenformin with/without

NaHCO3 (magnification × 200). Bars, 100 µm. Arrows indicate Ki67-positive nucleuses.

Images are representative of 3 independent experiments.

G, Representative photographs of lungs resected from mice treated with/without phenformin

and/or NaHCO3 for 21 days after the intravenous injection of B16F10 cells.

H, Numbers of metastases on the surfaces of lungs (n = 5). Bars = means ± SE. *P <0.05,

**P<0.01, N.S., not significant.

Figure 3. OXPHOS-inhibition causes the activation of caspase-dependent apoptosis at an




33

acidic pHe

A, Western blot analysis of cleaved caspase-3 p11 in A375 cells after treatment with/without

oligomycin (0.01 µg/ml) and/or Z-DEVD-FMK (10 µM) at pH 7.4 (N) or 6.7 (A) for 48 h; 9 μg

total protein extract was loaded in each lane.

B, Western blot analysis of cleaved caspase-6 in A375 cells after treatment with/without

oligomycin (0.01 µg/ml) and/or Z-VAD -FMK (100 µM) at pH 7.4 (N) or 6.7 (A) for 48 h; 9 μg

total protein extract was loaded in each lane.

C, D, Apoptosis (late apoptotic + early apoptotic) detected in A375 cells after treatment with

oligomycin (0.01 µg/ml), phenformin (1 mM) and/or Z-VAD-FMK (100 µM) at pH 6.7 for 48 h

(n = 3).

Images are representative of 3 independent experiments. Bars = means ± SE. *P <0.05, N.S., not

significant.

Figure 4. Intracellular Ca2+ level is increased in melanoma cells after treatment with

OXPHOSi at an acidic pHe, leading to cell death

A, A375 cells were treated with indicated drugs at pH 7.4 or pH 6.7 for 16 h and stained by

Fluo3/AM. Oligomycin: 0.01 μg/ml, Phenformin: 1 mM, TTFA: 0.1 mM


B, The ratio of A375 cells which show high (Ca-h) and low-intermediate (Ca-m) cytoplasmic




34

Ca2+ level after treatment with the OXPHOSi at pH 7.4 or pH 6.7. (n = 3).

C, D, A375 cells were cultured with oligomycin (0.01 μg/ml) at pH 6.7 for 16 h. Ca-m/Ca-h

cells were sorted and cultured at pH 7.4 (2000/well).

E, Sorted cells were seeded in 24 well plates (2000/well) and cultured at pH 7.4. The number of

viable cells was counted and normalized to that of day 1(n=3).


F, A375 cells were treated with BAPTA/AM (1 μM) 1 h before treatment with Oligomycin (0.01

μg/ml) for 24 h at pH 6.7 and stained by Fluo3/AM.

G, The ratio of dead cells in the cells treated with oligomycin and oligomycin + BAPTA/AM (n

= 12).

H, I, The ratio of Ca-h/Ca-m cells in the cells treated with oligomycin or oligomycin +

BAPTA/AM (n = 12).


Bars = means ± SE. *P <0.05, **P<0.01.

Figure 5. ROS scavenging synergizes with the cytotoxicity enhancement of OXPHOSi

elicited by acidity

A, Measurement of ROS production in A375 cells after treatment with/without oligomycin

(0.01 μg/ml) and phenformin (1 mM) with/without Trolox (100 µM) or Sodium pyruvate (10




35

mM) for 30 min at pHs of 7.4 and 6.7 (n = 3). The MFI of cells in each condition was

normalized to cells with no treatment at pH 7.4.

B to E, Apoptosis (late apoptotic + early apoptotic) detected in A375 cells after treatment

with/without oligomycin (0.01 µg/ml), phenformin (1 mM), TTFA (0.1 mM) with/without

Trolox (100 µM) (B, C) or Sodium pyruvate (10 mM) (D, E) at pH 6.7 for 48 h (n = 3). Trolox

and Sodium pyruvate were pre-treated 1 h before OXPHOS-inhibitors.

Images are representative of 3 independent experiments. Bars = means ± SE. *P <0.05,

**P<0.01, N.S., not significant.

Figure 6. The cytotoxicity enhancement of OXPHOSi elicited by acidity is distinct from

PGC1alpha-driven mitochondrial addiction and JARID1B-high slow-cycling cells

A, Western blot analysis of PGC1alpha and ATP synthase after acidification of melanoma cell

lines; 9 μg total protein extract was loaded in each lane.

B, MitoRed staining of melanoma cell lines cultured at pH 7.4 or 6.7 for 72 h (magnification ×

200). Bar, 100 µm.

C, D, Basal and maximal oxygen consumption rates in SK-MEL-28 or MEWO cells after

acidification for 72 h (n = 12).

E, Viability of SK-MEL-28 and SK-MEL-28-R cells after treatment with vemurafenib

(PLX4032, 10 µM) at the indicated concentration for 72 h. The value of vemurafenib-treated




36

cells was normalized to cells with no treatment (n = 6).

F, G, Apoptosis (late apoptotic + early apoptotic) in SK-MEL-28-R cells after treatment

with/without oligomycin (0.01 μg/ml) at pH 7.4 or 6.7 for 72 h (n = 3).

H, FACS analysis of JARID1B-positive cells after acidification of SK-MEL-28 and

SK-MEL-28-R cells for 72 h. The ratio of JARID1B-positive cells are shown.

I, FACS analysis for JARID1B-positive cells after treatment of SK-MEL-28-R and A375-R

cells with/without oligomycin (0.01 µg/ml) for 48 h at pH 6.7. The ratio of JARID1B-positive

cells are shown.

Images are representative of 3 independent experiments. Bars = means ± SE. *P <0.05,

**P<0.01.






















Published OnlineFirst February 21, 2017.Mol Cancer Ther Fumihito Noguchi, Shigeki Inui, Clare Fedele, et al. cytotoxicity of mitochondrial inhibitors against melanomaCalcium-dependent enhancement by extracellular acidity of the

Updated version

10.1158/1535-7163.MCT-15-0235doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2017/02/21/1535-7163.MCT-15-0235.DC1

Access the most recent supplemental material at:

Manuscript

Authoredited. Author manuscripts have been peer reviewed and accepted for publication but have not yet been

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mct.aacrjournals.org/content/early/2017/02/21/1535-7163.MCT-15-0235To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-15-0235

http://mct.aacrjournals.org/content/suppl/2017/02/21/1535-7163.MCT-15-0235.DC1

http://mct.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mct.aacrjournals.org/content/early/2017/02/21/1535-7163.MCT-15-0235


Yamada-oka, Suita-shi, Osaka 565-0871, Japan, Tel: 81 6 ......2017/02/21 · mitochondrial oxidative phosphorylation (OXPHOS) and were relatively resistant to various drugs including

Documents