Tirapazamine Sensitizes Hepatocellular Carcinoma Cells to Topoisomerase … · 2013. 12. 20. ·...

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Tirapazamine Sensitizes Hepatocellular Carcinoma Cells to

Topoisomerase I Inhibitors via Cooperative Modulation of

Hypoxia-Inducible Factor-1α

Tian-Yu Cai1, Xiao-Wen Liu1, Hong Zhu1, Ji Cao1, Jun Zhang1, Ling Ding1, Jian-Shu

Lou1, Qiao-Jun He1*, Bo Yang1*

1. Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, Institute of

Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang

University, Hangzhou, 310058, China

*Corresponding authors:

Professor Bo Yang, Ph.D.

Professor Qiao-Jun He, Ph.D.

#866 Yuhangtang Road, College of Pharmaceutical Sciences, Zhejiang University,

Hangzhou, 310058, China

Telephone: 86-(571)88208400; Fax: 86-(571)88208400

E-mail: [email protected], [email protected]

Running title: TPZ Sensitizes HCC to Topo I Inhibitors

Keywords

on June 21, 2021. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on December 20, 2013; DOI: 10.1158/1535-7163.MCT-13-0490

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Topoisomerase I Inhibitors, tirapazamine, hypoxia-inducible factor-1α, hypoxia,

hepatocellular carcinoma

Abbreviations

TPZ, tirapazamine; HIF-1α, hypoxia-inducible factor-1α; PARP, poly (ADP-ribose)

polymerase; HCC, Hepatocellular carcinoma; VEGF, vascular endothelial growth

factor; HRE, hypoxia response elements; PI, propidium iodide; CI, Combination

Index; DAPI, 4',6-diamidino-2-phenylindole

.

Grand Support

This work was supported by grants from National Natural Science Foundation of

China No. 81302789 (J. Lou), Zhejiang Provincial Program for The Cultivation of

High-Level Innovative Health Talents (Q. He), the Department of Education of

Zhejiang Province No. Y201226213 (L. Ding) and the Fundamental Research Funds

for the Central Universities No. 2012QNA7021 (L. Ding).

Competing Interests

The authors disclose no potential conflicts of interest.




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Abstract

Topoisomerase I inhibitors are a class of anti-cancer drugs with a broad spectrum of

clinical activity. However, they have limited efficacy in hepatocellular cancer. Here,

we present in vitro and in vivo evidence that the extremely high level of hypoxia-

inducible factor-1α (HIF-1α) in HCC is intimately correlated with resistance to

topoisomerase I inhibitors. In a previous study conducted by our group, we found that

TPZ could downregulate HIF-1α expression by decreasing HIF-1α protein synthesis.

Therefore, we hypothesized that combining TPZ with topoisomerase I inhibitors may

overcome the chemoresistance. In this study, we investigated that in combination with

TPZ, Topoisomerase I inhibitors exhibited synergistic cytotoxicity and induced

significant apoptosis in several HCC cell lines. The enhanced apoptosis induced by

TPZ plus SN-38 (the active metabolite of irinotecan) was accompanied by increased

mitochondrial depolarization and caspase pathway activation. The combination

treatment dramatically inhibited the accumulation of HIF-1α protein, decreased the

HIF-1α transcriptional activation and impaired the phosphorylation of proteins

involved in the homologous recombination repair pathway, ultimately resulting in the

synergism of these two drugs. Moreover, the increased anticancer efficacy of TPZ

combined with irinotecan was further validated in a human liver cancer Bel-7402

xenograft mouse model. Taken together, our data show for the first time that HIF-1α




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is strongly correlated with resistance to topoisomerase I inhibitors in HCC. These

results suggest that HIF-1α is a promising target and provide a rationale for clinical

trials investigating the efficacy of the combination of topoisomerase I inhibitors and

TPZ in hepatocellular cancers.

Introduction

Hepatocellular carcinoma (HCC) is the most frequently occurring primary liver

cancer, with an incidence of half a million cases per year worldwide (1). Although

surgery remains the treatment of choice for HCC, tumor size and hepatic functional

reservation might restrict surgical ablation. Conventional radio/chemotherapies are

generally not prescribed for advanced HCC due to their low efficacies (2). We and

other groups (3, 4) have found that the expression of HIF-1α is increased in most

HCCs and is associated with poor outcomes. Because fibrogenesis during the

development of cirrhosis ultimately destroys the normal blood supply to the liver, the

blood supply is limited in HCC with cirrhosis, which ultimately leads to local hypoxia

(5). The insufficient blood supply of the rapidly growing tumor tissues also induces

hypoxia in the central region of the tumor. Hypoxia leads to the activation of hypoxia-

inducible factor 1 alpha (HIF-1α), which acts as the transcriptional factor for a variety

of essential genes in the hypoxic microenvironment, including those encoding for




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vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (BFGF)

(6-8).

HIF-1α is a master regulator of tumor growth, angiogenesis, metastasis and

resistance to anticancer drugs (9-11). Although the proteasome pathway rapidly

degrades HIF-1α under normoxia, this protein is stable under hypoxia, where it

translocates to the nucleus and binds to hypoxia response elements (HREs) within the

promoter of its target genes. Significant evidence indicates that HIF-1α plays an

important role in the resistance to chemotherapy of HCC (12, 13). Hence, inhibition of

the HIF-1α pathway is a promising approach for the treatment of HCC.

Camptothecin (CPT), a natural product isolated from Camptotheca acuminata

Decne, was originally discovered because of its antitumor activity, and the mechanism

of action was later demonstrated to be the accumulation of Topoisomerase I–DNA

adducts, observed both in vitro and in vivo (14-17). CPTs bind the covalent 3-

phosphotyrosyl intermediate and specifically block DNA re-ligation, thus inducing

DNA damage. During DNA replication, the replication fork is thought to collide with

the ‘‘trapped’’ Topoisomerase I–DNA complexes, resulting in double-strand breaks

and ultimately cell death. Irinotecan is a topoisomerase I inhibitor registered for the

treatment of gastrointestinal cancer in humans and has been shown to exert activity on

many cancer cell lines in vitro, but it fails to provide definitive information in vivo,




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particularly through intravenous infusion in HCC patients (18). Similar to other

cancer types, HCC progresses and develops drug resistance via the upregulation of

stress-induced molecules such as HIF-1α. We surmised that HIF-1α may play an

important role in the resistance to topoisomerase I inhibitors in HCC.

Tirapazamine (TPZ, 3-amino-1,2,4-benzotriazine 1,4 dioxide, SR4233) is a

bioreductive drug belonging to the aromatic N-oxide family that has a selective

toxicity for hypoxic cells(19). In cell suspensions, TPZ is 50- to 500-fold more toxic

to hypoxic cells compared with cells in normoxia. The hypoxic selectivity of TPZ is

due to the rapid reoxidation of the initial radical to the parent prodrug by O2. Thus,

TPZ functions as a hypoxia-activated agent (20, 21), exhibiting the capability of

potentiating the antitumor efficacy of many anticancer drugs (22-25). The putative

mechanism may be attributed to its suppression of the accumulation of HIF-1α protein.

Thus, we hypothesized that the combination of TPZ (HIF-1α–downregulating agents)

with topoisomerase I inhibitors may be a potential therapeutic regimen for HCC.

In our study, we show for the first time that HIF-1α is strongly correlated with

resistance to topoisomerase I inhibitors in HCC. Using both in vitro and in vivo

models of HCC, we demonstrate the potential for synergistic activity of TPZ in

combination with topoisomerase I inhibitors based on the modulation of the HIF-1α

protein.




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Materials and Methods

Reagents and cell lines

CPT-11, SN-38, TPT, HCPT and MONCPT were kindly provided by Dr. Wei Lu (East

China Normal University, Shanghai, China), and TPZ (Tirapazamine) was purchased

from Topharman Shanghai Co. Ltd. All of the drugs were dissolved in

dimethylsulfoxide (DMSO) and stored in aliquots (20 mM) at -20 °C. Primary

antibodies to cleaved-Caspase 3, H2AX, γ-H2AX, Chk1, p-Chk1, Chk2, p-Chk2,

ATM, p-ATM, Rad51 and β-Actin were obtained from Cell Signaling Technologies.

Antibodies to Caspase-3, PARP, and HRP-labeled secondary anti-goat, anti-mouse,

and anti-rabbit antibodies were from Santa Cruz Biotechnology. Antibody to HIF-1α

was obtained from BD Biosciences. DMSO, propidium iodide (PI), sulforhodamine B

(SRB), DAPI (4', 6-diamidino-2-phenylindole), JC-1 and CoCl2 were obtained from

Sigma-Aldrich.

The five HCC cell lines, HepG2, Hep3B, Huh 7, Bel-7402 and SMMC-7721 were

purchased from Cell Bank of China Science. A normal liver cell line, Chang liver, was

obtained from Cell Bank of China Science. Following receipt, cells were grown and

frozen as a seed stock as they were available. All six cell lines were passaged for a

maximum of 2 months, after which new seed stocks were thawed. All six cell lines




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were authenticated using DNA fingerprinting (variable number of tandem repeats),

confirming that no cross-contamination occurred during this study. All six cell lines

were tested for mycoplasma contamination at least every month. The six cell lines

were cultured in RPMI 1640 medium/DMEM containing 10% fetal bovine serum and

1% antibiotics in a humidified atmosphere with 5% CO2 at 37 °C. Hypoxia treatment

was carried out by placing the cells in a CO2-Water Jacketed Incubator (Thermo

Forma) filled with a mixture of 0.6% O2, 5% CO2, and 94.4% nitrogen.

Cell proliferation assay

Cell proliferation was evaluated by the Sulforhodamine B (SRB) protein staining

assay (26). Briefly, treated cells in 96-well plates were fixed with 10% TCA and

stained with SRB. SRB in the cells was dissolved in 10 mM Tris-HCl and measured at

515 nm using a multiwell spectrophotometer (Thermo Electron Co.). The rate of

inhibition of cell proliferation was calculated for each well as (A515control cells-

A515treated cells)/A515control cells×100%. The average IC50 values were determined by the

logit method from at least three independent tests.

Flow cytometric analysis of apoptosis and mitochondrial membrane

potential (ΔΨm)




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Cells were treated with SN-38, TPZ or their combination under normoxia or hypoxia

for 12 h. After harvesting and washing twice with cold PBS buffer, the Annexin V-

FITC/PI apoptosis Detection Kit was used to analyze apoptotic cells. Analysis of the

sub-G1 phase after PI staining was also employed to assess apoptosis. For PI staining,

treated cells were harvested and fixed with 70% ethanol at -20 oC, then incubated with

RNaseA followed by PI in the dark for 30 min. For determination of mitochondrial

potential, cells were resuspended in PBS containing 0.1 µΜ JC-1 and were incubated

at 37 °C for 15 min in the dark. All samples were analyzed using a FACS-Calibur

cytometer (Becton Dickinson).

Clonogenic assays

Cells treated with TPZ or SN-38/TPT/HCPT/MONCPT or their combination were

plated in 60-mm dishes in triplicate on soft agar. Once set, the dishes were overlaid

with 2.5 ml of medium and incubated at 37 °C for 10 days in hypoxic conditions, at

which time the colonies were scored and photographed.

Quantitative real time RT–PCR (qRT–PCR) analysis

Total RNA was prepared with Trizol (Invitrogen) and cDNA was synthesized from 2

μg of RNA with Revertra Ace (Toyobo). qRT–PCR was performed with SYBR Green




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PCR kits (Qiagen Inc., Valencia, CA). The primers used were as follows: HIF-1α,

forward primer 5′-TCACCACAGGACAGTACAGGATGC-3′ and reverse primer 5′-

CCAGCAAAGTTAAAGCATCAGGTTCC-3′; VEGF, forward primer 5′-

AGGAGGGCAGAATCATCACG-3′ and reverse primer 5′-

CAAGGCCCACAGGGATTTTCT-3′; GAPDH, forward primer 5′-

GTCATCCATGACAACTTTGG-3′ and reverse primer 5′-

GAGCTTGACAAAGTGGTCGT-3′. In all experiments, two negative controls were

also performed. Values are expressed as fold increases relative to the reference sample.

Immunofluorescence

Cells were plated onto glass culture slides and incubated with SN-38/TPZ or SN-38 +

TPZ or vehicle [0.1% DMSO (v/v)] for different time periods. Cells were then fixed

with 4% paraformaldehyde and permeabilized with PBS containing 0.1% Triton X-

100. After blocking with 5% bovine serum albumin for 30 min, cells were incubated

with primary HIF-1α or γ-H2AX antibodies (1:100 dilution) for 1 h, washed 3x with

PBS and then incubated with Alexa Fluor 488–conjugated and rhodamine secondary

antibodies (Invitrogen) respectively in the dark. Nuclei were visualized by staining

with DAPI (4′6-diamidino-2-phenylindole). Fluorescence signals were analyzed using

an Olympus Fluorview 1000 confocal microscope.




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Plasmid based overexpression of HIF-1α

Transfection with HIF-1α plasmids or empty vector (EV) obtained form Origene was

performed using the FuGENE® HD Transfection Reagent (Roche Applied Science)

according to the manufacturer’s instructions.

shRNA knockdown of HIF-1α

shRNA targeting HIF-1α was purchased from Origene with a GFP-tag and puromycin

resistance. Transfection of shRNA into Bel-7402 cells was performed using

Lipofectamine 2000 (Invitrogen). Individual clones stably expressing the shRNA were

generated by single-cell sorting into 96 round-bottom wells and antibiotic selection

with puromycin (500 ng/mL). Western blot analysis and an Olympus Fluorview 1000

confocal microscope were used to select clones with stable knockdown of HIF-1α.

Immunoblot analysis

Immunoblotting of biomarkers in cell lysates and tumor tissue homogenates was

performed as previously described(27).

Luciferase reporter assays




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Bel-7402 cells were seeded into 96-well plates and then cotransfected with HRE-

luciferase-pGL3 and renilla luciferase (internal control for transfection efficiency)

plasmids using Lipofectamine 2000 according to the manufacturer's instructions.

Luciferase activity was measured using the Dual-Luciferase reporter assay system. In

the assay, firefly luciferase activity was normalized by renilla luciferase.

Measurement of in vivo activity

Tumors were established by injection of Bel-7402 cells (5×106 cells per animal,

subcutaneously into the armpit) into 5-to 6-week-old BALB/c male athymic mice.

Treatments were initiated when tumors reached a mean group size of about 100 mm3.

Tumor volume (mm3) was measured with calipers and calculated as (W2×L)/2, where

W is the width and L is the length. Athymic mice were intraperitoneally injected with

CPT-11 (2.5 mg/kg) dissolved in physiological saline once daily and TPZ (25 mg/kg)

dissolved in a cremophor:ethanol:0.9% sterile sodium chloride solution (1:1:8,

volume) every two days. Mouse weight and tumor volumes were recorded every 2

days until the animals were sacrificed. Animal care was in accordance with

institutional guidelines.

Statistical analyses




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The results are expressed as the mean ± SD of at least three independent experiments.

Differences between means were analyzed using Student’s t-test and were considered

statistically significant when P < 0.05.

Results

HIF-1α protein level is strongly correlated with resistance to

topoisomerase I inhibitors in HCC cell lines

In our study, we first investigated the strength of the association between

expression of HIF-1α and resistance to topoisomerase I inhibitors in HCC cell lines.

We measured Bel-7402 cell growth under hypoxic or normoxic culturing conditions in

the presence of topoisomerase I inhibitors. Compared to normoxia, the IC50s for SN-

38, TPT, HCPT and MONCPT were 7.6-, 6.2-, 5.4- and 4.3-fold higher, respectively,

under 0.6% O2. Then, we decided to investigate whether the accumulation of HIF-1α

induced by hypoxia was a key factor in this resistance; thus, an HIF-1α-deficient Bel-

7402 cell line was established. The transfection of a HIF-1α-targeting shRNA

construct almost completely suppressed the protein expression of HIF-1α even under

hypoxia, while HIF-1α expression in empty-vector-transfected Bel-7402 cells

remained intact (Fig. 1A). Compared to normoxia, the IC50s for SN-38, TPT, HCPT

and MONCPT were similar under 0.6% O2 for Bel-7402/HIF-1α (-), hypoxia-induced




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resistance disappeared in HIF-1α gene silencing cells (Fig. 1B and Fig. S1).

Meanwhile, cells treated with 200 µM CoCl2, which resulted in a robust induction of

intracellular HIF-1α, showed no statistical difference in the IC50 for SN-38 between

cells treated with CoCl2 and hypoxic cells (Fig. 1C). These data demonstrate that

hypoxia can induce resistance to topoisomerase I inhibitors in HCC cell lines and

HIF-1α may play an important role.

Tirapazamine synergistically increases topoisomerase I inhibitor-

inhibition of cell proliferation in HCC cell line

The above observations indicate the therapeutic potential treatment with the

combination of topoisomerase I inhibitors and HIF-1α inhibitors, which may

overcome the resistance to topoisomerase I inhibitors in HCC cell lines. It has been

reported that TPZ can inhibit the accumulation of HIF-1α protein (28), and we

confirmed this effect in our system using luciferase reporter gene assays and

immunoblot analysis. As shown in Fig. 2B, luciferase activity was markedly increased

under hypoxia compared with normoxia, while this activity was suppressed by TPZ in

a dose-dependent manner after 6 h treatment, ranging from 10.2% (0.31 μM) to

66.2% (10 μM). Similarly, the protein level of HIF-1α was reduced upon TPZ

treatment (Fig. 2A). Taken together, these data imply that TPZ is indeed inhibiting

HIF-1α accumulation.




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Because TPZ can inhibit the accumulation of HIF-1α protein, we determined the

cytotoxicity of TPZ and each of the four topoisomerase I inhibitors alone and of TPZ

combined with each of the other drugs at clinically achievable concentrations in the

Bel-7402 cell line using the SRB cytotoxicity assay. CI values were calculated at the

fixed-ratio drug concentrations used for cytotoxicity assays. Dose-response curves to

TPZ, the four topoisomerase I inhibitors and TPZ combined with each topoisomerase

I inhibitor are shown in Fig. 2C-F. The combinations of TPZ plus topoisomerase I

inhibitor all showed strong synergy (CI < 0.3) or synergy (CI < 0.7) in the Bel-7402

cell line, achieving about 80% cell death. In addition, we determined the cytotoxicity

of TPZ (2.5 μM) in combination with various concentrations of topoisomerase I

inhibitors in Bel-7402 cells. As shown in Fig. S2, treatment of cells with TPZ (2.5 μM)

significantly enhanced cell sensitivity to topoisomerase I inhibitors. To analyze the

antitumor effect of the combination treatment of TPZ with the four topoisomerase I

inhibitors, a clonogenic assay was performed. As shown in Fig. S3, TPZ and the

topoisomerase I inhibitors both weakly suppressed colony formation of Bel-7402

cancer cells (55–85%). Significantly, TPZ enhanced the anti-tumor effect of

topoisomerase I inhibitors and drastically suppressed colony formation (11–23%).

Thus, the combination effect of TPZ and topoisomerase I inhibitors was quite

dramatic, and this effect had to be confirmed in other HCC cell lines.




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Tirapazamine synergistically increases SN-38-induced cell proliferation

inhibition in different HCC cell lines

Because TPZ synergized most strongly with SN-38 (active metabolite of

irinotecan), and irinotecan is a widely used drug in clinical practice, we determined

the effects of TPZ, SN-38 and their combination on various HCC cell lines, including

Bel-7402, SMMC-7721, HepG2, Hep3B and Huh-7. Survival curves to TPZ, SN-38

and their combination are shown in Fig. 3A-E. As shown in Table S1, SN-38 plus

TPZ showed distinct synergy in all cancer cell lines tested, with all CI values below

0.8, indicating the much stronger antiproliferative abilities achieved by the

combination of SN-38 and TPZ. However, in a normal liver cell line, the combination

effect was minimal (Fig. 3F).

TPZ sensitizes HCC cells to SN-38-triggered caspase-dependent

apoptosis

To further understand mechanisms of enhanced cytotoxicity observed with the

combination of TPZ and SN-38, we examined their effects on apoptosis,

mitochondrial membrane depolarization, and caspase activation. We first investigated

whether the observed cytotoxicity was due to apoptosis. DAPI staining was used to

visualize the apoptosis induced by the co-treatment of TPZ and SN-38. After exposure

to SN-38 or/and TPZ of the indicated concentrations for 12 h, the nuclear DNA of




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Bel-7402 cells were permeabilized and stained with DAPI. As shown in Fig. 4A, 10

µM TPZ plus 0.1 µM SN-38 triggered more apoptosis, as indicated by the apoptotic

bodies, than the mono-treated cells. The PI (sub-G1) staining assay and Annexin V-PI

staining assay were also employed to detect levels of apoptosis (Fig. 4B and 4D).

After treatment with TPZ and/or SN-38 for 12 h, we found that cells treated with the

two agents together experienced 49% and 33% apoptosis in Bel-7402 cells using the

two assays. This was more apoptosis than either TPZ or SN-38 induced alone. We

next investigated the effects of treatment with the single agents or the combination on

mitochondrial membrane potential. Mitochondrial membrane depolarization, as

determined by the JC-1 mitochondrial probe, was induced by TPZ and SN-38 as

single agents 12 hours after treatment, and the combination resulted in a greater than

additive effect (P < 0.05) compared with the single agents (Fig. 4C).

Furthermore, we examined the effect of TPZ, SN-38 and their combination on the

activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP). We

found that although TPZ and SN-38 had little effect on caspase-3 and PARP, the two

together induced a more significant cleavage of PARP and caspase-3 (Fig. 4E).

Together, our results demonstrate that the combination of TPZ and SN-38 elicited

more apoptosis via mitochondrial membrane depolarization, resulting in increased cell

death.




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TPZ and SN-38 combination therapy suppresses HIF-1α expression

Our results indicate that high levels of HIF-1α confer resistance to SN-38; thus, we

were interested in examining the involvement of HIF-1α in the efficacy of the TPZ

and SN-38 combination treatment. Western blot analysis and immunofluorescence

microscopy (Fig. 5A and 5B) showed that TPZ and SN-38 alone weakly inhibited the

accumulation of HIF-1α protein in vitro. Strikingly, combination therapy resulted in

complete inhibition of HIF-1α protein accumulation. The combination did not have an

effect on the HIF-1β and HIF-2α proteins. Then, we analyzed the mRNA levels of

HIF-1α and VEGF by RT-PCR. The results showed that exposure to TPZ or SN-38

reduced the expression of VEGF, a target gene of HIF-1α (Fig. 5C). This effect was at

least additive when the two agents were administered together, similar to their effect

on HIF-1α protein levels. HIF-1α binds to the HRE cis-elements within the promoters

of hypoxia-responsive genes to regulate their expression. To investigate whether the

TPZ and SN-38 combination treatment could affect the activity of HIF-1α, we

examined its effect on HIF-1α binding to DNA using a reporter gene assay. After

transfection, cells were exposed to the single agents or the combination for 4 h under

hypoxia. As shown in Fig. 5D, luciferase activity was markedly induced under

hypoxia compared with normoxia, and this activity was both inhibited by increasing

doses of TPZ or SN-38. This inhibition was dramatically increased when TPZ and




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SN-38 were combined, and at the maximum combination dose (10 µM plus 0.1 µM)

there was a 92% decrease of HIF-1α transcriptional activity.

Next, we investigated the effect of the combination on HIF-1α posttranscriptional

regulation. As shown in Fig. S4A, the degradation rates of HIF-1α were similar in

treated and untreated cells, indicating the combination did not significantly affect

HIF-1α degradation. To exclude the possibility that the inhibition of HIF-1α

accumulation was attributed to HIF-1α protein degradation, Bel-7402 cells were

pretreated with MG132 (a specific proteasome inhibitor) or chloroquine (CQ, a

lysosome inhibitor) before the drugs were added to prevent HIF-1α degradation. As

shown in Fig. S4B, MG132 and CQ treatment failed to reverse the decrease of HIF-

1α protein levels in the combination treatment.

To more directly assess the effects of the combination on the rate of de novo

synthesis of the HIF-1α protein, cells were pretreated with cycloheximide (CHX) for

three hours under normoxia to inhibit new protein synthesis and then incubated in

fresh medium. At this point, most HIF-1α proteins were degraded, and a limited

amount of protein remained. These CHX-pretreated cells were then exposed to

hypoxic conditions, followed by treatment with TPZ, SN-38 or the combination for

different periods of time, and HIF-1α protein levels were analyzed by western blot.

As shown in Fig. 5E, significantly less HIF-1α protein accumulated in cells treated




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with the combination than in cells treated with TPZ or SN-38 alone at all times tested.

This result further confirmed that the combination decreases the rate of HIF-1α

protein synthesis.

There is a functional link between HIF-1α variation and DNA repair. To assess the

effect of TPZ, SN-38 and their combination on DNA, the formation of DSBs was

measured by immunostaining of γ-H2AX. The cells treated with TPZ or SN-38

showed several γ-H2AX foci after a 4-hour treatment. Cells treated with the

combination treatment for 4 hours showed a greater than 3-fold increase in the

number of γ-H2AX foci (Fig. 5B), which is similar to the results for γ-H2AX protein

levels (Fig. 5A). To investigate whether the effect of the combination treatment on

HIF-1α expression might affect DNA repair, shRNA-mediated repression of HIF-1α

was performed in Bel-7402 cell line. Bel-7402/HIF-1α(+) and Bel-7402/HIF-1α(-)

cells were pretreated with SN-38 for two hours under hypoxia to form DSBs and then

incubated in fresh medium without drug for six hours. The formation of DSBs was

measured by immunostaining. As shown in Fig. 5F, the Bel-7402/HIF-1α(-) cells

contained more DSB-indicative γ-H2AX than those Bel-7402/HIF-1α(+) cells,

indicating HIF-1α knockdown causes an inhibition of DNA repair, resulting in a larger

population of persistently unrepaired DSBs.

CPT-induced DSB are predominantly repaired by homologous recombination (HR)




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in mammalian cells (29-31). We attempted to detect which repair pathway was

inhibited by HIF-1α in the current model to repair the DSBs elicited by CPT. As

shown in Fig. 5A, ATM, Chk1 and Chk2 were phosphorylated after a 4 h of exposure

to SN-38 or TPZ. The Rad51 and phosphorylation of Chk1 at Ser317 were both

decreased in the combination treatment of TPZ and SN-38. The close correlation

between loss of RAD51 and DSB accumulation suggested that the inhibition of the

RAD51-mediated HR repair pathway was likely responsible for the accumulation of

DSBs in Bel-7402 cells.

The combination of TPZ and irinotecan arrests tumor growth.

Because the TPZ plus SN-38 exhibited the most effective synergistic effect with the

lowest CI values on human HCC Bel-7402 cells (Table S1), the in vivo efficacy of the

TPZ and irinotecan combination therapy was chosen to test against Bel-7402

xenografts in nude mice. As shown in Fig. 6A-B and Table S2, the i.p. administration

of TPZ at a dose of 25 mg/kg every two days or irinotecan at a dose of 2.5 mg/kg

every day produced no significant difference in mean RTV compared with that of the

control group. However, TPZ plus irinotecan caused marked tumor growth inhibition

(T/C value: 36.9%) that was significantly greater than that caused by TPZ (T/C value:

88.1%) or irinotecan treatment alone (T/C value: 86.4%). Furthermore, compared

with the initial body weights, the mice treated with the combination showed no




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significant body weight loss on day 26 (Fig. 6C). Thus, the combination of TPZ and

irinotecan exerted more potent tumor growth inhibitory effects compared with the

monotreatment groups, but caused no extended bodyweight loss to the animals.

The combination therapy induces apoptosis and downregulates HIF-1α

in tumor tissues.

Hematoxylin & eosin (H&E) staining for formalin-fixed paraffin embedded tissues

was used to distinguish tumor tissues from adjacent normal tissues. In mice treated

with TPZ and irinotecan, most tumor cells were severely damaged or destroyed (Fig.

6E). We next aimed to explore the effect of mono-treatment or combined treatment of

TPZ and irinotecan on the protein levels of apoptosis-related proteins in tumor tissues

from drug-administrated mice. Notably, the caspase cascade was activated in the

tissues from the nude mice treated with combination therapy (Fig. 6D). Importantly,

the expression of HIF-1α, as determined by western blot (Fig. 6D) and

immunohistochemistry (Fig. 6F), was consistent with the aforementioned cell culture

data (Fig. 5A and 5B), highlighting the involvement of these proteins in the tumor

growth inhibitory effects exerted by TPZ and irinotecan in vivo.

Discussion

Topoisomerase I inhibitors are a class of anti-cancer agents with a mechanism of




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action involving the disruption of DNA replication in cancer cells, the result of which

is cell death. They are important components in the current treatment of colorectal

cancer; however, in clinical studies of topoisomerase I inhibitors with advanced

hepatocellular carcinoma, they had modest activity in advanced hepatocellular cancer.

Accumulating evidence demonstrates that overexpression of HIF-1α plays an

important role in chemoresistance and that hypoxia has a protective effect against

DNA damage induced by topoisomerase inhibitors (32, 33). As shown in Fig. S5A,

the protein level of HIF-1α in Bel-7402 cells in hypoxia is much higher than the levels

in two colon cancer cell lines HT29 and SW480, which were reported to be

susceptible to SN-38 (34) in hypoxia owing to their decreased levels of HIF-1α. Thus,

we speculated that the HCC resistance to topoisomerase I inhibitors is correlated with

levels of HIF-1α. As expected, under hypoxia, Bel-7402 cells were resistant to SN-38,

TPT, HCPT and MONCPT. Furthermore, our results using short hairpin RNA or

cobalt chloride (CoCl2) are also consistent with our hypothesis that HIF-1α is an

important element in the limited efficacy of topoisomerase I inhibitors on HCC.

Accordingly, selective down-regulation of HIF-1α has been documented to be a

mechanism underlying increased sensitization to topoisomerase I inhibitors.

Tirapazamine is an anticancer drug that is activated to a toxic radical specifically in

hypoxic microenvironments. Cells in hypoxic areas are resistant to killing by most




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anticancer drugs, and TPZ has been shown to potentiate the antitumor efficacy of

many anticancer drugs in hypoxia. It was reported that TPZ can induce a reduction in

HIF-1α protein levels; thus the combination of TPZ with topoisomerase I inhibitors

was of particular interest to us.

As expected, in our in vitro study, synergistic anticancer effects of TPZ plus

topoisomerase I inhibitors were observed in human HCC cells. CI values and the

significant decline of the survival curves in the combination group strongly

demonstrated that TPZ potentiated the SN-38-induced cytotoxicity in HCC cells. In

addition, the data from both the flow cytometry and western blot analyses indicated

that TPZ plus SN-38 synergistically increased the execution of caspase-dependent

apoptosis. We also compared the cytotoxicity of TPZ plus SN-38 in HCC cell lines to

the Chang liver cell line (normal liver cells). The data suggest that the combination of

TPZ plus SN-38 kills HCC cells efficiently but minimally affects normal liver cells.

In our in vivo experiment, this synergistic effect of the drug combination was also

observed in the Bel-7402 xenograft nude mice model (Fig. 6B). The co-administration

of TPZ and irinotecan arrested tumor growth by 60.9%. Moreover, there was no

significant difference in body weight loss between combination and irinotecan

treatment groups. These results suggest that the TPZ and irinotecan combination

synergistically inhibited tumor growth and had minimal toxicity in vivo.




25

In this work, we have shown that the combination of TPZ and topoisomerase I

inhibitors has a major antitumor effect in vivo and induces massive cell death in vitro

under hypoxic conditions. These effects are correlated with a potent inhibition of HIF-

1α accumulation in tumor cells. Adding TPZ to SN-38-based therapy was

accompanied by inhibition of HIF-1α accumulation in vivo and in vitro as well as a

dramatic reduction of tumor volume and massive tumor cell death. This effect was

observed even at low doses of TPZ and SN-38 due to a cooperative effect of the two

agents on HIF-1α expression. The combination of TPZ and SN-38 did not affect the

mRNA levels of HIF-1α but did decrease the rate of HIF-1α protein synthesis and

inhibited HIF-1α transcriptional activation and the accumulation of HIF-1α protein,

resulting in a decrease in expression of target genes, such as VEGF.

The antitumor activity of topoisomerase I inhibitors is generally believed to result

from stabilization of covalent topoisomerase I-DNA complexes (35), which are

converted to DNA double-strand breaks upon collision with the replication fork. TPZ,

a hypoxia-selective cytotoxin, has demonstrated activity in cancer clinical trials.

Under hypoxic conditions, TPZ is reduced to a topoisomerase IIα poison that leads to

DNA double-strand breaks, single-strand breaks, and base damage (20). The typical

mechanism by which both TPZ and topoisomerase I inhibitors work is through

binding to the topoisomerase molecule, which blocks the topoisomerase from binding




26

to the DNA, causing the DNA damage and ultimately leading to cell death.

HIF-1α plays an important role in the prevention and repair of drug-induced DNA

damage and can cause chemoresistance. In Minori Koshiji’s work (36), transcript

levels of two DNA-PK complex members, DNA-PKcs and Ku80, were found to be

downregulated in HIF-1α-deficient cells that were previously found to have a higher

susceptibility to DNA DSB-inducing chemotherapeutics. L E Huang reported (37) that

HIF-1α upregulates DNA repair genes by counteracting c-Myc activities through c-

Myc displacement. In recent research (38), modulating expression of HIF-1α by small

interfering RNA or cobalt chloride markedly reduced or increased transcription of

XPA in lung cancer cell lines, and XPA has a dual role in sensing and recruiting other

DNA repair proteins to the damaged template. Therefore, we hypothesized that

modulation on HIF-1α is involved in the synergistic effect of the TPZ and SN-38

combination treatment. To test this hypothesis, we next asked whether the synergism

could be achieved in cells with forced HIF-1α over-expression. Bel-7402 cells forced

to overexpress HIF-1α were treated with TPZ, SN-38 or their combination. We found

that the synergistic effects were weakened when cells were transfected with the HIF-

1α plasmid (Fig. S6).

In our research, the well-established DNA damage marker γ-H2AX was used to

investigate genetic instability. Due to the presence of HIF-1α, TPZ or SN-38 by itself




27

in a low dose did not induce much DNA damage, but the combination treatment of

TPZ and SN-38 greatly increased the amount DNA damage observed in the cells. The

complete inhibition of HIF-1α accumulation may be responsible for γ-H2AX

induction in our model because HIF-1α can induce a DNA damage repair response.

Furthermore, the first phase occurred in both Bel-7402/HIF-1α (-) and Bel-7402/HIF-

1α (+) cells after 4 hours of SN-38 treatment, which represented a fast increase in foci.

This rapid increase in γ-H2AX foci suggests DNA damage is occurring in the tumor

cells. In the second phase, the γ-H2AX foci levels dramatically decreased in Bel-

7402/HIF-1α (+) cells but remained the same in Bel-7402/HIF-1α (-) cells. These data

may be explained by the following mechanistic model: the addition of SN-38 induced

DNA DSBs; DNA repair mechanisms were ongoing, which eliminated the increase of

foci production; after the cells had been placed in fresh medium without SN-38 for 4h,

the γ-H2AX foci were eliminated; the knockdown of HIF-1α by shRNA impaired

DNA repair, a larger population of persistently unrepaired DSBs remained. We also

observed that ATM, Chk1 and Chk2 were phosphorylated after 4 h exposure to SN-38

or TPZ. However, Rad51 and phosphorylation of Chk1 at Ser317 were decreased in

the combination treatment of TPZ and SN-38. The strong correlation between RAD51

removal and DSB accumulation suggested that the inhibition of the RAD51-mediated

HR repair pathway was most likely responsible for the accumulation of DSBs in Bel-




28

7402 cells. Therefore, our observations suggested that through cooperative inhibition

of HIF-1α accumulation, the combination of TPZ and SN-38 inhibited the RAD51-

mediated homologous recombination repair pathway, induced the accumulation of

topoisomerase inhibitor-induced DNA damage, exhibited synergistic cytotoxicity and

triggered significant apoptosis in HCC cell lines.

In summary, we report for the first time that HIF-1α is strongly correlated with

HCC cell line resistance to topoisomerase I inhibitors. The combination significantly

improved the anticancer activities of the drugs, as indicated by the synergistic

inhibitory effects on cancer cell proliferation, the sensitized execution of apoptosis,

and the enhanced in vivo antitumor efficiency. We believe these results can be

attributed to the downregulation of HIF-1α. Collectively, these data favor the regimen

of combining TPZ and topoisomerase I inhibitors as a promising therapeutic strategy,

and further preclinical and clinical studies of this novel combination are warranted.

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Figure legends

Figure 1. Depletion of HIF-1α could reverse hypoxia-induced SN-38 resistance. (A)

HIF-1α knockdown was achieved by transfection with a HIF-1α-targeting shRNA

construct. Bel-7402 cells were transfected with either the empty-vector or the GFP-

tagged shRNA-encoding constructs, followed by puromycin selection. Western blot

analysis with specific antibodies to HIF-1α and visualization of GFP-positivity with

an Olympus fluorview 1000 confocal microscope were used to select clones with

stable knockdown of HIF-1α. (B) Bel-7402-HIF-1α-positive or Bel-7402-HIF-1α-

negative cells obtained from (A) were treated with different concentrations of SN-38

(0-1 μM) for 24 h under normoxic or hypoxic conditions, followed by subsequent

proliferation inhibition assessment using the SRB assay. (C) Bel-7402 cells were

pretreated with CoCl2 (200 μM) for 30 min to allow for functional inhibition of HIF-

1α degradation under normoxic conditions. Cells were then exposed to different

concentrations of SN-38 (0-1 μM) for 24 h under normoxic or hypoxic conditions,

after which cell growth was determined by SRB assay. HIF-1α protein levels were

also determined by western blot analysis. β-Actin was measured as the loading

control. Each condition was studied in triplicate, and the error bars represent the

standard deviation around the mean.




33

Figure 2. TPZ enhanced the cytotoxicity elicited by topoisomerase I inhibitors under

hypoxic conditions. (A) Bel-7402 cells were exposed to hypoxia or normoxia and

treated with TPZ (0–10 μM) for 12 h. Protein levels of HIF-1α were detected by

western blot analysis. β-Actin was used as the loading control. (B) Bel-7402 cells

were exposed to hypoxia or normoxia and treated with TPZ (0–10 μM) for 6 h. An

HRE-dependent reporter assay was used to determine the effect of TPZ on HIF-1α

transcriptional activity. Five independent experiments were performed, and the values

are expressed as the mean ± SD. *P < 0.05, **P < 0.01 and ***P < 0.001, compared

to untreated controls in hypoxia. (C-F) Bel-7402 cells exposed to serial concentrations

of TPZ, topoisomerase I inhibitors (SN-38/ TPT/ HCPT/ MONCPT), or the constant-

ratio mixture of these two for 24 h under hypoxic condition. Cell growth was

determined by the SRB assay. Each condition was studied in triplicate, and the error

bars represent the standard deviation around the mean.

Figure 3. TPZ enhanced the cytotoxicity elicited by SN-38 in five HCC cell lines

under hypoxia. (A-E) Five HCC cell lines, Bel-7402, HepG2, SMMC-7721, Hep3B

and Huh 7, were treated with TPZ (0–20 μM) and/or SN-38 (0–0.2 μM) for 24 h

under hypoxia. Cell growth was determined by the SRB assay. The data are

representative of three independent experiments and are shown as the mean ± SD. (F)

Co-treatment with TPZ did not increase the toxicity of SN-38 in a normal human liver




34

cell line. A normal liver cell line, Chang liver cells, were treated with TPZ (0–20 μM)

and/or SN-38 (0–0.2 μM) for 24 h under hypoxia. Cell viability was determined by

the SRB assay. Three independent experiments were performed, and the values are

expressed as the mean ± SD.

Figure 4. TPZ enhanced the apoptosis induced by SN-38 in Bel-7402. (A-D) Bel-

7402 cells were exposed to SN-38 (0.1 μM) and/or TPZ (10 μM) for 12 h under

hypoxic conditions. (A) Apoptotic bodies were visualized by confocal microscopy

using DAPI staining. Scale bar, 50 μm. (B) Percentage of apoptosis was measured by

flow cytometry using PI staining. Three independent experiments were performed,

and the values are expressed as the mean ± SD. **P < 0.01, compared to group treated

with TPZ or SN-38 alone. (C) Loss of mitochondrial membrane potential (ΔΨm) was

measured by flow cytometry using the unique lipophilic cationic dye (JC-1). Three

independent experiments were performed, and the values are expressed as the mean ±

SD. *P < 0.05, compared to group treated with TPZ or SN-38 alone. (D) Percentage

of apoptosis was measured by flow cytometry using the Annexin V/PI apoptosis

detection kit. Three independent experiments were performed, and the values are

expressed as the mean ± SD. **P < 0.01, compared to group treated with TPZ or SN-

38 alone. (E) Bel-7402 cells were exposed to SN-38 (0.1μM) and/or TPZ (10 μM) for

0-24 h under hypoxic conditions. Protein levels of PARP, Caspase 3, and cleaved-




35

Caspase 3 were determined by western blot analysis. β-Actin was used as the loading

control.

Figure 5. The role of HIF-1α in the cellular apoptosis induced synergistically by SN-

38 and TPZ. (A-D) Bel-7402 cells were exposed to SN-38 (0.1 μM) and/or TPZ (10

μM) for 4 h under hypoxic conditions. (A) Protein levels of HIF-1α, HIF-1β, HIF-2α,

p-ATM, ATM, p-Chk1, Chk1, p-Chk2, Chk2, Rad51 and γ-H2AX were determined by

western blot analysis. β-Actin was used as the loading control. (B) Protein levels of

HIF-1α and γ-H2AX were analyzed by confocal microscopy. Scale bar, 20 μm. (C)

Total RNA was extracted and HIF-1α and VEGF mRNA expression was analyzed by

RT-PCR, using GAPDH as a housekeeping gene. Untreated controls in hypoxia were

scaled to 100%. Five independent experiments were performed, and the values are

expressed as the mean ± SD. *P < 0.05, compared to group treated with TPZ or SN-38

alone. (D) An HRE-dependent reporter gene assay was used to determine the effect of

SN-38 and/or TPZ on HIF-1α transcriptional activity. Five independent experiments

were performed, and the values are expressed as the mean ± SD. *P < 0.05, **P <

0.01 and ***P < 0.001, compared to group treated with TPZ or SN-38 alone. (E) Bel-

7402 cells were pre-incubated with CHX for 3 h in normoxic conditions and then

placed in fresh medium and treated with or without 10 µM TPZ plus 100 nM SN-38

for the indicated times under hypoxic conditions. The cells were harvested, and




36

lysates were immunoblotted with an anti-HIF-1α antibody. β-Actin was used as the

loading control. (F) HIF-1α deficient and wild type Bel-7402 cells were both exposed

to SN-38 (0.1 μM) for 2 h under hypoxic conditions and then placed in fresh medium

for 6 h under hypoxic conditions. Protein levels of HIF-1α and γ-H2AX at different

time points were analyzed by confocal microscopy. Scale bar, 20 μm.

Figure 6. The synergistic effect of SN-38 and TPZ on Bel-7402 human xenograft

models. (A-E) Nude mice bearing established Bel-7402 tumors were treated with

CPT-11 at 2.5 mg/kg by daily i.p. injections, and/or TPZ at 25 mg/kg by i.p. injection

every two days for 27 days. (A) Tumor volumes are expressed as the mean ± SEM.

***P < 0.001 versus vehicle-treated controls (n = 6 per group). (B) Relative tumor

volumes are expressed as the mean ± SEM. **P < 0.01 versus vehicle-treated controls

(n = 6 per group). (C) Body weights are expressed as the mean ± SEM. (D)

Expression of PARP, Caspase 3, cleaved-Caspase 3 and HIF-1α extracted from tumor

tissues from drug-administrated mice of the four groups were detected by western blot.

β-Actin was used as the loading control. (E, F) Effects of CPT-11 and/or TPZ on

expression levels of HIF-1α in Bel-7402 cell-derived tumors were determined by

hematoxylin and eosin (H&E) staining and immunohistochemical and

immunofluorescence analysis.




Published OnlineFirst December 20, 2013.Mol Cancer Ther Tian-Yu Cai, Xiao-Wen Liu, Hong Zhu, et al.

αHypoxia-Inducible Factor-1Topoisomerase I Inhibitors via Cooperative Modulation of Tirapazamine Sensitizes Hepatocellular Carcinoma Cells to

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