Redox Factor-1 Mediates Inflammatory Response during Tumor Promotion in Skin Epidermal JB6 Cells

doi:10.4236/ojapo.2012.13003

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Open Journal of Apoptosis, 2012, 1, 19-24

http://dx.doi.org/10.4236/ojapo.2012.13003 Published Online October 2012 (http://www.SciRP.org/journal/ojapo)

Redox Factor-1 Mediates Inflammatory Response during

Tumor Promotion in Skin Epidermal JB6 Cells

Delira Robbins1, Wenjuan Li1, Katie Humphrey2, Yunfeng Zhao1*

1Department of Pharmacology, Toxicology & Neuroscience, Louisiana State University Health Sciences Center,

Shreveport, USA

2Southwood High School, Shreveport, USA

Email: [email protected]

Received August 20, 2012; revised September 30, 2012; accepted October 19, 2012

ABSTRACT

Recently, redox factor-1 (Ref-1) has received considerable attention as an enzyme for stimulating tumor cell growth.

We hypothesized that Ref-1 is upregulated during the early stage of tumorigenesis. Utilizing both tumor promotion sen-

sitive P+ and promotion resistant P− mouse skin epidermal JB6 cells, we found that Ref-1 expression was only induced

in tumor promotion sensitive P+ cells following TPA treatment. Consistent with that, Ref-1 knockdown suppressed skin

cell transformation. Interestingly, Ref-1 knockdown suppressed nuclear translocation of NF-kappaB subunit p65, and

inhibited production of proinflammatory cytokines. These results suggest Ref-1 may promote early tumorigenesis and

serve as a target for chemoprevention.

Keywords: Ref-1; Tumorigenesis; JB6 Cells; Tumor Promotion; Chemoprevention

1. Introduction

Apurinic/apyrimidinic (AP) sites can potentially block

DNA replication and are cytotoxic and mutagenic to cells

[1,2]. Human apurinic (apyrimidinic) endonuclease 1

(APE1) plays a key role in the base excision repair path-

way (BER), and is a major repair protein for abasic sites

[3]. APE1 was later coined redox factor-1 (Ref-1) due to

its ability to sense changes in the intracellular redox

status and activate transcription factors of the stress re-

sponse pathway. APE1/Ref-1 is a ubiquitously expressed

multifunctional protein also known to regulate the tranp-

scription of stress inducible genes such as NF-



B, p53,

HIF-1



and AP-1.

APE1/Ref1 expression levels have been found to be

elevated in a number of cancers such as ovarian, cervical,

prostate, rhabdomyosarcoma and germ cell tumors and

correlated with the radiosensitivity of cervical cancers [4].

It has been suggested that APE1/Ref-1 provides an elo-

quent link between cancer, DNA repair, transcription

factor regulation, oxidative signaling and cell cycle con-

trol which suggest APE1/Ref-1 as a potential chemopre-

ventive target in tumorigenesis. In order to determine the

role of Ref-1 in tumor promotion, we used tumor promo-

tion-sensitive JB6 P+ and promotion-resistant JB6 P−

mouse skin epidermal cells to observe the differential

expression of APE1/Ref-1 following TPA treatment, and

to determine how Ref-1 plays its role in early tumori-

genesis.

2. Materials and Methods

2.1. Cell line, Reagents, Treatment

The murine skin epidermal tumor promotion sensitive

JB6 P+ and promotion resistant JB6 P− cells were pur-

chased from American Type Culture Collection (ATCC,

Rockville, MD). Cells were grown in EMEM medium

containing 4% fetal bovine serum (Hyclone), 2 mM

L-glutamine (Invitrogen), and 50 µg/ml penicillin/strep-

tomycin (Invitrogen) in a 37°C incubator under 5% CO2.

The tumor promoter phorbol ester, 12-O-tetradecanoyl-

phorbol-13-aceteate (TPA; Sigma) was prepared as a 100

mM stock solution and directly diluted in cell culture

medium, with the final concentration being 100 nM.

2.2. Total Cell Lysate

Collected JB6 P+ and P− cells were suspended in 250 µl

of phosphate buffered saline (PBS, pH 7.4) containing a

proteinase inhibitor cocktail (Calbiochem). The JB6 cells

were collected by centrifugation and resuspended in

RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS,

0.5% Na. deoxycholate and 1% Triton X-100) supple-

mented with the proteinase cocktail (5 µg/ml each of

pepstatin, leupeptin and aprotinin). Cells were sonicated

on ice for two strokes (10 sec per stroke) using a Fisher

*Corresponding author.

D. ROBBINS ET AL.

Sonic Dismembrator (Model 100, Scale 4). After incu-

bating on ice for 30 min, cell lysate was centrifuged at

18,000 × g for 20 min, and the supernatant was collected

and designated as Total Cell Lysate.

2.3. Western Blot Analysis

Protein concentrations of the samples were determined

using a colorimetric assay (BioRad Laboratories). Thirty

micrograms of total cell lysate were separated on 10%

SDS-PAGE gels. Proteins were then transferred to nitro-

cellulose membranes. The membranes were incubated

against p65, Ref-1, β-actin, and SDHB proteins (Santa

Cruz Biotechnology) to obtain the results.

2.4. Ref-1 siRNA Transfection

Cells were seeded at 2 × 105 cells per well in six-well

tissue culture plates. The cells were incubated at 37˚C in

a 5% CO2 incubator until they became 70% - 80% con-

fluent. For each transfection, 3 µl of the Ref-1 siRNA du-

plex (Santa Cruz Biotechnology,) were diluted into 100

µl of siRNA transfection medium (sc-36868, Santa Cruz

Biotechnology) and labeled as Solution A. Solution B

consisted of 6 µl of transfection reagent (Santa Cruz

Biotechnology) diluted into 100 µl of siRNA transfection

medium. Solution A and B were mixed gently and incu-

bated for 30 minutes at room temperature. The cells were

washed once with 2 ml of siRNA transfection medium.

For each transfection, 0.8 ml of siRNA transfection me-

dium were added to each tube containing the solution

A/B mixture, mixed and directly added to the washed

cells. Cells were incubated for 24 hours at 37˚C in a 5%

CO2 incubator. Immediately following, the transfection

mixture was removed and replaced with 2 ml of 1 × nor-

mal growth medium. The cells were incubated for an

additional 24 h and assayed via Western blot analysis.

Fluorescein conjugated control siRNA (Santa Cruz Bio-

technology) was used to monitor transfection efficiency.

2.5. Soft Agar Assay

The soft agar cell transformation assay was carried out in

six-well plates. The bottom agar was made using 1.25%

agar, 2 × EMEM medium, 10% FBS, PBS, glutamine,

and penicillin and was incubated in a hot water bath for

15 min. The mix was then divided and treated with vari-

ous treatments. In each well, 3.5 mL of the agar mix was

added and allowed to harden for 30 min. The top agar

mix contained 0.5% bottom agar mix and 2 × 105 of cells.

The cell treatments were added at 2 × concentration to

the top agar mix and 1 ml of each was added to each well.

The agar was allowed to solidify and incubated in a 37˚C

incubator under 5% CO2 for 14 d. Cells were stained

with neutral red dye (0.25 mg/ml) 2-(p-Iodophenyl)-3-

(p-nitrophenyl)-5-phenyl tetrazolium chloride hydrate

(Aldrich #I-1,040-6) containing 1 mL per well. The dye

was sonicated before added to cells. Cells were allowed

to stain for twenty-four hours.

2.6. Quantification of Cytokine and Chemokine

Proteins

JB6 P+ cells were transfected with siRNA to Ref-1 as

previously mentioned. Cells were collected and lysed

using RIPA buffer to isolate the total cell lysate as pre-

viously mentioned. One hundred micrograms of each cell

lysate was used for cytokine and chemokine detection

and quantification by the Quantibody Mouse Cytokine

Array 1 kit (RayBiotech, Inc.) according to the manufac-

turer’s protocol. The signal (Cy3) was captured using an

Axon Genepix laser scanner (The Research Core Fa-

cility at Louisiana State University Health Sciences Cen-

ter, Shreveport, LA). Quantitative data and statistical

analysis were performed using Prism 3.0 software.

2.7. Statistical Analysis

One-way ANOVA followed by the Newman-Keuls post-

test were used for multi-group comparisons. Experiments

were repeated at least three times. Data were reported as

mean ± standard error (S.E.M.) p < 0.05 was considered

statistically significant.

3. Results

3.1. Ref-1 Expression Was Only Induced in

Promotable JB6 P+ cells Following TPA

Treatment

We first tested whether the tumor promoting phorbol

ester, TPA, could induce Ref-1 expression in mouse skin

epidermal tumor promotion sensitive P+ and tumor pro-

motion resistant P− cells. JB6 P+ and P− cells were

treated with TPA (100 nM) for 24 hours. Ref-1 expres-

sion was assessed via Western blot analysis. Our results

(Figure 1) demonstrated that Ref-1 expression was only

induced in JB6 P+ cells compared to JB6 P− cells as

early as 24 h post TPA treatment.

3.2. Ref-1 Knockdown Suppressed TPA-Induced

Skin Cell Transformation in Tumor

Promotion-Sensitive Jb6 P+ Cells via

Suppression of TPA-Induced P65 Nuclear

Translocation

To determine whether APE1/Ref-1 plays an important

role in skin cell transformation, we knocked down

APE1/Ref-1 utilizing siRNA and observed the effects of

APE1/Ref-1 knockdown on anchorage-independent gro-

wth in soft agar. We found that knockdown of APE1/

D. ROBBINS ET AL. 21

Ref-1 expression significantly decreased TPA-induced

skin cell transformation (Figure 2).

Ref-1 is known as a redox-sensitive upstream regulator

of NF-



B. We investigated the effect of TPA-induced

Ref-1 expression on p65 expression, the transactivational

subunit of NF-



Similar to Ref-1 expression, we observed higher levels

of p65 protein expression in JB6 P+ cells compared to P−

cells. In Figure 3, we labeled phosphorylated p65 (Ser

536) with FITC staining and stained the nucleus with

DAPI to detect p65 nuclear translocation induced by

TPA treatment. To further reveal the potential mecha-

nism of Ref-1 involvement in early stage TPA-induced

tumor promotion, we knocked down Ref-1 expression

via siRNA and investigated its effect on phosphorylated

p65 TPA-induced nuclear translocation. We found fol-

lowing TPA 24 h treatment that phosphorylated p65

(Ser536) translocated to the nucleus. Interestingly, Ref-1

knockdown suppressed phosphorylated p65 nuclear tran-

slocation.

Figure 1. TPA-induced Ref-1 activation was higher in pro-

motable JB6 P+ cells compared to non-promotable JB6 P−

cells. JB6 P+ and P− cells were treated with TPA (100 nM)

for 1 or 24 h (T/1h; T/24 h). Whole cell lysate was prepared

for the experiments. DMSO: 0.1% DMSO for 24 h. Western

blot analysis of Ref-1, and p65 protein expression. β-actin,

was used as a loading control. Experiments were repeated

three times and a representative result is shown.

Figure 2. Knockdown of Ref-1 suppressed TPA-induced

skin cell transformation. (A) Soft agar colony formation

assay was performed using siRNA-transfected cells. (B)

Quantification of soft agar assay. siDMSO: siRNA Ref-1

transfected cells treated with 0.1% DMSO; siTPA: siRNA

Ref-1 transfected cells treated with TPA (5 nM). * p< 0.05

significantly different from DMSO treatment group; ** p<

0.05. significantly different from TPA treatment group.

Experiments were repeated three times and a representa-

tive result is shown.

Figure 3. Knockdown of Ref-1 suppressed TPA-induced

phosphorylated p65 nuclear translocation. JB6 P+ cells

were treated with TPA (100 nM) for 24 h. (A) Western blot

analysis of Ref-1 siRNA transfection. (B) Immunofluores-

cence of JB6 P+ cells. FITC green (phosphorylated p65);

DAPI (nuclear staining). Experiments were repeated three

times and a representative result is shown. 20X magnifica-

tion.

3.3. Ref-1 Knockdown Suppressed Cytokine

Expression in Tumor Promotion-Sensitive

JB6 P+ Cells

Previous reports that investigated the influence of in-

flammation on carcinogenesis were concerned with the

early stages of tumor development (i.e. tumor initiation

and promotion). In addition, the pathogenesis of tumor

development has been generally thought to occur either

intrinsically through genetic alterations or extrinsically

through external factors to stimulate tumor cells to re-

lease inflammatory mediators [5]. To gain further insight

into how APE1/Ref-1 may affect early stage tumor pro-

motion, we progressed forward by assessing the effects

of APE1/Ref-1 knockdown on NF-



B-mediated cyto-

kine/chemokine expression following TPA treatment in

tumor promotion-sensitive JB6 P+ cells. Interestingly,

we found that TPA treatment induced an inflammatory

response of pro-inflammatory cytokines/chemokines, ma-

inly those involved in leukocyte infiltration. In Figure 4,

TPA 24 h treatment significantly increased KC, mono-

cyte chemotactic protein-1 (MCP-1) and Regulated upon

Activation, Normal T-cell Expressed and Secreted (RAN

TES), IL-3 and IL-4. KC, also known as CXC1, is se-

creted by human melanoma cells and has mitogenic

properties. This cytokine is a main attractor of neutron-

phils and expression is mediated by NF-



B in mice [6].

MCP-1 expression has been observed in various inflame-

matory diseases [7-9] and tumors [10]. This chemokine is

directly induced by TPA treatment and plays a role in the

chemotaxis of macrophages [11]. Lastly RANTES, is a

chemokine for T cells, eosinophils and basophils and

plays an active role in reactive oxygen species produc-

ntion and recruitment of leukocytes to inflammatory sites

[12]. Interestingly, knockdown of Ref-1 in both DMSO

and TPA treated groups significantly suppressed the ex-

pression of these cytokines/chemokines. Nevertheless,

knockdown of Ref-1 significantly suppressed expression

of both proinflammatory cytokines/chemokines and de-

reased anchorage-independent growth of JB6 P+ cells. c

D. ROBBINS ET AL.

Figure 4. Knockdown of Ref-1 suppressed TPA-induced cytokine release in tumor promotable JB6 P+ cells. Quantification of

pro-inflammatory cytokines/chemokines. siDMSO: siRNA Ref-1 transfected cells treated with 0.1% DMSO; siTPA: siRNA

Ref-1 transfected cells treated with TPA (5 nM). *p< 0.05 significantly different from DMSO treatment group; **p< 0.05 sig-

nificantly different from TPA treatment group. Experiments were repeated at least three times and a representative result is

shown.

4. Discussion cal cysteine residues of NF-



B [17], we further investi-

gated the correlation of APE1/Ref-1 and NF-



B expres-

sion in early stage skin tumor promotion by comparing

the expression of the NF-



B transactivation subunit p65

in both promotion-sensitive (P+) and promotion-resistant

(P−) cells. Elevated levels of p65 following TPA treat-

ment were consistent with elevated levels of APE1/Ref-1

that occurred only in promotion-sensitive P+ cells, sug-

gesting that APE1/Ref-1 and NF-



B are involved in

early stage tumor promotion in skin carcinogenesis. We

demonstrated that targeting APE1/Ref-1 in early stage

tumor promotion can have direct effects on proinflam-

matory signaling and skin cell transformation. In addi-

tion, knockdown of APE1/Ref-1 significantly reduced

the expression of cytokines/chemokines and the colony

forming ability of promotion-sensitive (P+) cells follow-

ing TPA treatment. There are two common possibilities

to explain the differential expression of APE1/Ref-1 in

promotion-sensitive (P+) and promotion-resistant (P−)

cells and the ability of APE1/Ref-1 knockdown to de-

crease TPA-induced colony formation: 1) the current

antioxidant status of the cell type; 2) a decrease in the

stress response. Our previous studies have shown that

chemoprevention via an antioxidant approach is a novel

yet practical strategy to suppress early stage skin car-

cinogenesis [18]. In addition, we have shown that pro-

motion-resistant P− cells have higher MnSOD expression

APE1/Ref-1 is ubiquitously ex pressed in cells with cell

type specific cellular localization [13]. In addition, both

expression and subcellular localization of APE1/Ref-1

are altered in hyperproliferative disorders such as aging

and tumors. It has also been suggested that APE1/Ref-1

expression is inversely correlated with apoptosis sug-

gesting that APE1/Ref-1 may contribute to early stage

tumor promotion. We chose to assess the redox activity

of APE1/Ref-1 in modulating NF-



B nuclear transloca-

tion and its effect on cytokine release and skin cell

transformation. We utilized the murine JB6 skin epider-

mal cell system to determine the role of APE1/Ref-1 in

early stage tumorigenesis and whether APE1/Ref-1 is a

potential novel target for chemoprevention. The mouse

Balb/C JB6 cells [14] are the only well-characterized

model to study tumor promotion. JB6 cells have two

clone variants: tumor promotion sensitive P+ cells and

promotion resistant P− cells. Herein, we found that tumor

promotion sensitive JB6 P+ cells expressed higher APE1/

Ref-1 protein levels following TPA treatment which cor-

relates with the current literature that suggests that

APE1/Ref-1 expression is higher in cancer cells com-

pared to their normal counterparts [15,16].

NF-



B is a known transcription factor that is redox

regulated by APE1/Ref-1 through reduction of the criti-

D. ROBBINS ET AL. 23

and activity when compared with promotion-sensitive P+

cells. In addition, several studies have shown that MnSOD

may act as a novel tumor suppressor gene and that over-

expression of MnSOD can significantly decrease tumor

promotion and key oncogenic survival and pro-inflam-

matory signaling pathways such as activator protein 1

(AP-1) and NF-



B [19-21]. Therefore, regulating the int-

racellular redox status has the potential to augment the

effects of current chemotherapeutic treatments and also

block survival and pro-inflammatory pathways in tumor

promotion sensitive cancer cells.

On the contrary, several studies have suggested that

knockdown of APE1/Ref-1 can lead to apoptosis. APE1

/Ref-1 is known to be induced by oxidative stress [22].

However, APE1/Ref-1 expression has been shown to be

inversely correlated with apoptosis, suggesting an anti-

apoptotic function of the multifunctional protein [23].

Unnikrishnan and colleagues presented data that oxida-

tive stress can alter the function of APE1/Ref-1 and the

apoptotic response was increased in APE/Ref-1 haploin-

sufficient mice [24]. Within this study heterozygous de-

letion of APE1/Ref-1 resulted in decreased NF-



B DNA

binding activity, accompanied by increases in GADD45

gene expression, p53 stability and caspase activity [24].

Previous studies have also shown that loss of APE1

/Ref-1 resulted in increased TNF-induced apoptosis [25].

In addition, homozygous deletion of the APE1/Ref-1

gene is embryonic lethal, suggesting the importance of

APE1/Ref-1 expression in cell survival. Therefore, mo-

dulating APE1/Ref-1 expression can play either a cell

survival or apoptotic role in tumorigenesis, but yet iden-

tifies APE/Ref-1 as a key controller of intracellular redox

changes in chemoprevention. Clinically, APE1/Ref-1 va-

riants have been identified in the human population [26];

and these variants have been associated with increased

cancer risk. Together, these data point toward an ap-

proach to improve drug target specificity and personal-

ized medicine in the prevention and treatment of tumors

with upregulated APE1/Ref-1 expression. Nevertheless,

further studies are ongoing to determine whether the sig-

nificant decrease seen in the colony-forming ability of

JB6 P+ cells was due to apoptosis or changes in mito-

chondrial function. So far, we have not found any associ-

ated toxicity following siRNA transfection and no indi-

cations of an apoptotic phenotype in cells that underwent

transfection. Moreover, we will continue to assess the

role of APE1/Ref-1 redox activity in early tumor promo-

ntion by utilizing known antioxidant-inducing natural

products to determine whether antioxidant expression

can modulate the redox functions of APE1/Ref-1 in early

stage skin carcinogenesis. In conclusion, our results sug-

gest that Ref-1 promotes early tumorigenesisby via con-

tributing to NF-κB activation and the pro-inflammatory

response. Therefore, Ref-1 might be able to serve as a

novel target for chemoprevention.

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Abbreviations

AP, Apurinic/apyrimidinic; AP-1, activator protein 1;

APE1, apurinic (apyrimidinic) endonuclease 1; DMSO,

dimethyl sulfoxide; FBS, fetal bovine serum; MCP-1,

monocyte chemotactic protein-1; MnSOD, manganese

superoxide dismutase; NF-κB, nuclear factor kappa B;

PBS, phosphate buffered saline; Ref-1, redox factor-1;

ROS, reactive oxygen species; SDHB, succinate dehy-

drogenase subunit B; siRNA, small interfering RNA;

TNF, tumor necrosis factor; TPA, 12-O-tetradecanoy-

lphorbol-13-aceteate.