Connexin 43 Enhances Sunitinib-Induced

Cytotoxicity in Malignant Mesothelioma Cells

コネキシン 43 は悪性中皮腫におけるスニチニブ感受性を増強する

2017

Miaki UZU

1

Contents

List of abbreviations ................................................................................................... 2

Backgrounds .............................................................................................................. 4

Chapter 1 Cx43 enhances SU-induced cytotoxicity in corporation with Bax ............. 8

Introduction ............................................................................................................. 8

1-1 Effect of Cx43 on anti-proliferative effect of SU ............................................... 10

1-1-1 Materials and Methods .............................................................................. 10

1-1-2 Results...................................................................................................... 15

1-2 Involvement of GJ function in enhancement of SU-induced cytotoxicity .......... 24

1-2-1 Materials and Methods .............................................................................. 24

1-2-2 Results...................................................................................................... 26

1-3 Involvement of Bax in enhancement of SU-induced cytotoxicity by Cx43 ........ 29

1-3-1 Materials and Methods .............................................................................. 29

1-3-2 Results...................................................................................................... 32

1-4 Discussion ...................................................................................................... 41

1-5 Summary ........................................................................................................ 46

Chapter 2 JNK intermediates interaction between Cx43 and Bax .......................... 47

Introduction ........................................................................................................... 47

2-1 Materials and Methods .................................................................................... 48

2-2 Results ............................................................................................................ 49

2-3 Discussion ...................................................................................................... 55

2-4 Summary ........................................................................................................ 59

Conclusion ............................................................................................................... 60

References ............................................................................................................... 63

List of publication ..................................................................................................... 73

Acknowledgements .................................................................................................. 74

Examiners ................................................................................................................ 75

2

List of abbreviations

Bak: Bcl-2 homologous Antagonist/Killer

Bax: Bcl-2 associated X protein

Bcl-2: B-cell lymphoma 2

BH: Bcl-2 homology

BSA: bovin serum albumin

6-CF: 6-carboxyfluorescein

Cx: connexin

DMSO: dimethyl sulfoxide

DTMR: dextran, tetramethylrhodamine

ERK: extracellular signal-regulated kinase

FBS: fetal bovine serum

GA: 18-glycyrrhetinic acid

GJ: gap junction

GJIC: gap junctional intercellular communication

JC-1: 5, 5’, 6, 6’-tetrachloro-1, 1’, 3, 3’-tetraethylbenzimidazolocarbocyanine iodide

JNK: c-jun N-terminal kinase

MAPK: mitogen-activated protein kinase

MM: malignant mesothelioma

MTT: 3-(4, 5-dimetylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide

PBS: phosphate buffered saline

PDGF(R): platelet-derived growth factor (receptor)

PKC: protein kinase C

RNA: ribonucleic acid

ROS: reactive oxygen species

3

RTK: receptor tyrosine kinase

SAPK: stress-activated protein kinase

siRNA: short-interfering RNA

SU: sunitinib

TBS(-T): Tris buffered saline (and tween 20)

VEGF(R): vascular endothelial growth factor (receptor)

4

Backgrounds

Connexin (Cx) is a four-transmembrane protein and forms an intercellular channel

called gap junction (GJ). Cellular homeostasis, growth, and differentiation are

mediated via GJ intercellular communication (GJIC) (1). GJ allows a direct transfer of

several second messengers and small molecules between apposed cells, including

Ca2+ , cyclic adenosine 3’, 5’-monophosphate (cAMP), cyclic guanosine 3’, 5’-

monophosphate (cGMP), inositol 1, 4, 5-triphosphate (IP3), and glutathione (2).

Twenty-one Cx subtypes are expressed in human (3), with some members being

only expressed in specific tissues, while others including connexin 43 (Cx43) and

Cx32, expressed ubiquitously (4). Each subtype makes GJ and maintains the cellular

network in the expressed tissue. For example, it has been reported that molecular

transfer between astrocytes via GJ made up of Cx43 and Cx30, which are major Cx

subtypes in these cells, affects the synaptic activity of nearby neurons in the brain

(5). Recently, it has been recognized that Cx is not just a component of GJ, but also

it interacts with various proteins including Src (6), extracellular signal-regulated

kinase (ERK) (7), S-phase kinase associated protein (Skp) 2 (8), B-cell lymphoma 2

(Bcl-2) associated X protein (Bax) (9), and -catenin (10), and affects them in their

expressions or activities (11). From these facts, it is surmised that aberrant function

of Cx and GJ would be caused by or lead to many types of diseases.

Frequently, it has been observed that Cx expression is decreased accompanied by

decreased GJ function in many cancer cells (4). Several Cx gene transfection

experiments have suggested that Cx-mediated GJIC works as a tumor suppressor in

cells where a specific Cx subtype was originally expressed (12). It has also been

shown that Cx43 reduces cell growth in the absence of GJIC (13), suggesting that

direct molecular interaction of Cx has a crucial role in its tumor suppressive effect.

5

However, restoring Cx expression is not always sufficient to inhibit tumor

proliferation. In our previous study, transfection of Cx43 gene in malignant

mesothelioma (MM) cells did not change the ability of anchorage-independent growth

and migration. Thus, we combined increased Cx43 expression with a treatment of

cisplatin and showed that Cx43 enhanced its cytotoxicity in MM cells (14). Another

group reported that transfection of Cx43 gene increased tumor suppressive effect of

docetaxel in a prostate cancer xenograft model (15). Moreover, we confirmed that

Cx32 enhanced cytotoxicity of vinblastine and vinorelbine in renal and lung cancer

cell lines, respectively (16, 17). These studies indicate that combination of Cx and

anti-cancer agents would be a promising strategy for cancer chemotherapy. Although

our previous works have already found a proto-oncogenic tyrosine kinase Src, as a

key molecule for both Cx43 and Cx32 to enhance drug sensitivity in cancer cells,

further investigation into other proteins with which Cx interacts can broaden the

potential of Cx for a target of cancer therapy. Especially, targeting direct modulators

of cell survival/death will be beneficial.

Therefore, we turned our attention to the report that showed a direct interaction

between Cx43 and Bax, as this interaction promoted induction of apoptosis in

pancreatic cancer cells (9). Bax is a major proapoptotic protein of the Bcl-2 family,

which causes mitochondrial dysfunction followed by caspase activation in process of

apoptosis. Bax is a clinically important protein, since it has been reported that the

higher Bax expression level is correlated with the favorable clinical outcomes in

patients with gastric and breast tumors (18, 19). With a view to increasing Bax

function, we investigated a beneficial combination of Cx43 with an anti-cancer agent

in MM cells, whose original mesothelial cells express Cx43 as the major Cx subtype

(14).

6

MM is an insidious tumor, whose main risk factor is inhalation of asbestos. MM

displays a long latency period of over 40 years following the first exposure to

asbestos (20). Asbestos is a fire-resistant material that was widely used in various

industries between the 1940s and the 1970s, with MM incidence increasing

throughout the world (21). For example, the incidence of MM is currently 2000-3000

per year in the United States, and approximately 70,000 new MM cases are expected

over the next 20 years (22). In Europe, the peak year of MM incidence is anticipated

to fall between 2015 and 2020, with estimated 250,000 new cases over the next 40

years (22). Surgery is desirable for MM patients in clinical stage I; however, the

majority of patients are diagnosed at an advanced stage, and the only treatment

option for them is systemic therapy (23). Unfortunately, MM is usually a chemo- and

radio-resistant tumor, which contributes to its poor prognosis (23). Although a

platinum-based chemotherapy has been used for the MM patients now, its impact on

the survival of these patients is modest (24). What is worse, there is no second-line

treatment. Such unmet clinical need and progression in the identification of key

growth factors, glycoproteins, and genetic mutations in MM tumorigenesis have

generated an interest in the development of novel agents to target these oncogenic

abnormalities (20). It has been reported that both vascular endothelial growth factor

(VEGF) and platelet-derived growth factor (PDGF) play an important role in the

growth of MM cells via the autocrine loops (25, 26). Moreover, the higher VEGF

expression in MM tissues was correlated with a poor prognosis for the patients (27).

Based on these observations, it is rational to target these signaling pathways. Thus,

we turned our attention to sunitinib (SU). SU is a multi-targeted tyrosine kinase

inhibitor, which suppresses the activation of VEGF receptors (VEGFRs 1-3), PDGF

receptors (PDGFRs α and β), and other growth factor receptors (28).

7

This study describes the effect of increased Cx43 expression on SU-induced

cytotoxicity in MM cells. This thesis is composed of two chapters; in the first chapter,

the efficacy of a combination of Cx43 and SU in MM cells is described, while the

second chapter describes our investigation into the detailed mechanism by which

Cx43 enhanced SU-induced cytotoxicity.

8

Chapter 1 Cx43 enhances SU-induced cytotoxicity in corporation with Bax

Introduction

Cell death is deeply related to cancer therapy. Originally, cell death had been

recognized as “necrosis”, which is caused by noxious stimuli and often accompanied

by atrophy of tissues and organs. In 1972, Kerr et al. defined the cell death

characterized by nuclear and cytoplasm condensation as “apoptosis” in distinction

from necrosis (29). Cells exhibiting such distinct structure were observed in damaged

and healthy tissues of adult and neonatal rats by electron microscopy, which

suggests that apoptosis is involved in tissue development and maintenance. The

mechanism of how this process is regulated had been unknown for a long time since

that discovery. In 1985, Tsujimoto et al. isolated Bcl-2 gene at the chromosomal

breakpoint of t(14;18)-bearing follicular B cell lymphomas (30), and it was shown that

overexpression of Bcl-2 enabled certain hematopoietic cell lines to survive following

apoptosis induced by the growth factor withdrawal (31, 32). It was also found that it is

localized at the mitochondrial membrane, which was a unique characteristic of a

protein with a proto-oncogenic role at that time. Presently, Bcl-2 is widely recognized

as a cell death repressor protein. In 1993, Bax was identified as a 21 kilodalton

protein, which was coimmunoprecipitated with Bcl-2 (33). Bax shows homology with

Bcl-2 with 20.8% identity and 43.2% similarity in amino acid sequence.

Overexpression of Bax in murine lymphoid progenitor cells enhanced apoptosis

caused by the growth factor withdrawal, and it was shown that expression ratio of

Bax to Bcl-2 affected the sensitivity of that cell line to apoptotic stimulation. Whereas

many types of conventional cytotoxic agents cause apoptosis in cancer cells (34), the

Bcl-2/Bax ratio is considered an important indicator of the sensitivity of cancer cells to

anti-cancer agents. Now, proteins which contain the conserved Bcl-2 homology (BH)

domain are classified into the Bcl-2 family member and further subdivided into 3

9

groups according to their structures and functions: anti-apoptotic Bcl-2-like proteins,

proapoptotic multidomain proteins including Bax and Bcl-2 homologous

Antagonist/Killer (Bak), and proapoptotic BH3-only proteins (35). These proteins are

expected to regulate apoptosis arising from mitochondrial dysfunction by interacting

with one another.

Since increasing Bax function can be beneficial for the treatment of any types of anti-

cancer agents, this chapter describes our investigations of whether Cx43 enhances

SU-induced cytotoxicity, and the potential contribution of Bax to the enhanced

cytotoxicity.

10

1-1 Effect of Cx43 on anti-proliferative effect of SU

In chapter 1-1, it was investigated whether Cx43 enhances the growth inhibitory

effect of SU in MM cells. In addition, the activity of receptor tyrosine kinase (RTK)

signaling was evaluated, because it is a target of SU.

1-1-1 Materials and Methods

Reagents

All cultures and reagents were purchased from Sigma (St. Louis, MO, USA) unless

otherwise indicated.

・30% Acrylamide solution (Bio-Rad, Hercule, CA, USA)

・Bovine serum albumin (BSA)

・Bromophenol blue (ICN Biomedicals, Eschwege, Germany)

・3-(4,5-Dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Dojindo

Laboratories, Kumamoto): 5 mg/mL stock solution in distilled water was used.

・Dimethyl sulfoxide (DMSO; Wako Pure Chemicals, Osaka)

・Dulbecco’s PBS (-) (PBS; Nissui Pharmaceutical Co., Ltd., Tokyo)

・Ethanol (99.5) (Wako Pure Chemicals)

・Ethylenediaminetetraacetic acid (Dojindo Laboratories)

・Fetal bovine serum (FBS; Biowest, Kansas City, MO, USA)

・β-Glycerophosphate

・2-Mercaptoethanol (Wako Pure Chemicals)

・Penicillin-Streptomycin (Gibco, Palo Alto, CA, USA)

・Phenylmethane sulfonyl fluoride

・Polyoxyethylene (10) Octylphenyl Ether (TritonX-100; Wako Pure Chemicals)

・Polyoxyethylene (20) Sorbitan Monolaurate (Tween 20; Wako Pure Chemicals)

11

・Propidium iodide (PI; Invitrogen, Carlsbad, CA, USA): 1 mg/mL stock solution in

distilled water was used.

・Protease inhibitor cocktail

・RNaseA (MP Biomedicals, Santa Ana, CA, USA): 10 mg/mL stock solution in 10

mM sodium acetate buffer (pH 5.2) was used.

・RPMI-1640 medium

・Skim milk (Megmilk Snow Brand, Co., Ltd., Tokyo)

・Sodium acetate buffer solution

・Sodium chloride (NaCl; Wako Pure Chemicals)

・Sodium dodecyl sulfate (SDS; Wako Pure Chemicals)

・Sodium orthovanadate

・Sucrose (Wako Pure Chemicals)

・Sunitinib malate (SU): 10 or 100 mM stock solution in DMSO was used.

・5, 5’, 6, 6’-tetrachloro-1, 1’, 3, 3’-tetraethylbenzimidazolocarbocyanine iodide (JC-

1) (Santa Cruz Biotechnology, Santa Cruz, CA, USA): 1.5 mM stock solution in

DMSO was used.

・0.25% Trypsin-EDTA Solution

・UltraPure™ Tris (Tris; Invitrogen)

Cell culture, construct creation, and transfection

H28, a representative human MM cell line, was obtained from ATCC (Manassas, VA,

USA) and grown in RPMI-1640 medium supplemented with 10% FBS, 100 units/mL

penicillin/100 μg/mL streptomycin at 37°C in 5% CO2. Cx43-transfected H28 cells

(H28-T) were produced as previously described (14). In brief, a human Cx43 cDNA

containing the entire coding region was inserted into the expression vector pcDNA™

3.1 D/V5-His-TOPO® (Invitrogen), and it was transfected into H28 cells by using

FuGENE® HD Transfection Reagent (Roche Applied Science, Pleasanton, CA,

12

USA), and the cells were then selected in a culture medium containing 0.7 mg/mL

G418. Wild-type cells (H28-W) were used as a control.

Cell viability assay

A total of 5.0 × 103 cells were seeded in a 96-well plate. After 24 h incubation, the

cells were treated with 0.38-10 μM SU or DMSO (control) followed by culturing for 24

h. At the end of the period, 0.25 mg/mL MTT was added to each well and the plate

was incubated for 1 h. The supernatant was removed and DMSO was added to each

well. The absorbance at a wavelength of 540 nm and a reference wavelength of 650

nm was measured with a Multiscan™ JX microplate reader (Thermo Labsystems,

Cheshire, UK). Cell viability (%) was calculated as follows: [optical density (OD) of

the treated cells] / (OD of the control cells) × 100.

Cell cycle analysis

A total of 1.5 × 105 H28-W cells and 3.0 × 105 H28-T cells were seeded in 60 mm

dishes. After 6 h incubation, the cells were cultured in medium containing 0.1% FBS

for 24 h followed by treatment with 10 μM SU or DMSO (control) for each indicated

period. Then, the cells were collected and fixed in 80% ethanol. Before analysis, the

cells were incubated in PBS containing 50 μg/mL PI and 200 μg/mL RNaseA for 30

min at 37°C. The cell suspension was filtered through a nylon mesh filter, and the

filtrate was analyzed using a BD FACSCanto™ II flow cytometer (BD Biosciences,

San Jose, CA, USA).

Measurement of mitochondrial membrane potential

A total of 3.2 × 105 cells were seeded in a 6-well plate. After 24 h incubation, the cells

were treated with 5 or 10 M SU or DMSO (control) followed by culturing for 8 h.

Then, cells were trypsinized and incubated with 1 M JC-1 for 20 min at 37°C. After

13

that, cells were washed and resuspended in PBS. Fluorescence of JC-1 monomers

was measured at 485 nm excitation and 535 nm emission, and that of JC-1

aggregates was measured at 550 nm excitation and 600 nm emission by using

SpectraMax™ i3 (Molecular Devices, Sunnyvale, CA, USA).

Western blot analysis

A total of 1.5 × 105 H28-W cells and 3.0 × 105 H28-T cells were seeded in 60 mm

dishes. After 6 h incubation, the cells were cultured in 0.1% FBS medium for 24 h

followed by treatment with 10 μM SU or DMSO (control) for each indicated period.

Cells were collected by scraping and dissolved in ice-cold lysis buffer [50 mM Tris

(pH 7.4), 150 mM NaCl, 1% Triton X-100, 10 mM β-glycerophosphate, 1 mM sodium

orthovanadate, 1 mM ethylenediaminetetraacetic acid, 1mM phenylmethane sulfonyl

fluoride, and 1% protease inhibitor cocktail]. Protein concentrations were determined

by using a DC protein assay kit (Bio-Rad). Total cell lysates (10 μg or 25 μg) were

diluted in sample buffer [250 mM Tris (pH 6.8), 40% sucrose, 20% 2-

mercaptoethanol, 8% SDS, and 0.002% bromophenol blue] and separated using a

7.5% or 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel. The

separated proteins were then transferred to a polyvinylidene difluoride membrane

(ATTO, Tokyo). After that, the membranes were blocked with 5% skim milk in tris-

buffered saline and tween 20 (TBS-T) [13.7 mM NaCl, 25 mM Tris, and 0.05%

Tween 20] or 5% BSA in TBS-T for 1 h at room temperature or overnight at 4°C. The

membranes were then incubated with primary antibodies for 1 h at room temperature

or overnight at 4°C followed by incubation with secondary horse radish peroxidase

(HRP)-conjugated antibodies diluted in TBS-T containing 1% skim milk for 1 h at

room temperature. The primary antibodies are shown in Table 1. The detection was

accomplished using an Immobilon™ Western Chemiluminescent HRP Substrate

14

(Merck Millipore, Darmstadt, Germany) and an ImageQuant™ LAS 4000 (GE

Healthcare). β-Actin was used as the internal standard.

Statistical analysis

Statistical analyses were performed by using the Student’s t-test, the Aspin-Welch’s

t-test, or the Tukey-Kramer test. A value of P < 0.05 was considered statistically

significant.

Table 1 The primary antibodies used in western blot analysis

Target Source Cat. No. dilution TBS-T with Incubation Species

VEGFR2Cell Signaling Technologies

(Danvers, MA, USA)#2479 1000 5% BSA on, 4°C rabbit

PDGFR Santa Cruz Biotechnology sc-53872 1000 5% BSA on, 4°C mouse

p44/42 MAPK (Erk1/2)

(137F5): ERKCell Signaling Technologies #4695 5000 1% skim milk on, 4°C rabbit

Phopho-p44/42 MAPK (Erk1/2)

(Thr202/Tyr204): p-ERKCell Signaling Technologies #9101 2000 5% BSA on, 4°C rabbit

Akt (pan) (C67E7): Akt Cell Signaling Technologies #4691 5000 1% skim milk on, 4°C rabbit

Phopho-Akt (Ser473): p-Akt Cell Signaling Technologies #9271 2000 5% BSA on, 4°C rabbit

-Actin Sigma A5441 10000 1% skim milk 1 h, rt mouse

rt: room temperature; on: overnight

15

1-1-2 Results

First, the effect of Cx43 on the sensitivity of MM cells to SU was examined. It was

shown that H28-W cells were resistant to SU; approximately 80% of the cells

survived even after a 24 h exposure to 10 M SU (Fig. 1-1-1). In contrast, the viability

of Cx43-transfected H28-T cells was significantly lower than that of H28-W cells in

the presence of SU in a 0.38-10 M range. Particularly, less than half of the control

cells survived after 24 h of treatment with 10 μM SU.

Next, we examined the effect of Cx43 expression on the cell cycle. The S-phase

population in H28-T cells was significantly higher than in H28-W cells at every time

point (Fig. 1-1-2A). SU had almost no impact on cell cycle distribution except for a

significant decrease in the S-phase population in H28-W cells treated with SU for 24

h. On the other hand, SU increased the subG1 population, suggesting an increase in

the apoptotic population in both cells (Fig. 1-1-2B). It should be noted that the ratio of

increase in the subG1 population was significantly higher in H28-T cells than in H28-

W cells.

To verify that SU treatment activated the mitochondrial apoptotic pathway, followed

by a significant increase in the subG1 population, mitochondrial membrane potential

(⊿φM) was measured. JC-1 forms an aggregate at the mitochondria with a red

fluorescence (600 nm) and redistributes to the cytosol as a monomer with a green

fluorescence (535 nm) following the decrease in ⊿φM. A Change in the ratio of the

fluorescent intensity reflects a change in ⊿φM. As expected, there was no alteration

in H28-W cells treated with SU for 8 h, while ⊿φM in H28-T cells exposed to 10 M

SU was significantly decreased (Fig. 1-1-3).

16

Subsequently, the activity of the RTK signaling pathway was investigated. VEGFR2

and PDGFRβ were expressed in both H28-W and H28-T cells (Fig. 1-1-4A). The

expression levels of these receptors were slightly lower in H28-T cells than that

observed in H28-W cells, although there were no significant differences. The effect of

SU on Akt and ERK, which are downstream factors of these receptors, was then

investigated. In H28-W cells, phosphorylation of Akt was decreased after 24 h of SU

treatment, followed by a slight decrease in the total expression after 48 h of SU

treatment in H28-W cells, although the change was not significant (Fig. 1-1-4B). In

contrast, in H28-T cells, the total expression of Akt was dramatically decreased in a

time-dependent manner, which was associated with its decreased phosphorylation in

H28-T cells. SU had no effect on both total and phosphorylated ERK expressions

(Fig. 1-1-4C). In H28-T cells, phosphorylation of ERK significantly decreased after 24

h of SU treatment, followed by a slight decrease in the total expression after 48 h of

SU treatment.

17

Fig. 1-1-1 SU sensitivity of Cx43-transfected cells. Cells were treated with the indicated

concentrations of SU or DMSO (SU 0 μM) for 24 h, and cell viability was assessed by MTT

assay. Each bar represents the mean cell viability normalized to control in each cell group,

and vertical lines indicate S.D. of 3 independent experiments. **P < 0.01, ***P < 0.001:

Significant difference between H28-W and H28-T cells at every SU concentration by using

the Student’s t-test or the Aspin-Welch’s t-test.

18

Fig. 1-1-2 Estimation of cell cycle distribution and apoptosis. Cells were treated with 10 μM

SU or DMSO (SU 0 M) for the indicated period, and each of 20,000 cells was analyzed by

flow cytometry. (A) Alteration in cell cycle distribution in H28-W and H28-T cells in the

presence of SU. Each bar represents the mean percentage of cells in each population, and

vertical lines indicate S.D. of 3 independent experiments. *P < 0.05: Significant difference

from control in each cell group, +P < 0.05,

++P < 0.01,

+++P < 0.001: Significant difference

between H28-W and H28-T cells in each control by using the Tukey-Kramer test. (B) Effect

of SU on the induction of apoptosis detected as the pre-G0-G

1 (subG

1 population) in cell

cycle distribution. Each bar represents the mean percentage of cells in subG1 population,

and vertical lines indicate S.D. of 3 independent experiments. *P < 0.05, **P < 0.01, ***P <

0.001: Significant difference from control in each cell group, and ##

P < 0.01, ###

P < 0.001

between H28-W and H28-T cells treated with 10 μM SU by using the Tukey-Kramer test.

19

Fig. 1-1-3 Effect of SU on mitochondrial membrane potential. Cells were treated with the

indicated concentrations of SU or DMSO (SU 0 M) for 8 h followed by incubation with 1

M JC-1. Each bar represents the ratio of fluorescent intensity of JC-1 aggregates at the

mitochondria (600 nm) to JC-1 monomers in the cytosol (535 nm) normalized to control

in each cell group, and vertical lines indicate S.D. of 8 wells. ***P < 0.001: Significant

difference from control in each cell group by using the Tukey-Kramer test.

20

21

22

23

Fig. 1-1-4 Effect of SU on RTK signaling detected by western blotting. (A) Total expressions

of VEGFR2 and PDGFRβ in H28-W and H28-T cells cultured for 48 h. Each bar represents

the intensity of the protein bands normalized to the internal standard β-actin, and vertical lines

indicate S.D. of 3 independent experiments. (B) Total Akt (Akt) and phosphorylated Akt (p-

Akt), and (C) total ERK (ERK) and phosphorylated ERK (p-ERK) expressions after treatment

with 10 μM SU or DMSO (Cont) for each indicated period. Each bar represents the intensity

of the protein bands normalized to the internal standard β-actin and is presented as a ratio

relative to 24 h control. ERK and p-ERK includes 2 bands derived from ERK1 (✤) and ERK2

(✤✤), and their intensities were collectively quantified. Vertical lines indicate S.D. of 3

independent experiments. *P < 0.05, ***P < 0.001: Significant difference of control at each

time point in each cell group by using the Tukey-Kramer test.

24

1-2 Involvement of GJ function in enhancement of SU-induced cytotoxicity

In chapter 1-2, it was examined whether GJ function contributes to the enhancement

of SU-induced cytotoxicity by using a GJ inhibitor, 18β-glycyrrhetinic acid.

1-2-1 Materials and Methods

Reagents

All cultures and reagents were purchased from Sigma unless otherwise indicated.

・6-Carboxyfluorescein (6-CF): 10 mg/mL stock solution in distilled water was used.

・Dextran tetramethylrhodamine (DTMR; Invitrogen): 50 mg/mL stock solution in

distilled water was used.

・Dulbecco’s phosphate buffered saline with calcium chloride and magnesium

chloride

・18β-Glycyrrhetinic acid (GA): 40 mM stock solution in DMSO was used.

・4% Paraformaldehyde phosphate buffer solution (Wako Pure chemicals)

Refer to Chapter 1-1-1 for the used other reagents.

Cell culture

Refer to Chapter 1-1-1.

Scrape loading and dye transfer assay

A total of 5.0 × 105 H28-W cells and 1.0 × 106 H28-T cells were seeded in 35 mm

dishes. After 24 h incubation, the cells were rinsed twice with PBS. PBS containing

0.05% 6-CF (a GJ-permeable fluorescent dye) and 0.05% DTMR (a GJ-impermeable

fluorescent dye) was then added to the cells before scrape loading at room

temperature. The dye solution was left on the cells for 3 min, after which it was

25

discarded, and the cells were rinsed 3 times with PBS with calcium and magnesium.

After that, the cells were fixed with 4% paraformaldehyde phosphate buffer solution,

after which it was discarded, and PBS was added. Finally, the cells were observed

under an Olympus FV500 confocal microscope (Olympus, Tokyo). GJ activity was

assessed using 6-CF dye transfer, and the scraped edge was stained with DTMR.

GJIC inhibition

Firstly, to determine the effect of GA on GJ activity, the cells were treated with 40 μM

GA or DMSO (control) for 24 h. Subsequently, GJ activity was assessed by using the

scrape loading and dye transfer assay. Furthermore, to determine the effect of GJIC

inhibition on SU cytotoxicity, the cells were treated with 40 μM GA and 0.38-10 μM

SU for 24 h followed by the assessment of cell viability by using the MTT assay.

Refer to Chapter 1-1-1 for the method of MTT assay.

Statistical analysis

Refer to Chapter 1-1-1.

26

1-2-2 Results

At first, it was verified that a GJ-permeable 6-CF spread from the scraped edge

stained with DTMR, suggesting the functional GJ in H28-T cells (Fig. 1-2A).

Incubation with 40 μM GA for 24 h resulted in the inhibition of GJ function. On the

other hand, 6-CF and DTMR were almost completely overlapped, which represents

no GJ function in H28-W cells, and it was unchanged by GA treatment. When GA

was combined with SU, H28-W cell viability was not altered as compared to SU alone

(Fig. 1-2B). GA had little influence on the H28-T cell viability in conjunction with low

doses (less than 3.3 μM) of SU, however, the combined treatment with GA and 6 or

10 μM SU caused a significant decrease in the cell viability as compared to SU

alone.

27

28

Fig. 1-2 Effect of GJ blockage on SU-induced cytotoxicity. (A) Effect of 18β-glycyrrhetinic

acid (GA) on GJ function. Cells were treated with 40 μM GA or DMSO (Cont) for 24 h, and

scrape loading and dye transfer assay. 6-CF dye transfer (green) was used to assess GJ

function, scraped edge was stained with DTMR (red), and locations where 6-CF and DTMR

overlapped are shown in yellow (Merge). (B) Effect of GA on SU-induced cytotoxicity. Cells

were treated with the indicated concentrations of SU or DMSO (SU 0 μM) and 40 μM GA

(with GA) or DMSO (without GA) simultaneously for 24 h, and cell viability was assessed by

MTT assay. Each bar represents the mean cell viability normalized to control in each cell

group, and vertical lines indicate S.D. of 3 independent experiments. *P < 0.05, **P < 0.01:

Significant difference between GA-treated cells (with GA) and untreated cells (without GA)

at every SU concentration in each cell group by using the Student’s t-test.

29

1-3 Involvement of Bax in enhancement of SU-induced cytotoxicity by Cx43

In chapter 1-1, we showed that SU caused a decrease in mitochondrial membrane

potential and increased the subG1 population in Cx43-transfected cells, suggesting

that Cx43 expression might promote the apoptosis caused by mitochondrial

dysfunction. Considering these results, the involvement of Bax was focused on; Bax

is a member of the Bcl-2 family, which regulates the mitochondrial apoptotic pathway

and is reported to directly interact with Cx43. In this chapter 1-3, it was examined

whether Bax interacts with Cx43 and contributes to the enhancement of SU-induced

cytotoxicity in MM cells.

1-3-1 Materials and Methods

Reagents

・Fluoromount™ (Diagnostic Biosystems, Pleasanton, CA, USA)

・Lipofectamine® 3000 Reagent (Invitrogen)

・Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen)

・MitoTracker® Red CM-H2XRos (MitoTracker Red; Invitrogen): 1 mM stock solution

in DMSO was used.

・Saponin Practical Grade (Saponin; MP Biomedicals)

Refer to Chapter 1-1-1 for the used other reagents.

Cell culture

Refer to Chapter 1-1-1.

Immunoprecipitation and western blot analysis

A total of 1.5 × 105 H28-W cells and 3.0 × 105 H28-T cells were seeded in 60 mm

dishes. After 6 h incubation, the cells were cultured in 0.1% FBS medium for 24 h

30

followed by treatment with 10 μM SU or DMSO (control) for each indicated period.

Cells were collected and protein concentrations were determined as mentioned in

Chapter 1-1. For immunoprecipitation, total cell lysates (100 μg) pre-cleared with

protein sepharose G (GE Healthcare, Buckinghamshire, UK) for 1 h at 4°C, were

immunoprecipitated with 3.2 μg anti-Cx43 antibody (Sigma, C6219), 1 g anti-Bax

(N-20) antibody (Santa Cruz Biotechnology, sc-493) or anti-rabbit antibody (negative

control) for 1 h at 4°C followed by incubation with protein sepharose G for 1 h at 4°C.

Immunoprecipitates were washed 5 times with lysis buffer followed by resuspension

in sample buffer and boiling for 5 min at 100°C. Then, immunoprecipitates were

purified by centrifugation at 2300 g. Refer to Chapter 1-1-1 for the method of the

following western blotting. The dilution/incubation conditions for the used primary

antibodies were as follows: Cx43 (1:10000/1 h at room temperature), Bax (1:10000/1

h at room temperature for total cell lysates, 1:2000/overnight at 4°C for

immunoprecipitates). Both antibodies were diluted in TBS-T containing 1% skim milk.

Immunofluorescence staining

A total of 3.2 × 105 cells were seeded in a 6-well plate. After 24 h incubation, the cells

were treated with 10 μM SU or DMSO (control) for 8 h followed by incubation with

MitoTracker Red for 20 min at 37°C. Then, cells were fixed in 4% paraformaldehyde

phosphate buffer solution for 20 min at 37°C. After blocking with PBS containing

0.1% saponin and 3% BSA for 30 min at room temperature. The cells were then

incubated with primary antibodies for 1 h at room temperature followed by incubation

with secondary Alexa Fluor 405- or Alexa Fluor 647-conjugated antibodies diluted in

PBS containing 0.1% saponin for 1 h at room temperature. Finally, the cells were

observed under a Zeiss LSM 780 confocal microscope (Carl Zeiss MicroImaging

GmbH, Jena, Germany). The dilution conditions for the used primary antibodies were

as follows: Cx43 (1:100); Bax (6A7) (Santa Cruz Biotechnology, sc-23959) (1:50).

Both antibodies were diluted in PBS containing 0.1% saponin and 3% BSA. Obtained

31

images were analyzed by using ImageJ2 software (National Institute of Health,

Bethesda, MD, USA).

Short-interfering RNA (siRNA) transfection

For the Cx43 knockdown experiment, a total of 3.2 × 105 cells were seeded in a 6-

well plate. After 24 h incubation, cells were cultured in FBS-free medium shortly

before siRNA transfection using Lipofectamine™ RNAiMAX Transfection Reagent,

according to the manufacturer’s protocol. The following siRNAs were used: a Cx43-

specific siRNA (Hs_GJA1_5); a negative control siRNA (AllStars Negative Control

siRNA). Cells were transfected with a final amount of 50 pmol siRNA for 24 h, and

then used for the immunofluorescence staining as mentioned above.

For the Bax knockdown experiment, a total of 2.2 × 105 H28-T cells were seeded in a

6-well plate. After 6 h incubation, the cells were cultured in medium containing 0.1%

FBS shortly before siRNA transfection using Lipofectamine® 3000 Reagent,

according to the manufacturer’s protocol. The following siRNAs were used: a Bax-

specific siRNA (Hs_BAX_10); a negative control siRNA. Cells were transfected with a

final concentration of 10 nM siRNA for 24 h, and then used for the western blotting

and the flow cytometry. Refer to Chapter 1-1-1 for the method of each experiment.

All the used siRNAs were obtained from Qiagen (Valencia, CA, USA).

Statistical analysis

Refer to Chapter 1-1-1.

32

1-3-2 Results

Bax (23 kilodaltons) and Cx43 (~43 kilodaltons) were immunoprecipitated from the

total cell lysates of SU-treated and untreated H28-T cells by using an anti-Cx43

antibody or an anti-Bax (N-20) antibody, respectively (Fig. 1-3-1). The total Bax

expression level was slightly higher in H28-T cells compared with H28-W cells,

although the difference was not statistically significant (Fig. 1-3-2A). SU had almost

no effect on the total Bax expression level in both cells (Fig. 1-3-2B). However, a

putative Bax dimer band over 35 kilodaltons was detected only in H28-T cells, and

SU treatment increased the density of this band. Bax mainly exists in the cytosol as

an inactive monomer in intact cells, and apoptotic stimuli cause Bax to change

conformation with its N-terminus exposure (active Bax) and then to translocate to the

mitochondria and to form dimers/oligomers; finally, the Bax oligomers result in

mitochondrial dysfunction.

To investigate whether the SU treatment of MM cells promotes the process of Bax

activation, immunofluorescence staining was performed by using an anti-Bax (6A7)

antibody that specifically recognizes N-terminus of active Bax. As shown in Fig. 1-3-

3, no expression of active Bax was detected in both SU-treated and untreated H28-W

cells, which do not express Cx43 protein. In contrast, in H28-T cells, SU treatment

caused cell shrinkage, increased active Bax expression and its accumulation at the

mitochondria. More importantly, the observation of dozens of H28-T cells showed that

both total expression level and the ratio of mitochondrial localization of active Bax

were significantly increased by SU treatment, and these were suppressed by the

knockdown of Cx43 (Fig. 1-3-4).

33

Finally, the knockdown of Bax was performed to assure that Bax activation

contributes to induction of apoptosis. As expected, the depletion of Bax expression

significantly reduced the ratio of subG1 population in the presence of SU, though cell

cycle distribution was not affected (Fig. 1-3-5).

34

Fig. 1-3-1 Interaction between Cx43 and Bax. Cells were treated with 10 μM SU or DMSO

(Cont) for 24 h, and immunoprecipitation (IP) from H28-T cell extract was performed using

an anti-Cx43 antibody, an anti-Bax antibody or an anti-rabbit antibody (negative control,

NC) followed by western blotting (WB). (A) Bax was detected, and an arrow represents its

band. (B) Cx43 was detected, and each arrow represents a non-phosphorylated (✤) and a

phosphorylated (✤✤) form, respectively. Each photo is representative of 3 independent

experiments.

35

Fig. 1-3-2 Difference of Bax behaviors in H28-W and H28-T cells detected by western

blotting. (A) Total Bax expression in naïve H28-W and H28-T cells. Each bar represents the

intensity of the protein bands normalized to the internal standard β-actin, and vertical lines

indicate S.D. of 3 independent experiments. (B) Effect of SU on Bax expression in H28-W

and H28-T cells. Cells were treated with 10 μM SU or DMSO (Cont) for 24 h. β-Actin is

shown as the internal standard. Each arrow represents a monomer (✤) and a dimer (✤✤)

form, respectively. Each photo is representative of 3 independent experiments.

36

Fig. 1-3-3 Effect of SU on Bax activation in H28-W and H28-T cells detected by

immunofluorescence staining. Cells were treated with 10 μM SU or DMSO (Cont) for 8 h

followed by incubation with MitoTracker Red to be stained for mitochondria (red). After

fixation, cells were treated with anti-Cx43 antibody (blue) and anti-Bax (6A7) antibody

(green). Anti-Bax (6A7) antibody recognizes active Bax. Yellow areas represent where the

mitochondria and active Bax are overlapped.

37

38

Fig. 1-3-4 Effect of Cx43 knockdown on SU-induced Bax activation in H28-T cells. Cells

were transfected with a final amount of 50 pmol Cx43-specific siRNA (siCx43) or negative

control (NC) siRNA for 24 h followed by treatment with 10 μM SU or DMSO (Cont) for 8 h

and incubation with MitoTracker Red to be stained for mitochondria (red). After fixation,

cells were treated with anti-Cx43 antibody (blue) and anti-Bax (6A7) antibody (green). Anti-

Bax (6A7) antibody recognizes active Bax. Yellow areas represent where the mitochondria

and active Bax are overlapped. (A) Each photo is representative of the observed cells in

each group. (B) Box plots show the total expression of Cx43 (n = 17-20). (C) Box plots show

the total expression of active Bax (left) and the ratio of active Bax localized at the

mitochondria to total active Bax expression (right) (n = 27-30). **P < 0.01, ***P < 0.001:

Significant difference from control in each siRNA treatment group, and ##

P < 0.01, ###

P <

0.001: Significant difference between siBax and NC treated with 10 μM SU by using the

Tukey-Kramer test.

39

40

Fig. 1-3-5 Effect of Bax knockdown on SU-induced alteration in cell cycle distribution and

induction of apoptosis in H28-T cells. (A) Effect of siRNA treatment on Bax protein

expression. Cells were transfected with a final concentration of 10 nM Bax-specific siRNA

(siBax) or negative control (NC) siRNA for 24 h, and protein expression was detected by

western blotting. β-Actin is shown as the internal standard. (B, C) Effect of siRNA treatment

on cell cycle distribution and induction of apoptosis detected as the pre-G0-G

1 (subG

1

population) in cell cycle distribution in the presence of SU. Cells were transfected with a

final concentration of 10 nM Bax-specific siRNA or NC siRNA for 24 h followed by treatment

with 10 μM SU or DMSO (SU 0 M) for 48 h. Each of 20,000 cells was analyzed by flow

cytometry. Each bar represents the mean percentage of cells in each population, and

vertical lines indicate S.D. of 3 independent experiments. ***P < 0.001: Significant difference

from control in each siRNA treatment group, and ###

P < 0.001: Significant difference between

siBax and NC treated with 10 μM SU by using the Tukey-Kramer test.

41

1-4 Discussion

In this chapter, the potential of Cx43, the well-studied Cx subtype, to enhance the

activity of SU in MM cells was investigated. We used a human MM cell line H28,

which has no detectable Cx43 protein expression and functional GJ. The sensitivity

to SU was compared between H28-T cells (Cx43-transfected cells) and H28-W cells

(parental cells).

First, we assessed the difference in SU-induced cytotoxicity between H28-W and

H28-T cells. The cell viability test showed that the cytotoxic effect of SU was greater

in H28-T cells than in H28-W cells (Fig. 1-1-1). In particular, the difference in cell

viability was the greatest in the presence of 10 μM SU. This concentration is high

compared to clinical plasma concentration after oral administration (0.125-0.25 M),

although it falls within the commonly used range in vitro study (36). It is reported that

tumor tissues from human and mouse administered SU contained approximately 10

M SU (37). SU is a multi-targeted tyrosine kinase inhibitor, which is hydrophobic

(logP = 5.2) and a weak base (pKa = 8.95). These characteristics allow SU to easily

pass through the plasma membrane and to be trapped in the acidic lysosome,

leading to its accumulation in cells. Thus, we considered that the concentration was

reasonable, and 10 M SU was mainly used in the following experiments.

In the evaluation of the effect of SU on the cell cycle, we observed that SU had

almost no influence except for a significant decrease in S-phase population and the

corresponding increase in G1-phase population after 24 h of SU treatment of H28-W

cells (Fig. 1-1-2A). Interestingly, when the cell cycle population was compared

between untreated H28-W and H28-T cells, a significantly higher S-phase population

was observed in H28-T cells compared with the H28-W cells at every time point. This

tendency was also observed in the previous work (14). SU treatment caused a

42

significant increase in the size of the subG1 population in both cells, and that

population of H28-T cells treated with 10 μM SU was significantly larger than in that

of SU-treated H28-W cells (Fig. 1-1-2B). Treatment with low concentrations (less

than 3.3 μM) of SU had no effect on the induction of apoptosis and cell cycle

distribution in both cells (data not shown). Based on these results, we infer that the

greatest difference in cell viability between H28-W and H28-T cells in the presence of

10 μM SU is derived from the efficient induction of cell death in H28-T cells.

Furthermore, 10 μM SU resulted in a significant decrease in the mitochondrial

membrane potential in H28-T cells, while no alteration was detected in H28-W cells

(Fig. 1-1-3). It is suggesting that SU treatment causes mitochondrial dysfunction in

H28-T cells, but not in H28-W cells.

Next, we assessed the effect of SU on RTK signaling. In H28-T cells, total Akt

expression was almost undetectable, and phosphorylation of ERK was significantly

suppressed by SU treatment (Fig. 1-1-4B, C). These results indicate that increased

Cx43 expression enables SU to effectively suppress RTK signaling. The decrease in

Akt expression most likely occurred due to the induction of apoptosis, since it is

known that Akt is cleaved and inactivated by caspases in process of apoptotic cell

death (38). Inhibition of ERK phosphorylation is thought to have contributed to the

growth inhibition, because ERK normally promotes cell cycle progression from G1- to

S-phase through upregulation of cyclin D, which contributes to cell proliferation (39).

Previously, we confirmed that total and phosphorylated ERK expressions were higher

in naïve H28-T cells than in H28-W cells (data not shown). Taken together the report

that ERK signaling is activated by GJIC (40), we infer that the GJIC-activated ERK

signaling results in a higher S-phase population in H28-T cells than in H28-W cells. In

contrast, it has been pointed out that sustained activation of ERK promotes apoptosis

(41). Liu et al. showed that ERK increased the expression of proapoptotic factors

43

such as Bax and p53-upregulated modulator of apoptosis (PUMA), through p53

phosphorylation (42). In fact, total Bax expression was higher in H28-T cells than in

H28-W cells (Fig. 1-3-2A), which is thought to be related to the rapid apoptotic

process in H28-T cells exposed to SU.

We then investigated the mechanism by which Cx43 enhances SU-induced

cytotoxicity. As mentioned above, Cx appears to have an influence on the drug

sensitivity of cancer cells through both GJ-dependent and GJ-independent

mechanisms; the GJ-dependent mechanism is known as a “bystander effect”. This is

the effect where an anti-cancer agent itself or a death signal is transmitted

throughout a tumor via GJ channels. The combination of herpes simplex virus gene

therapy and ganciclovir administration working through this effect is well documented

with a positive outcome in malignant glioma patients in a phase I clinical trial (43).

Moreover, it has been shown that the restoration of GJ function improves the anti-

proliferative effects of various types of agents including gemcitabine (44), cisplatin

(45), and sorafenib (46). The fact that GJ can pass molecules up to 1200 daltons

(47), and SU is a small molecule of approximately 400 daltons, suggests that SU

may be directly transferred between cells via GJs. To investigate the involvement of

GJ function in enhancing the cytotoxic effect of SU, a pharmacological GJ inhibitor,

18β-glycyrrhetinic acid (GA) was used in the present study. It was expected that GA

treatment would attenuate the cytotoxicity of SU in H28-T cells. However, GJ

inhibition did not modulate the viability of H28-T cells treated with low concentrations

of SU (Fig. 1-2B). The combined treatment with GA and 6 or 10 M SU rather

decreased H28-T cell viability in comparison to SU alone. The result obtained may

come from the ambiguous characteristic of GJ; GJ normally contributes to

maintaining intracellular homeostasis, and it is believed that cell damage causes

transfer of survival signals or dilution of death signals through GJ (48). In other

44

words, GJ provides both cell survival and cell death signals depending on the

situation (49). In the present study, GJ seems to play a protective role against the cell

damage that is caused by high concentrations of SU. However, there remains the

possibility that GA caused off-target effect, and it might relate to the reported

mechanism by which GA inhibits GJ function by modulating the phosphorylation

status of Cx proteins, leading to GJ disassembly (50). It relates to the activation of

protein kinase C (PKC), which has a potential to affect the function of various

proteins. Thus, while the contribution of GJ cannot be completely excluded by this

experiment, additional GJ inhibitors should be investigated in combination with SU to

conclude the involvement of GJ.

In investigating the GJ-independent mechanism, we hypothesized that Cx43

modulates the expressions or functions of some pro or antiapoptotic proteins

including Src (6) and Bax (9) in a GJ-independent manner. In the present work, we

focused on the proapoptotic factor Bax, since the induction of apoptosis was notable.

In fact, Bax was confirmed to directly interact with Cx43 (Fig. 1-3-1). A Cx43-specific

antibody reacted to several Cx43 proteins with different molecular size from H28-T

cell lysate immunoprecipitated using a Bax-specific antibody. The upper band

observed may be phosphorylated forms of Cx43, although further investigations are

needed. As shown in Fig. 1-3-2B, a Bax dimer was detected only in H28-T cells, and

SU treatment increased the expression of this form of dimeric Bax. Bax forms

dimers/oligomers at the mitochondria and results in the decrease in mitochondrial

membrane potential (51), the appearance of dimer forms indicates that Bax was

activated and promoted apoptosis in H28-T cells. The results exhibited in Fig. 1-3-2

are interesting findings; the bigger size Bax among the 2 detected bands was more

clearly expressed in H28-T cells than in H28-W cells, and a Bax dimer was detected

even in intact H28-T cells. Bax has at least 9 splice variants, and the Bax-specific

45

antibody used can detect alpha, beta, and delta isoforms. Considering that Bax alpha

is the common 21 kilodalton isoform, and only beta and omega transcripts would

yield products with higher molecular size than the alpha isoform, the bigger size

molecule may be a 24 kilodalton Bax beta isoform (52). It is reported that the Bax

beta is constantly degraded by the proteasomal system, although it has a greater

potential to cause apoptosis than the alpha isoform; it naturally translocates from the

cytosol to the mitochondria and forms oligomers there without any stimulus. It is

corresponding to the appearance of dimer in naïve H28-T cells shown in Fig. 1-3-2B.

Moreover, it seems that Cx43 interacts with the 24 kilodalton Bax isoform (Fig. 1-3-

1A). These results suggest that Cx43 interacts with Bax beta and stabilizes its

expression. To verify that Bax changes its conformation and translocates to the

mitochondria, an antibody detecting the N-terminus region of Bax, which is exposed

only in the active form, was used in immunofluorescence staining. As shown in Fig.

1-3-3, both total expression and ratio of localization at the mitochondria clearly

increased in H28-T cells after SU treatment, while there was no detectable active

Bax in H28-W cells. Moreover, the increased total expression and the subsequent

translocation to the mitochondria induced by SU in H28-T cells were significantly

decreased by the knockdown of Cx43 (Fig. 1-3-4A, C). These results suggest that

the presence of Cx43 was crucial for the process of Bax activation.

Finally, the results of the knockdown of Bax expression reduced the ratio of subG1

population in H28-T cells treated with SU, which indicates that Bax is involved in SU-

induced apoptosis (Fig. 1-3-5). It is reported that H28 cells express another

proapoptotic Bcl-2 family protein Bak (53), and it may also contribute to induction of

apoptosis in both H28-W and H28-T cells. However, the Bax knockdown in H28-T

cells showed approximately 50% reduction in apoptotic ratio, which suggests that

Bax is the major proapoptotic protein in these cells.

46

1-5 Summary

In Cx43-transfected H28 cells, the following results were obtained;

Growth inhibitory effect of SU was enhanced

Inhibitory effect of SU on RTK signaling was enhanced

Apoptosis was notably induced by SU

GJ function had little effect on enhancement of SU-induced cytotoxicity

Mitochondrial dysfunction was observed after SU treatment

Process of Bax activation was detected

(Conformational change, translocation to mitochondria, and oligomerization)

The presence of Cx43 was crucial for process of Bax activation

Bax was involved in induction of apoptosis

47

Chapter 2 JNK intermediates interaction between Cx43 and Bax

Introduction

In the previous chapter, we elucidated that the interaction between Cx43 and Bax

was a key to enhance SU-induced cytotoxicity in MM cells. To investigate the

mechanism by which Cx43 affects Bax activity, localization of Cx43 is important, as it

forms an intercellular channel and is mainly localized at the plasma membrane. On

the other hand, several studies have suggested that it is also localized at the

mitochondrial membrane (54, 55). These reports suggest that approximately 4% of

total Cx43 was localized at the mitochondria obtained from human umbilical vein

endothelial cells (HUVEC) and a whole rat ventricles, respectively. Our preliminary

data showed that Cx43 proteins were localized at the mitochondria in H28-T cells,

while most of them seemed to be near the plasma membrane. Taken the result from

Fig. 1-3-1 together, two possible pathways can be assumed as mechanisms by

which Bax was activated by Cx43; (1) Cx43 at the plasma membrane directly actives

Bax, or it serves as a docking platform for Bax and another protein for the promotion

of Bax translocation to the mitochondria. (2) Cx43 at the mitochondrial membrane

directly or indirectly stabilizes localization of Bax at the mitochondria. This study

focused on the former hypothesis, since active Bax was frequently detected not only

at the mitochondria, but also near the plasma membrane.

In this chapter, a potential involvement of c-jun N-terminal kinase (JNK), a member of

the mitogen-activated protein kinase (MAPK) family, in the interplay of Cx43 and Bax

was examined; it is known that this serine/threonine kinase phosphorylates

threonine167 in Bax and then promotes Bax translocation to the mitochondria (56). In

addition, because another MAPK family member ERK directly interacts with Cx43

(57), it is plausible that JNK may also interact with Cx43.

48

2-1 Materials and Methods

Reagents

・SP600125 (SP; Wako Pure Chemicals): 50 mM stock solution in DMSO was used.

Refer to Chapter 1-1-1 and 1-3-1 for the used other reagents.

Cell culture

Refer to Chapter 1-1-1.

Western blot analysis

Refer to Chapter 1-1-1 for the method. The dilution/incubation conditions for the used

primary antibodies were as follows: JNK2 (56G8) (Cell Signaling Technologies,

#9258) (1:5000 diluted in TBS-T containing 1% skim milk /1 h at room temperature);

Phospho-SAPK/JNK (Thr183/Tyr185) (81E11) (Cell Signaling Technologies, #4668)

(1:1000 diluted in TBS-T containing 5% BSA /overnight at 4°C).

Immunofluorescence staining

For the inhibition of JNK activity, 24 h after seeding, cells were treated with 5 or 10

μM SP for 30 min before exposure to 10 μM SU or DMSO (control) for 8 h. Refer to

Chapter 1-3-1 for the following procedure. The dilution condition for the used primary

antibody was as follows: JNK1+JNK2 (phospho T183+Y185) (Abcam, Cambridge,

UK, ab4821) (1:50).

siRNA transfection

Refer to Chapter 1-3-1 for the method of the Cx43 knockdown experiment.

Statistical analysis

Refer to Chapter 1-1-1.

49

2-2 Results

Changes in both the phosphorylation status and the total expression of JNK were

investigated by western blotting. There were 2 different bands on the membrane

detecting both phosphorylated and total JNK. In H28-W cells, no alteration was

observed due to SU treatment in both the phosphorylation status and the total

expression (Fig. 2-1). In contrast, in H28-T cells, SU treatment induced obvious

changes: increase in the total expression of JNK with smaller molecular weight and

the phosphorylation of both JNK proteins, clearly identifying JNK activation only in

Cx43-transfected H28-T cells.

Then, the effect of Cx43 knockdown on JNK activation in H28-T cells was observed.

Fig. 2-2A shows the representative images in negative control siRNA-treated cells

exposed to SU for 1 h. Weak signals derived from phosphorylated JNK were

detected in the nucleus, and stronger punctate signals were scattered in the cytosol.

Spotted signals derived from active Bax was also detected. The merged image

shows that phosphorylated JNK and active Bax were partly colocalized. More

importantly, phosphorylation of JNK was decreased, and active Bax was almost

undetectable in Cx43-specific siRNA-treated cells (Fig. 2-2B).

Finally, the relationship between JNK activation and Bax activation was investigated

by using a JNK inhibitor, SP600125 (SP). After 8 h of SU treatment, phosphorylated

JNK was strongly expressed in the nucleus. Pretreatment with SP efficiently

suppressed SU-induced JNK activation (Fig. 2-3A, B). Additionally, SP treatment also

suppressed Bax activation in a dose-dependent manner, accompanied by a slight

decrease in localization ratio at the mitochondria (Fig. 2-3C).

50

Fig. 2-1 Effect of SU on JNK activity in H28-W and H28-T cells detected by western blotting.

Total JNK (JNK) and phosphorylated JNK (p-JNK) expressions after treatment with 10 μM

SU or DMSO (Cont) for 8 h are shown. β-Actin is shown as the internal standard. Each

photo is representative of 2 or 3 independent experiments.

51

52

Fig. 2-2 Effect of Cx43 knockdown on SU-induced JNK and Bax activation in H28-T cells.

Cells were transfected with a final amount of 50 pmol Cx43-specific siRNA (siCx43) or

negative control (NC) siRNA for 24 h followed by treatment with 10 μM SU for 1 h. After

fixation, cells were treated with anti-phosphorylated JNK (p-JNK) antibody (magenta) and

anti-Bax (6A7) antibody (green). Anti-Bax (6A7) antibody recognizes active Bax. (A) NC

siRNA-treated group. Areas surrounded by quadrangles in upper images were magnified

shown as lower ones. Arrows represent locations where p-JNK and active Bax are

overlapped (white). (B) Cx43-specific siRNA-treated group. Each photo is representative

of 2 independent experiments.

53

54

Fig. 2-3 Effect of JNK inhibition on SU-induced Bax activation in H28-T cells. Cells were

treated with 5 or 10 M SP600125 (SP) or DMSO (Cont) followed by treatment with 10

μM SU or DMSO (Cont) for 8 h and incubation with MitoTracker Red to be stained for

mitochondria (red). After fixation, cells were treated with anti-phosphorylated JNK (p-JNK)

antibody (magenta) and anti-Bax (6A7) antibody (green). Anti-Bax (6A7) antibody

recognizes active Bax. Yellow areas represent where the mitochondria and active Bax are

overlapped. (A) Each photo is representative of the observed cells in each group. (B) Box

plots show the total expression of p-JNK (n = 3-9). (C) Box plots show the total expression

of active Bax (left) and the ratio of active Bax localized at the mitochondria to total active

Bax expression (right) (n = 3-9). **P < 0.01: Significant difference between Cont and SU,

and #P < 0.05,

##P < 0.01,

###P < 0.001: Significant difference from SU by using the Tukey-

Kramer test.

55

2-3 Discussion

In general, treatment with anti-cancer agents causes oxidative stress, which is one of

the major inducers of mitochondrial dysfunction. Drug-induced oxidative stress

activates JNK and p38, both of which are known as stress-activated protein kinases

(SAPKs) in the MAPK family (58, 59). This family includes ERK other than JNK and

p38, and these serine/threonine kinases have the common structure including an A-

loop (activation site phosphorylated by upstream kinases), a D-domain (allosteric

site) and a kinase domain (60). Each kinase is activated by upstream MAPK kinases,

which phosphorylates substrates like transcription factors, c-Jun and c-Fos (61). As

mentioned in the introduction of this chapter, Bax is one of the substrates for JNK

(56). Thus, we hypothesized that JNK is involved in SU-induced Bax activation.

Investigating the effect of SU on JNK activity, we verified that JNK phosphorylation

was notably increased in H28-T cells, while no alteration was observed in H28-W

cells (Fig. 2-1). Detection of JNK protein by western blotting showed 2 different

bands in both phosphorylated and total JNK. JNKs are encoded by 3 genes, and

each product produces low and high molecular weight splice variants (46-49 and 54-

57 kilodaltons, respectively) (62). JNK3 is expressed specifically in the brain (63),

and a JNK2-specific antibody was used to detect total expression, therefore, it is

rational to think that products derived from JNK2 were detected as total JNK in the

present study. Almost the same total expression level of high molecular weight

product was detected in both cells, while that of low molecular weight product was

demonstrably higher in naïve H28-T cells than in H28-W cells, and SU treatment

increased this expression in H28-T cells. There are 4 isoforms derived from JNK2, 2

of which give the high molecular weight products and the others give low molecular

weight ones. It is reported that there was a difference in the binding potential for c-

Jun among the isoforms, while there was no relationship between the binding

56

potential and molecular size (64). Moreover, all products from both JNK1 and JNK2

have activation motif (threonine-proline-tyrosine), whose dual phosphorylation

products were detected as phosphorylated JNK by the antibody used in the present

study. Thus, there is the possibility that the detected phosphorylated JNKs contain

products from both genes. Such diversity in JNK expression makes it difficult to

identify which isoform reacted to SU-induced stress without further investigations.

There still remains a question; why JNK was not activated in H28-W cells? A

potential reason may be due to the difference in metabolism between H28-W and

H28-T cells. It is reported that H28 depends on glycolysis to obtain adenosine

triphosphate (ATP) (65). The glycolytic pathway branches to the pentose phosphate

pathway, which supplies reduced nicotinamide adenine dinucleotide phosphate

(NADPH). This may result in an abundance of NADPH and cause the resistance to

SU-induced oxidative stress and the following JNK activation in H28-W cells, as

NADPH is required for scavenging reactive oxygen species (ROS) (66). On the other

hand, it is also suggested that mitochondrial Cx43 is involved in mitochondrial

respiration, which consumes oxygen (67). From these reports, it can be thought that

increased Cx43 expression at the mitochondria shifts cell metabolism preference

from anaerobic glycolysis to aerobic respiration following the increased sensitivity to

oxidative stress. Totally, the presence of Cx43 seems to promote JNK activation.

In the next step, the interaction between the phosphorylated form of JNK (derived

from both JNK1 and JNK2) and active Bax was investigated by immunofluorescence

staining. After 1 h of SU treatment, phosphorylated JNK was localized in both the

cytosol and nucleus, although expression level was low in the nucleus (Fig. 2-2A). It

can be surmised that JNK was activated in the cytosol and then translocated to the

nucleus, since active JNK was mainly localized in the nucleus after 8 h of SU

57

treatment (Fig. 2-3A). Considering that MAPKs are activated by their upstream

kinases in the cytosol and transduce the signal to transcription factors, the observed

dynamic change of phosphorylated JNK seems reasonable. Active Bax was also

sparsely detected after 1 h of SU treatment. Phosphorylated JNK and active Bax was

partly colocalized, which suggests that they directly contacted. The most important

observation in these investigations is that JNK activation was decreased and Bax

activation was inhibited by the Cx43 knockdown (Fig. 2-2B). These results suggest

that Cx43 enhances activation of both JNK and Bax at an early point. Direct inhibition

of JNK activity by a pharmacological JNK inhibitor clearly decreased active Bax

expression (Fig. 2-3B, C), suggesting that activated JNK has an impact on Bax

conformational change. It is not so surprising that the decrease in mitochondrial

localization was modest; it is assumed that once activated, Bax rapidly translocates

to the mitochondria, and therefore, JNK inhibition had a little impact on the

localization of active Bax.

Based on the results of this chapter, it is expected that Cx43 may promote activation

of JNK and the interaction of it with Bax. In future studies, the direct interaction

between Cx43 and JNK should be verified, and the mechanism by which Cx43

activates JNK needs to be investigated. Oxidative stress causes oxidation of various

kinds of proteins followed by the alteration in their function. For example, oxidation of

PKC gamma increases its kinase activity and promotes its translocation to the

plasma membrane and the interaction with Cx43 (68). Thus, it is reasonable to

expect that another kinase including PKC gamma activates JNK in cooperation with

Cx43. In addition, it should be verified that the detected “active Bax” is equivalent to

the reported “Bax phosphorylated at threonine167”. As discussed in the previous

chapter, it is important to clarify which Bax isoform interacts with Cx43, since Bax

alpha and Bax beta have a different amino acid sequence in C-terminus region, and

58

Bax beta lacks threonine167. Instead, it has threonine185, which is a candidate residue

to be phosphorylated, and there is a lack of information regarding the

phosphorylation sites in Bax beta. As for phosphorylation, there remains the

possibility that modulation of phosphorylation of serine184, which is another critical

phosphorylation site in Bax alpha, is also a key for Cx43-mediated Bax activation

(51). Thus, more investigation into the involvement of kinases other than JNK may

clarify the mechanism of the interaction between Cx43 and Bax.

59

2-4 Summary

JNK was activated by SU treatment only in Cx43-transfected cells

The presence of Cx43 was crucial for early activation of both JNK and Bax

after SU treatment

JNK activation promoted Bax activation

60

Conclusion

In this study, the significance of the interaction between Cx43 and Bax in drug-

induced apoptosis was evaluated by using a combination of Cx43-transfected MM

cells and SU. The findings obtained are summarized below.

The effect of Cx43 on SU-induced cytotoxicity was evaluated in chapter 1. 10 M SU

caused apoptotic cell death in MM cells, and it was enhanced by increased Cx43

expression. The enhancement of SU cytotoxicity due to inhibition of the RTK

signaling pathway was augmented in Cx43-transfected cells, while SU failed to

suppress that pathway in parental cells with no detectable Cx43 protein.

Furthermore, GJ function had little contribution to the mechanism by which Cx43

enhanced the cytotoxicity of SU.

Thus, the present study focused attention to Bax, a master regulator of apoptosis.

The Cx43 knockdown experiment showed that Cx43 was important for SU-induced

Bax conformational change and translocation to the mitochondria. Moreover, a Bax

dimer was detected only in Cx43-transfected cells. In Bax-specific siRNA-treated

cells, the apoptotic ratio was decreased to approximately 50% of the control post-SU

treatment, which indicates that Bax largely contributed to the promotion of apoptosis

in Cx43-transfected cells.

Taken together, these results highlight the importance of the interaction between

these proteins for induction of apoptosis.

A detailed mechanism was investigated in chapter 2. Treatment with anti-cancer

agents generally causes ROS production, which activates JNK also known as SAPK.

Considering the report that showed that phosphorylation of Bax by JNK promotes

Bax activation, we hypothesized that JNK may affect the interplay between Cx43 and

61

Bax. Investigating this aspect, we found that SU treatment increased both total

expression and phosphorylation of JNK only in Cx43-transfected cells. The

subsequent Cx43 knockdown experiment showed that Cx43 had a crucial role in

early JNK activation caused by SU. JNK inhibition efficiently reduced expression of

active Bax, which suggests that activated JNK contributed to Bax activation.

Fig. 3 illustrates the role of Cx43 in SU-induced apoptosis. The current investigations

have identified a novel Cx43-JNK-Bax apoptotic axis that enhances the cytotoxicity of

SU in MM cells.

Although a combination of other Cx subtypes and anti-cancer agents should be

investigated to generalize this axis, the current finding suggests the important role of

Cx in protein-protein interactions that are beyond its original role in GJs. Increasing

Bax function is corresponding the latest chemotherapeutic strategy to directly target

the Bax protein, and some direct Bax activators have been already identified and

showed promising results both in vitro and in vivo (69).

Fig. 3 Scheme of the mechanism by which Cx43 enhances SU-induced cytotoxicity.

62

An overview of our research depicted in Fig. 4 shows that both Cx43 and Cx32 can

enhance the drug-induced cytotoxicity. While these studies have mainly focused on

the molecular interaction of Cx protein, at the same time, the involvement of GJ also

seems to partly contribute to the enhancement of cytotoxicity of several anti-cancer

agents as shown in earlier studies by us as well as others (16, 17, 44, 45). Recently,

some groups have reported that GJ allows microRNAs to be transferred between

cancer cells or cancer cells and the neighboring normal cells, which affects

phenotypes of cancer cells including the potential of invasion and angiogenesis (70,

71). GJ-mediated direct transfer of small molecules may overcome problems,

especially in vivo, since the center of a tumor is generally far from a blood vessel and

is difficult to be directly perfused by anti-cancer agents.

There are many challenges to be resolved for application of Cx to clinical cancer

therapy: for instance, the technical challenge to selectively enhance expression or

function of Cx in target tumors. Its pluripotency raises the concern regarding off-

target and side effects. Nevertheless, Cx is one of the attractive targets for cancer

therapy, and further investigations into the role of Cx in protein-protein interactions as

well as the role in GJs will promote its application in clinical therapeutic benefits.

Fig. 4 Overview of whole our study regarding combination of Cxs and anti-cancer agents.

63

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73

List of publication

This thesis for the doctorate in pharmaceutical sciences is written based on the

original article below.

1. Uzu M, Sato H, Yamada R, Kashiba T, Shibata Y, Yamaura K, Ueno K. Effect of

Enhanced Expression of Connexin 43 on Sunitinib-Induced Cytotoxicity in

Mesothelioma Cells. J Pharmacol Sci. 128:17-26, 2015.

<Other original articles>

1. Sato H, Siddig S, Uzu M, Suzuki S, Nomura Y, Kashiba T, Gushimiyagi K, Sekine

Y, Uehara T, Arano Y, Yamaura K, Ueno K. Elacridar enhances the cytotoxic

effects of sunitinib and prevents multidrug resistance in renal carcinoma cells. Eur

J Pharmacol. 746:258-266, 2015.

2. Sato H, Uzu M, Kashiba T, Suzuki R, Fujiwara T, Okuzawa H, Ueno K. Sodium

butyrate enhances the growth inhibitory effect of sunitinib in human renal cell

carcinoma cells. Oncol Lett. (in press)

<Review Article>

1. Sato H, Uzu M. Attractive target for cancer, gap junction and its components,

connexin. Nihon Yakurigaku Zasshi. 145:74-79, 2015.

74

Acknowledgements

First and foremost, I would like to express my sincere gratitude and appreciation to

my supervisor, Professor Akihiro Hisaka for his critical advice throughout this entire

work. Many thanks for his efforts in reviewing and correcting this thesis manuscript.

Second, I would like to appreciate the great support from my teacher, Assistant

Professor Hiromi Sato. Her useful suggestions and incisive comments helped me to

complete this work. She devoted a considerable portion of her time to reading and

correcting this thesis manuscript.

After that, I would like to sincerely thank Assistant Professor Hiroto Hatakeyama for

his valuable advice.

Then, I would like to deeply thank Professor Koichi Ueno (Center for Preventive

Medical Science, Chiba University) for his continuous advice and encouragement

throughout my studies.

In addition, I would like to thank all my mates and friends at laboratory of Clinical

Pharmacology and Pharmacometrics, and Geriatric Pharmacology and Therapeutics

for their companionship and assistance.

Last but not least, I would like to express the deepest thanks to my parents for their

encouragement and support for all my life. I would never complete my work without

their dedicated assistance, and I greatly appreciate it.

75

Examiners

This thesis for the doctorate in pharmaceutical sciences was examined by the

following referees authorized by Graduate School of Medical and Pharmaceutical

Sciences, Chiba University, Japan.

Professor of Chiba University (Graduate School of Pharmaceutical Sciences)

Naoto Yamaguchi, Ph.D. (Pharm. Sci.) －Chief examiner—

Professor of Chiba University (Graduate School of Medical Sciences)

Naohiko Anzai, M.D. & Ph.D.

Professor of Chiba University (Graduate School of Medical Sciences)

Itsuko Ishii, Ph.D. (Pharm. Sci.)

Professor of Chiba University (Graduate School of Pharmaceutical Sciences)

Mahmood Ashfaq, Ph.D.