DNA-PKcs deficiency sensitizes the human hepatoma HepG2 cellsto cisplatin and 5-fluorouracil through suppression of the PI3K/Akt/NF-jB pathway

Yuan Fang • Zongtao Chai • Dansong Wang •

Tiantao Kuang • Wenchuan Wu • Wenhui Lou

Received: 11 July 2014 / Accepted: 17 October 2014

� Springer Science+Business Media New York 2014

Abstract The aim of the present study was to investigate

the effects of DNA-PKcs deficiency on the chemosensi-

tivity of human hepatoma HepG2 cells to cisplatin (CDDP)

and 5-fluorouracil (5-Fu), and to explore the underlying

molecular mechanism. After transfection with DNA-PKcs

siRNA or control siRNA, HepG2 cells were exposed to

combination treatment of CDDP and 5-Fu. The cell via-

bility, DNA damage, cell apoptosis, intracellular reactive

oxygen species and glutathione (GSH) level, expression of

apoptosis related proteins, activity of phosphatidylinositol

3-kinase/protein kinase B (PI3K/AKT) pathway, and

nuclear factor-jB (NF-jB) pathways were assessed. The

combination of CDDP and 5-Fu had a synergistic cytotoxic

effect in HepG2 cells in terms of the cell viability, DNA

damage, apoptosis, and oxidative stress level. DNA-PKcs

siRNA could sensitize the HepG2 cells to the combined

treatment. DNA-PKcs suppression further reduced the Akt

phosphorylation level and Bcl-2 expression in HepG2 cells

exposed to CDDP and 5-Fu, but enhanced the expression of

pro-apoptotic proteins p53 and caspase-3. Moreover,

CDDP could inhibit the transcriptional activity of NF-jB

through degradation of IkB-a, while 5-Fu alone seemed in

some extent increases the NF-jB activity. The combined

treatment with CDDP and 5-Fu resulted in significantly

decrease of the transcriptional activity of NF-jB, which

was further aggravated by DNA-PKcs siRNA treatment. In

conclusion, DNA-PKcs suppression had complementary

effects in combination with CDDP and 5-Fu treatment in

HepG2 cells, which was associated with suppression of

NF-jB signaling pathway cascade, activation of caspase-3

and p53, as well as down-regulation of Bcl-2 and GSH.

Keywords DNA-PKcs � HepG2 cells � Apoptosis �Oxidative stress � NF-jB � PI3K/AKT pathway � Bcl-2 �GSH

Introduction

Hepatocellular carcinoma (HCC) is one of the most com-

mon malignancies worldwide and the third leading cause of

cancer-induced mortality. Hepatocellular carcinoma has an

increasing incidence in recent years, especially in the

developing Asian countries [1]. Clinically, most patients

are not suitable for surgical resection due to the advanced

stage at presentation, extensive disease, or poor liver

function [2]. Therefore, the systemic chemotherapy is a

widely used treatment option for HCC, and current che-

motherapy agents include fluorouracil (5-Fu), cisplatin

(CDDP), doxorubicin, mitomycin C, and so on [3, 4].

However, the effectiveness of chemotherapy using

individual antitumor agent is practically unsatisfactory

because of the low response rate usually below 20 % and

the severe chemoresistance [5, 6]. Recently, several groups

have used combination of CDDP and 5-Fu for the

advanced HCC, indicating favorable results with an overall

20–30 % response rate [7]. It was reported that CDDP and

5-Fu were synergistic in vitro [8], and CDDP could

Yuan Fang and Zongtao Chai have contributed equally to this work.

Y. Fang � D. Wang � T. Kuang � W. Wu � W. Lou (&)

Department of General Surgery, Zhongshan Hospital, Fudan

University, Shanghai 200032, People’s Republic of China

e-mail: louzsh@126.com

Z. Chai

Key Laboratory of Carcinogenesis and Cancer Invasion,

Ministry of Education, Zhongshan Hospital, Liver Cancer

Institute, Fudan University, Shanghai 200032

People’s Republic of China

123

Mol Cell Biochem

DOI 10.1007/s11010-014-2253-6

enhance the cell growth inhibition by 5-Fu through

increasing the intracellular folate pool and activating a Fas-

mediated receptor signaling pathway [9]. Practically, the

combination of 5-Fu and CDDP has become a conventional

chemotherapy strategy for the advanced HCC [10, 11].

However, the antitumor efficiency of the combined treat-

ment still needs to be elevated, and the underlying

molecular mechanisms should be better understood.

The PI3K/Akt pathway is frequently activated in various

malignancies, which consequently activates the prosurvival

pathways and enhances the chemoresistance through NF-

jB activation and p53 inhibition [12, 13]. Arenobufagin

induces apoptosis and autophagy in the HCC cells through

inhibition of PI3K/Akt pathway [14]. The combination of

5-Fu and wogonin could inhibit cyclooxygenase-2

expression and increase the chemosensitivity through reg-

ulation on the PI3K/Akt signaling pathway [15]. CDDP

also exerts the anticancer effects through inhibition of NF-

jB transcriptional activity [16]. The interactions between

PI3K/Akt and NF-jB signaling pathways have become

research focus to explore effective complementary

approaches for cancer therapy [17, 18].

DNA-dependent protein kinase catalytic subunit (DNA-

PKcs) is the key component of DNA-PK and mediates

DNA double-strand break (DSB) repair in response to

DNA damage. Suppression of DNA-PKcs through DNA-

PKcs inhibitor (NU7441) resulted in defects of DNA-PKcs

autophosphorylation at Ser 2056, sensitizing the chronic

lymphocytic leukemia cells to mitoxantrone treatment [19].

The ATM-mediated DNA-PKcs activation is involved in

sustained survival of breast cancer MCF-7 cells under

genotoxic stress induced by CDDP treatment [20]. Panta

et al. also proved that DNA-PKcs activation could induce

the IKK/NF-jB activity and promote cell survival in

murine embryo fibroblasts upon anthracycline doxorubicin

treatment [21]. Thus, we hypothesized that DNA-PKcs

suppression might have an adjuvant therapeutic signifi-

cance in improving the chemotherapy efficiency for HCC

with the existing agents CDDP and 5-Fu.

Materials and methods

Chemicals and reagents

Cisplatin (CDDP) and 5-fluorouracil (5-Fu) were pur-

chased from Selleck Chem (Houston, Texas, USA). Dul-

becco’s modified Eagle’s medium (DMEM), fetal calf

serum (FBS) were obtained from Invitrogen (Paisley, UK).

Cell counting kit-8 (CCK-8) was from Dojindo (Kuma-

moto, Japan). Mammalian protein extraction reagent (M-

PER), nuclear and cytoplasmic extraction reagent kit (NE-

PER), and bicinchoninic acid (BCA) protein assay kit were

from Thermo Fisher Scientific Inc (Waltham, MA, USA).

Nitrocellulose membrane was purchased from Millipore

(Darmstadt, Germany). DCFH-DA (20,70-dichlorofluores-

cein diacetate) was from Sigma (Saint Louis, MO, USA).

GSH assay kit was purchased from Jiancheng (Nanjing,

China). Chemiluminescent EMSA Kit and Biotin-labeled

NF-jB probes were from Beyotime (Wuhan, China). NF-

jB p65, phospho-IKKa/b, and IKKa/b, p-IjBa and IjBa,

p-Akt (Ser473), and Akt, p53, Caspase-3, and Bcl-2 anti-

bodies were from Cell Signaling (Beverly, MA, USA);

DNA-PKcs antibody was from Santa Cruz (Dallas, Texas,

USA). GAPDH antibody was from Multisciences Bio-

technology (Hangzhou, China); anti-Rabbit IgG (H ? L)/

HRP and anti-Mouse IgG (H ? L)/HRP were from Ding-

guo Biotechnology (Beijing, China). All other analytical

grade reagents were obtained from Sigma (Saint Louis,

MO, USA) unless specified otherwise. Absorbance was

recorded using a Multiskan Mk3 plate reader (Thermo

Electron Corporation, Waltham, MA, USA).

Cell culture and treatments

Human hepatoma HepG2 cells were obtained from the

American Type Culture Collection (ATCC, Manassas, VA)

and cultured in DMEM (high glucose concentration) sup-

plemented with 10 % FBS, 0.33 % sodium bicarbonate,

and antibiotics (100 units/ml penicillin and 0.1 mg/ml

streptomycin) at 37 �C in a 95:5 air/CO2 water-saturated

atmosphere. The cells were passaged every 3 days and

used for experiments when in the exponential growth

phase. CDDP and 5-Fu were dissolved in dimethyl sulf-

oxide (DMSO), and controls were treated with the vehicles

only. HepG2 cells were treated with different concentra-

tions of CDDP (0, 1, 2, 5, 10, 20, and 50 mg/L) or/and 5-Fu

(0, 5, 10, 20, 50, 100, and 200 mg/L) for 24 h. All mea-

surements were performed in triplicate.

RNA interference

The DNA-PKcs siRNA (h) (containing a pool of two

target-specific 19–25 nt siRNAs designed to knockdown

the expression of human DNA-PKcs) and a control siR-

NA were purchased from Santa Cruz (Dallas, Texas,

USA). Cells were seeded overnight and then transfected

with siRNA for 48 h using Lipofectamine-2000 (Invitro-

gen, Paisley, UK) according to the manufacturer’s

instruction. The negative control cells (HepG2-Sc) were

transfected with a non-targeting siRNA (control siRNA)

with the same transfection procedure. Cells were then

treated with CDDP and/or 5-Fu for 24 h, and harvested

for following analysis.

Mol Cell Biochem

123

Cell viability

HepG2 cells were seeded in 96-well plates at a density of

3,000 cells per well. Exponentially growing cells were

allowed to attach overnight before being exposed to vari-

ous concentrations of CDDP and 5-Fu, and incubated for

another 24 h. Cell viability was measured using the cell

counting kit-8 (CCK-8) assay according to the manufac-

turer’s instruction. Absorbance was measured with a

spectrophotometric plate reader at 450 nm.

Comet assay

DNA single-strand breaks (SSB) were evaluated with the

alkaline single-cell gel electrophoresis [22]. Briefly, normal

melting point agarose (NMA, 1 %) was prepared on the

frosted microscope slides. Then cells were mixed with low

melting point agarose (LMA, 1 %) and were dripped onto the

pre-coated NMA layers. The third LMA layers were coated

on the cell mixture layer. After lysis for 1 h, the cell-con-

taining slides were soaked into the electrophoresis solution

(300 mM NaOH, 1 mM Na2 EDTA, pH [ 13) for 40 min,

and then subjected to electrophoresis (25 V, *300 mA) for

20 min and subsequent neutralization with Tris–HCl

(400 mM, pH 7.5). Finally, cells were stained with PI (5 mg/

L), and the tail moment (% Tail DNA) was evaluated under a

fluorescence microscope (Olympus BX-51, Japan).

Apoptosis analysis

The apoptosis of HepG2 cells were measured by Hoechst

33342 staining and flow cytometry using Annexin V-FITC/

PI assay according to the operating instructions. After

treatment, approximate 106 cells were harvested and washed

twice with cold PBS solution. Then cells were stained with

1 mg/L of Hoechst 33342 for 15 min at 37 �C. Cells with

apoptotic nuclei (i.e., condensed or fragmented) were

counted in comparison with total population (n = 300 cells).

For the flow cytometry analysis, cells were suspended with

400 lL binding buffer, incubated with 5 lL of Annexin

V-FITC at 37 �C for 15 min and 10 lL of PI at 37 �C for 5

min in the dark. The stained cells were immediately analyzed

by flow cytometry (Becton–Dickinson, San Jose, USA).

Determination of reactive oxygen species (ROS)

and glutathione (GSH)

The intracellular ROS level was assessed by the fluorescent

probe DCFH-DA according to the manufacturer’s instruc-

tion. The fluorescence intensity was observed under a

fluorescence microscope (Olympus BX-51, Tokyo, Japan)

and analyzed by Image-pro plus 6.0 software. GSH activity

was detected by the dithio-bis-nitrobenzoic acid (DTNB)

assay using colorimetry at 420 nm. Absorbance was

recorded using a Multiskan Mk3 plate reader. All experi-

ments were carried out at least three times.

Western blotting

HepG2 cells after various treatments were lysed to obtain dif-

ferent cell extracts using the NE-PER kit or M-PER kit. Protein

concentrations were determined by the BCA protein assay.

Equal amounts of protein samples were separated by sodium-

dodecylsulfate-polyacrylamide gel electrophoresis, and trans-

ferred onto nitrocellulose membranes using the Trans-Blot SD

Electrophoretic Transfer Cell (Bio-Rad Hercules, CA, USA).

The membranes were then blocked and incubated with primary

antibodies and corresponding second antibodies according to

the manual. Finally, blots were visualized through enhanced

chemiluminescence and quantized using the Chemi-Imager

digital imaging system (Alpha Innotech, San Leandro, USA).

GAPDH was used as the internal inference, and each experi-

ment was repeated at least three times.

Electrophoretic mobility-shift assay (EMSA)

Nuclear extracts were prepared with the NE-PER Kit, and

the DNA–protein binding reactions were conducted using

the chemiluminescent EMSA Kit according to the manu-

facturer’s instructions. NF-jB consensus oligo sequences

were as follows: 50-AGT TGA GGG GAC TTT CCC AGG

C-30 and 30-TCA ACT CCC CTG AAA GGG TCC G-50.Sample response system (a total volume of 10 lL) inclu-

ded 5 lL of nuclease-free water, 2 lL of EMSA/Gel-Shift

binding buffer (59), 2 lL of purified nuclear protein, and

0.5 lL of biotin-labeled NF-jB probe. The reaction mix-

tures were loaded on 4 % non-denaturing polyacrylamide

gels, which were then blotted onto nylon membranes and

subjected to chemiluminescence detection.

Statistical analyses

All experiments were performed in triplicate, and data

were expressed as mean ± SD. All data were subjected to

Analysis of Variance (ANOVA) and Tukey’s post hoc test

for comparisons. A p value of \0.05 was considered to

indicate statistical significance.

Results

DNA-PKcs suppression inhibited cell survival

of HepG2 exposed to CDDP and 5-Fu

HepG2 cells were exposed to different concentrations of

CDDP or/and 5-Fu, and CCK-8 assay was conducted to

Mol Cell Biochem

123

Fig. 1 DNA-PKcs suppression sensitizes HepG2 cells to CDDP and

5-Fu. HepG2 cells were treated with different concentrations of

CDDP and 5-Fu for 24 h, and the cell survival rates were measured

with CCK-8 assay. a Cells were treated with various concentrations of

CDDP (0, 1, 2, 5, 10, 20, and 50 mg/L). b Cells were treated with

different concentrations of 5-Fu (0, 5, 10, 20, 50, 100, and 200 mg/L).

c HepG2 cells were transfected with DNA-PKcs siRNA, and then

exposed to 2 mg/L CDDP or/and 10 mg/L 5-Fu. Cont HepG2 cells

treated with vehicles only. Sc HepG2 cells transfected with the

negative control siRNA. Si HepG2 cells transfected with the DNA-

PKcs siRNA. C-2 HepG2 cells were exposed to 2 mg/L CDDP for

24 h. F-10 HepG2 cells were exposed to 10 mg/L 5-Fu for 24 h.

C ? Sc Cells transfected with the control siRNA were treated with

2 mg/L CDDP. C ? Si Cells transfected with the DNA-PKcs siRNA

were treated with 2 mg/L CDDP. F ? Sc Cells transfected with the

control siRNA were treated with 10 mg/L 5-Fu. F ? Si Cells

transfected with the DNA-PKcs siRNA were treated with 10 mg/L

5-Fu. C ? F HepG2 cells were treated with 2 mg/L CDDP and

10 mg/L 5-Fu for 24 h. C ? F?Sc HepG2 cells were transfected with

the control siRNA, and then treated with 2 mg/L CDDP and 10 mg/L

5-Fu for 24 h. C ? F?Si HepG2 cells were transfected with the

DNA-PKcs siRNA, and then treated with 2 mg/L CDDP and 10 mg/L

5-Fu for 24 h. *p \ 0.05, **p \ 0.01 versus Cont group; #p \ 0.05

versus C-2 or F-10 group; ^p \ 0.05 versus C ? F?Sc group

Mol Cell Biochem

123

determine the cell viability. As shown in Fig. 1a and b,

CDDP and 5-Fu induced decrease of cell viability in a

concentration-dependent manner. The cell viability rates

were approximately 78.6 and 80.3 % under treatment of

2 mg/L CDDP and 10 mg/L 5-Fu, respectively. Compared

with the individual treatment group, the combined treat-

ment group with CDDP and 5-Fu showed a more severe

inhibition on cell viability with an average survival rate

about 65.2 %. In addition, transfection of DNA-PKcs

siRNA or control siRNA alone had no obvious inhibiting

effect on the cell viability. However, DNA-PKcs siRNA

(not control siRNA) resulted in further decrease of cell

survival (with an elevated efficiency by 20–30 %) induced

by CDDP and/or 5-Fu treatment, as shown in the C ? Si,

F ? Si, and C ? F?Si groups (Fig. 1c). In the following

experiments, we mainly focused on the effects of DNA-

PKcs siRNA in the combined treatment of CDDP and 5-Fu

to HepG2 cells.

DNA-PKcs suppression increased CDDP and 5-Fu

induced apoptosis and DNA damage

Hoechst 33342 staining and Annexin V-FITC/PI double

staining assay were used to measure the cell apoptosis

ratio. As shown in Fig. 2, the cell apoptosis ratio of 2 mg/L

CDDP and 10 mg/L 5-Fu groups were significantly higher

than that of control groups (p \ 0.01). The combination of

CDDP and 5-Fu treatment had a synergistic effect on the

cell apoptosis (p \ 0.01), and DNA-PKcs siRNA further

increased the apoptosis ratio (p \ 0.01). Similarly, the

results of comet assay showed that CDDP and 5-Fu could

induce DNA damage, combined treatment with CDDP and

Fig. 2 DNA-PKcs suppression increased the cell apoptosis induced

by CDDP and 5-Fu. a Hoechst 33342 staining was used to measure

the cell apoptosis ratio. Cells with apoptotic nuclei (i.e., condensed or

fragmented) were counted in comparison with total population

(n = 300 cells). b Annexin V-FITC/PI double staining assay was

used to measure the cell apoptosis ratio. Data were expressed as

Mean ± SD from three independent flow cytometry experiments.

c The represented figures of annexin V-FITC/PI double-staining

assay. Cont HepG2 cells treated with vehicles only. Sc HepG2 cells

transfected with the negative control siRNA. Si HepG2 cells

transfected with the DNA-PKcs siRNA. C-2 HepG2 cells were

exposed to 2 mg/L CDDP for 24 h. F-10 HepG2 cells were exposed

to 10 mg/L 5-Fu for 24 h. **p \ 0.01 versus Cont group; ##p \ 0.01

versus C-2 or F-10 group; ^p \ 0.05, ^^p \ 0.01 versus C ? F?Sc

group

Mol Cell Biochem

123

5-Fu synergistically increased the DNA damage

(p \ 0.01). DNA-PKcs siRNA (not control siRNA) further

aggravated the DNA damage in HepG2 cells exposed to

CDDP and 5-Fu (p \ 0.05 Fig. 3).

DNA-PKcs suppression mediated the intracellular ROS

and GSH content

To investigate whether ROS level in HepG2 cells exposed

to combined CDDP and 5-Fu treatment was influenced by

DNA-PKcs siRNA, fluorescent analysis was performed

with a cell-permeable fluorescent probe DCFH-DA. Com-

pared with the control group, ROS production was mark-

edly elevated by CDDP and 5-Fu individual or combined

treatment, and combination of DNA-PKcs inhibition

resulted in further higher level of intracellular ROS

(Fig. 4a). As one of the free radical scavengers, however,

the GSH content was markedly reduced by CDDP and 5-Fu

treatment. DNA-PKcs siRNA could induce further

decrease of GSH level, indicating that alteration of oxi-

dative stress response might be involved in chemo-sensi-

tization mediated by DNA-PKcs siRNA (Fig. 4b).

DNA-PKcs suppression reduced Akt phosphorylation

and altered expression of apoptosis related proteins

Phosphorylation of Akt was investigated to determine

whether the PI3K/Akt signaling pathway was involved in

the chemo-sensitivity of HepG2 cells to CDDP and 5-Fu;

Fig. 3 DNA-PKcs siRNA augments DNA damage induced by CDDP

and 5-Fu. DNA single-strand breaks (SSB) were evaluated with

alkaline single-cell gel electrophoresis. The cell slides were stained

with PI (5 mg/L) and Tail moments were evaluated under a

fluorescence microscope. a Representative images of comet assay

performed on HepG2 cells exposed to CDDP or/and 5-Fu. b DNA

damage was evaluated by the tail moment. Each bar represents

mean ± SD from 300 cells of a representative experiment.

**p \ 0.01 versus Cont group; ##p \ 0.01 versus C-2 or F-10 group;

^p \ 0.05 versus C ? F?Sc groupFig. 4 DNA-PKcs suppression mediated ROS production and GSH

content in HepG2 cells exposed to CDDP and 5-Fu. a DNA-PKcs

inhibition promoted ROS production in HepG2 cells treated with

indicated concentrations of CDDP and 5-Fu. DCFH-DA fluorescent

analysis was performed to assess the ROS level. Data presented were

mean ± SD of three independent experiments. b DNA-PKcs inhibi-

tion induced further decrease of GSH level in HepG2 cells after

CDDP and 5-Fu treatment. Data were mean ± SD of three indepen-

dent experiments. *p \ 0.05, **p \ 0.01 versus Cont or Sc group;

#p \ 0.05, ##p \ 0.01 versus C-2 or F-10 group; ^p \ 0.05 versus

C ? F?Sc group

Mol Cell Biochem

123

Western blot analysis showed that CDDP and 5-Fu indi-

vidual or combined treatment reduced the phosphorylation

level of Akt in HepG2 cells after 24 h exposure, which was

most evident in the combined treatment group under

depression of DNA-PKcs, although no obvious change was

found in the total amount of Akt protein. At the same time,

the expression of anti-apoptotic protein Bcl-2 was signifi-

cantly down-regulated, while the pro-apoptotic factors p53

and caspase-3 were obviously elevated in the combined

treatment group (Fig. 5).

DNA-PKcs suppression modulated the activity of NF-

jB signaling pathway

As a primary transcription factor, NF-jB activation could

lead to fairly rapid changes in the cell growth-related gene

expressions when respond to harmful cellular stimuli [23].

In the present study, we measured the transcriptional

activity of NF-jB using the chemiluminescent EMSA

assay. In HepG2 cells, CDDP individual treatment inhib-

ited the transcriptional activity of NF-jB, while 5-Fu

induced slightly increase of NF-jB activity. In the com-

bined treatment group, the transcriptional activity of NF-

jB obviously reduced, which was consistent with the

protein expression of NF-jB in the nuclei (Fig. 6b).

Moreover, DNA-PKcs suppression further depressed the

NF-jB transcriptional activity in the combined treatment

group (Fig. 6a).

The phosphorylation of IjB kinases (IKK-a/b) and IjB-

a is prerequisite for the activation of NF-jB [23]. As

shown in Fig. 6b, the phosphorylated IKK-a/b and IjB-aobviously reduced in the individual CDDP-treated HepG2

group, but not in the individual 5-Fu-treated group. The

combined treatment with CDDP and 5-Fu significantly

reduced the phosphorylation level of IKK-a/b and IjB-a,

which was lower than both these two individual treatment

groups. DNA-PKcs suppression further reduced the phos-

phorylation of IKK-a/b and IjB-a in the HepG2 cells

exposed to CDDP and 5-Fu treatment.

Discussion

Numerous experimental and clinical studies have revealed

that combination of CDDP and 5-Fu has a pronounced

Fig. 5 Influence of DNA-PKcs suppression on Akt phosphorylation

and expression of apoptosis related proteins. HepG2 cells were

exposed to 2 mg/L CDDP or/and 10 mg/L 5-Fu for 24 h. Protein

expressions were analyzed by Western blot. GAPDH was used as the

internal reference. Cont HepG2 cells were treated with vehicles only.

Sc HepG2 cells were transfected with the negative control siRNA. Si

HepG2 cells were transfected with the DNA-PKcs siRNA. C-2

HepG2 cells were treated with 2 mg/L of CDDP for 24 h. F-10

HepG2 cells were treated with 10 mg/L of 5-Fu for 24 h

Fig. 6 DNA-PKcs suppression reduced the activity of NF-jB

signaling pathways in HepG2 cells exposed to CDDP and 5-Fu.

HepG2 cells were exposed to 2 mg/L of CDDP or/and 10 mg/L of

5-Fu for 24 h. a NF-jB transcriptional activity was assayed by EMSA

assay. b Proteins expression was analyzed by Western blot. GAPDH

was used as the internal reference. Presented images were from the

representative results of three independent experiments. Cont HepG2

cells were treated with vehicles only. Sc HepG2 cells were transfected

with the negative control siRNA. Si HepG2 cells were transfected

with the DNA-PKcs siRNA. C-2 HepG2 cells were treated with 2 mg/

L of CDDP for 24 h. F-10 HepG2 cells were treated with 10 mg/L of

5-Fu for 24 h

Mol Cell Biochem

123

antitumor activity in various types of human tumors, but

the academia has not form consensus about the synergistic

mechanism at present [7–9]. In this study, HepG2 cells

were transfected with DNA-PKcs siRNA or negative con-

trol siRNA, and then exposed to different concentrations of

CDDP and 5-Fu. The cell viability, apoptosis, oxidative

stress response, and DNA damage were measured and

compared, indicating that CDDP and 5-Fu individual

treatment had toxic effects in the HepG2 cells, and com-

binatory treatment with CDDP and 5-Fu had a synergistic

effect. Compared with the individual treatment group, the

combined treatment group showed even lower cell survival

rate, elevated apoptosis ratio and DNA damage, increased

ROS production, and reduced intracellular GSH level.

Combination of DNA-PKcs siRNA obviously augmented

the cytotoxicity of CDDP and 5-Fu in HepG2 cells.

Reactive oxygen species originated from the aerobic

cells are well recognized to be involved in various cell

processes such as cell proliferation, apoptosis, and DNA

damage [24, 25]. We described in the present study that

CDDP and 5-Fu treatment resulted in a higher level of ROS

production and DNA damage in HepG2 cells as compared

with the CDDP/5-Fu individual treatment group, which

was further aggravated by combination of DNA-PKcs

siRNA. Meanwhile, the antioxidant glutathione reduced

most significantly in HepG2 cells exposed to the combined

treatment group. Glutathione plays a vital role in protecting

cells from oxidative stress and controlling apoptotic gene

expression, membrane transportation, and chemotherapy

resistance [26–28]. The Bcl-2 expression is also closely

related to GSH in that Bcl-2 modulates the GSH production

and nucleus relocalization under oxidative stress [29]. In

the present study, DNA-PKcs suppression in HepG2 cells

exposed to CDDP and 5-Fu treatment resulted in Bcl-2 and

GSH reduction, accompanying by increased expression of

p53 and caspase-3, indicating activation of the mitochon-

drial apoptotic pathways.

Overexpression of DNA-PKcs is frequently found in

various cancers, which is closely associated with the tumor

cell growth, poor prognosis, and clinical therapeutic out-

come [30–32]. Depletion of DNA-PKcs through shRNA

transfection resulted in inhibition of cell proliferation and

increased apoptosis in human cervical cancer HeLa cell

line exposed to cisplatin [33]. Moreover, besides the

capability of the non-homologous-end-joining repair for

DNA double strands breaks (DSBs), DNA-dependent pro-

tein kinase (DNA-PK) is involved in processing the oxi-

datively induced non-DSB clustered DNA lesions, which is

important for the therapeutic efficiency of cancer treat-

ments based on the induction of oxidative stress [34].

Mutant forms of DNA-PKcs at serine/threonine residues in

the 2,609–2,647 cluster or specific inhibitor KU57788

could completely block or markedly reduce the efficiency

of end joining [35]. In the present study, DNA-PKcs defi-

ciency through small interfering RNA transfection signifi-

cantly sensitized the HepG2 cells to CDDP and 5-Fu in

terms of the cell viability, apoptosis, oxidative stress, and

DNA damage.

DNA-PKcs is a member of the phosphatidylinositol

3-kinase (PI3K) family, and the PI3K/AKT signaling

pathway is implicated in essential cellular functions such

as survival, proliferation, migration, and differentiation.

Wang et al. confirmed that suppression of the PI3K/AKT

signaling can effectively sensitize osteosarcoma cells to

cisplatin [36]. Our results also showed that combination of

CDDP and 5-Fu inhibited the activity of PI3K/Akt path-

way; DNA-PKcs siRNA could effectively suppress the

expression of DNA-PKcs and further reduced the activity

of PI3K/Akt signaling, which may be associated with

sensitization of HepG2 cells to combination treatment of

CDDP and 5-Fu.

The PI3K/Akt/NF-jB signaling pathway is known to be

involved in stimulation of tumor cell survival, invasive

behavior, and chemosensitivity in various malignancies

[37]. Two serine threonine IjB kinases (IKK-a/-b) are

responsible for phosphorylation and degradation of IjB

molecules, which may result in release of NF-jB from its

binding with IjB. Free NF-jB is then translocated into the

nuclei and exerts transcriptional activity to regulate phys-

iological processes such as cell proliferation and cell death

[23]. CDDP is a potent inhibitor to prevent NF-jB acti-

vation, while 5-Fu could induce NF-jB activation in many

cancers. Hartojo et al. reported that the combination of a

specific NF-jB inhibitor curcumin with 5-Fu and CDDP

could increase apoptosis and enhance both 5-FU- and

CDDP-mediated chemosensitivity in esophageal adeno-

carcinoma [38]. Interferon-gamma could also sensitize

hepatitis B virus (HBV)-expressing HCC cells to 5-Fu

through inhibition of HBV-mediated NF-jB activation

[39]. The Western blotting results of the present study

showed that CDDP led to reduced nuclear translocation of

NF-jB/p65 protein, NF-jB transcriptional activity, and

phosphorylation levels of IKK-a/-b. However, 5-Fu medi-

ated an opposite protein expression profile of NF-jB sig-

naling pathway, as well as the NF-jB transcriptional

activity. Moreover, the activation of NF-jB from 5-Fu

treatment was inversed by the concomitant treatment of

CDDP and further suppressed by combination of DNA-

PKcs siRNA, which may make HepG2 cells more vulner-

able to chemotherapeutic agents.

Taken together, combination with CDDP and 5-Fu had

synergistic effects on HepG2 cells in inducing proliferation

inhibition, ROS production, DNA damage, and cell apop-

tosis. DNA-PKcs suppression could augment the cytotox-

icity induced by CDDP and 5-Fu, which was associated

with blocking of PI3K/Akt/NF-jB signaling pathway,

Mol Cell Biochem

123

increased expression of caspase-3 and p53, and down-

regulation of Bcl-2 and GSH.

Acknowledgments The study was supported by Special Research

Fund for public welfare from Ministry of Health of China

(201202007)

Conflict of interest The authors declare that there are no conflicts

of interest.

References

1. Mendizabal M, Reddy KR (2009) Current management of

hepatocellular carcinoma. Med Clin North Am 93(4):885–900

2. Llovet JM, Burroughs A, Bruix J (2003) Hepatocellular carci-

noma. Lancet 362:1907–1917

3. Lin DY, Lin SM, Liaw YF (1997) Non-surgical treatment of

hepatocellular carcinoma. J Gastroenterol Hepatol 12(9–10):

S319–S328

4. Ueno H, Okada S, Okusaka T, Ikeda M, Kuriyama H (2002)

Phase I and pharmacokinetic study of 5-fluorouracil administered

by 5-day continuous infusion in patients with hepatocellular

carcinoma. Cancer Chemother Pharmacol 49:155–160

5. Stehlin JS Jr, de Ipolyi PD, Greeff PJ, McGaff CJ Jr, Davis BR,

McNary L (1988) Treatment of cancer of the liver. Twenty years’

experience with infusion and resection in 414 patients. Ann Surg

208(1):23–35

6. Falkson G, Ryan LM, Johnson LA, Simson IW, Coetzer BJ,

Carbone PP, Creech RH, Schutt AJ (1987) A random phase II

study of mitoxantrone and cisplatin in patients with hepatocel-

lular carcinoma. An ECOG study. Cancer 60(9):2141–2145

7. Ellis PA, Norman A, Hill A, O’Brien ME, Nicolson M, Hickish

T, Cunningham D (1995) Epirubicin, cisplatin and infusional

5-fluorouracil (5-FU) (ECF) in hepatobiliary tumours. Eur J

Cancer 31A(10):1594–1598

8. Kogure T, Ueno Y, Iwasaki T, Shimosegawa T (2004) The effi-

cacy of the combination therapy of 5-fluorouracil, cisplatin and

leucovorin for hepatocellular carcinoma and its predictable fac-

tors. Cancer Chemother Pharmacol 53:296–304

9. Kim R, Tanabe K, Inoue H, Toge T (2002) Mechanism(s) of

antitumor action in protracted infusion of low dose 5-fluorouracil

and cisplatin in gastric carcinoma. Int J Oncol 20(3):549–555

10. Woo HY, Bae SH, Park JY, Han KH, Chun HJ, Choi BG, Im HU,

Choi JY, Yoon SK, Cheong JY (2010) A randomized compara-

tive study of high-dose and low-dose hepatic arterial infusion

chemotherapy for intractable, advanced hepatocellular carci-

noma. Cancer Chemother Pharmacol 65(2):373–382

11. Yoshikawa M, Ono N, Yodono H, Ichida T, Nakamura H (2008)

Phase II study of hepatic arterial infusion of a fine-powder for-

mulation of cisplatin for advanced hepatocellular carcinoma.

Hepatol Res 38(5):474–483

12. Grandage VL, Gale RE, Linch DC, Khwaja A (2005) PI3-kinase/

Akt is constitutively active in primary acute myeloid leukaemia

cells andregulates survival and chemoresistance via NF-kappaB,

Mapkinase and p53 pathways. Leukemia 19(4):586–594

13. Pellegrino R, Calvisi DF, Neumann O, Kolluru V, Wesely J,

Chen X, Wang C, Wuestefeld T, Ladu S, Elgohary N, Bermejo

JL, Radlwimmer B, Zornig M, Zender L, Dombrowski F, Evert

M, Schirmacher P, Longerich T (2014) EEF1A2 inactivates p53

via PI3K/AKT/mTOR-dependent stabilization of MDM4 in

hepatocellular carcinoma. Hepatology 59(5):1886–1899

14. Zhang DM, Liu JS, Deng LJ, Chen MF, Yiu A, Cao HH, Tian HY,

Fung KP, Kurihara H, Pan JX (2013) Arenobufagin, a natural

bufadienolide from toad venom, induces apoptosis and autophagy

in human hepatocellular carcinoma cells through inhibition of

PI3K/Akt/mTOR pathway. Carcinogenesis 34(6):1331–1342

15. Zhao L, Sha YY, Zhao Q, Yao J, Zhu BB, Lu ZJ, You QD, Guo

QL (2013) Enhanced 5-fluorouracil cytotoxicity in high COX-2

expressing hepatocellular carcinoma cells by wogonin via the

PI3K/Akt pathway. Biochem Cell Biol 91(4):221–229

16. Nagai H, Sumino Y (2008) Therapeutic strategy of advanced

hepatocellular carcinoma by using combined intra-arterial che-

motherapy. Recent Pat Anticancer Drug Discov 3(3):220–226

17. Kavitha K, Kowshik J, Kishore TK, Baba AB (1830) Nagini S

(2013) Astaxanthin inhibits NF-jB and Wnt/b-catenin signaling

pathways via inactivation of Erk/MAPK and PI3K/Akt to induce

intrinsic apoptosis in a hamster model of oral cancer. Biochim

Biophys Acta 10:4433–4444

18. Thevenod F, Lee WK (2013) Cadmium and cellular signaling

cascades: interactions between cell death and survival pathways.

Arch Toxicol 87(10):1743–1786

19. Elliott SL, Crawford C, Mulligan E, Summerfield G, Newton P,

Wallis J, Mainou-Fowler T, Evans P, Bedwell C, Durkacz BW,

Willmore E (2011) Mitoxantrone in combination with an inhib-

itor of DNA-dependent protein kinase: a potential therapy for

high risk B-cell chronic lymphocytic leukaemia. Br J Haematol

152(1):61–71

20. Yoon JH, Ahn SG, Lee BH, Jung SH, Oh SH (2012) Role of

autophagy in chemoresistance: regulation of the ATM-mediated

DNA-damage signaling pathway through activation of DNA-

PKcs and PARP-1. Biochem Pharmacol 83(6):747–757

21. Panta GR, Kaur S, Cavin LG, Cortes ML, Mercurio F, Lothstein

L, Sweatman TW, Israel M, Arsura M (2004) ATM and the

catalytic subunit of DNA-dependent protein kinase activate NF-

kappaB through a common MEK/extracellular signal-regulated

kinase/p90 (rsk) signaling pathway in response to distinct forms

of DNA damage. Mol Cell Biol 24(5):1823–1835

22. An J, Zou W, Chen C, Zhong YF, Yu ZQ, Wang QJ (2013) The

cytological effects of HBCDs on human hepatocyte L02 and the

potential molecular mechanism. J Environ Sci Health Part A

48(11):1333–1342

23. Birbach A, Gold P, Binder BR, Hofer E, de Martin R, Schmid JA

(2002) Signaling molecules of the NF-kappa B pathway shuttle

constitutively between cytoplasm and nucleus. J Biol Chem

277:10842–10851

24. Buttke TM, Sandstrom PA (1994) Oxidative stress as a mediator

of apoptosis. Immunol Today 15:7–10

25. Fleury C, Mignotte B, Vayssiere JL (2002) Mitochondrial reac-

tive oxygen species in cell death signaling. Biochimie

84:131–141

26. Anderson ME (1998) Glutathione: an overview of biosynthesis

and modulation. Chem Biol Interact 111–2:1–14

27. Hammond CL, Lee TK, Ballatori N (2001) Novel roles for glu-

tathione in gene expression, cell death, and membrane transport

of organic solutes. J Hepatol 34:946–954

28. Chen X, Carystinos GD, Batist G (1998) Potential for selective

modulation of glutathione in cancer chemotherapy. Chem Biol

Interact 111–2:263–275

29. Franco R, Cidlowski JA (2012) Glutathione efflux and cell death.

Antioxid Redox Signal 17(12):1694–1713

30. Beskow C, Skikuniene J, Holgersson A, Nilsson B, Lewensohn

R, Kanter L, Viktorsson K (2009) Radioresistant cervical cancer

shows upregulation of the NHEJ proteins DNA-PKcs, Ku70 and

Ku86. Br J Cancer 101:816–821

31. Xing J, Wu X, Vaporciyan A, Spitz MJG (2008) Prognostic

significance of ataxia-telangiectasia mutated, DNA-dependent

protein kinase catalytic subunit, and Ku heterodimeric regulatory

complex 86-kD subunit expression in patients with nonsmall cell

lung cancer. Cancer 112:2756–2764

Mol Cell Biochem

123

32. Willmore E, Elliott S, Mainou-Fowler T, Summerfield G, Jackson

G, O’Neill F, Lowe C, Carter A, Harris R, Pettitt A, Cano-

Soumillac C, Griffin RJ, Cowell IG, Austin CA, Durkacz BW

(2008) DNA-dependent protein kinase is a therapeutic target and

an indicator of poor prognosis in B-cell chronic lymphocytic

leukemia. Clin Cancer Res 14:3984–3992

33. Tian X, Chen G, Xing H, Weng D, Guo Y, Ma D (2007) The

relationship between the down-regulation of DNA-PKcs or Ku70

and the chemosensitization in human cervical carcinoma cell line

HeLa. Oncol Rep 18(4):927–932

34. Peddi P, Loftin CW, Dickey JS, Hair JM, Burns KJ, Aziz K,

Francisco DC, Panayiotidis MI, Sedelnikova OA, Bonner WM,

Winters TA, Georgakilas AG (2010) DNA-PKcs deficiency leads

to persistence of oxidatively induced clustered DNA lesions in

human tumor cells. Free Radic Biol Med 48(10):1435–1443

35. Povirk LF, Zhou RZ, Ramsden DA, Lees-Miller SP, Valerie K

(2007) Phosphorylation in the serine/threonine 2609–2647 cluster

promotes but is not essential for DNA-dependent protein kinase-

mediated nonhomologous end joining in human whole-cell

extracts. Nucleic Acids Res 35(12):3869–3878

36. Wang K, Zhuang Y, Liu C, Li Y (2012) Inhibition of c-Met

activation sensitizes osteosarcoma cells to cisplatin via suppres-

sion of the PI3K-Akt signaling. Arch Biochem Biophys

526(1):38–43

37. Azijli K, Weyhenmeyer B, Peters GJ, de Jong S, Kruyt FA (2013)

Non-canonical kinase signaling by the death ligand TRAIL in

cancer cells: discord in the death receptor family. Cell Death

Differ 20(7):858–868

38. Hartojo W, Silvers AL, Thomas DG, Seder CW, Lin L, Rao H,

Wang Z, Greenson JK, Giordano TJ, Orringer MB, Rehemtulla

A, Bhojani MS, Beer DG, Chang AC (2010) Curcumin promotes

apoptosis, increases chemosensitivity, and inhibits nuclear factor

kappaB in esophageal adenocarcinoma. Transl Oncol

3(2):99–108

39. Chung C, Park SG, Park YM, Joh JW, Jung G (2007) Interferon-

gamma sensitizes hepatitis B virus-expressing hepatocarcinoma

cells to 5-fluorouracil through inhibition of hepatitis B virus-

mediated nuclear factor-kappaB activation. Cancer Sci

98(11):1758–1766

Mol Cell Biochem

123