Biomedicine & Pharmacotherapy 66 (2012) 563–567

Short communication

Gef gene therapy enhances the therapeutic efficacy of cytotoxics in coloncancer cells

Raul Ortiz a, Jose Prados b,*, Consolacion Melguizo b, Ana R. Rama a, Pablo J. Alvarez b,Fernando Rodrıguez-Serrano b, Octavio Caba a, Houria Boulaiz b, Antonia Aranega b

a Department of Health Sciences, University of Jaen, Jaen, Spainb Institute of Biopathology and Regenerative Medicine (IBIMER), Department of Human Anatomy and Embryology, School of Medicine, University of Granada, 18071 Granada, Spain

A R T I C L E I N F O

Article history:

Received 25 April 2012

Accepted 28 May 2012

Keywords:

Colon carcinoma

Gef gene

Gene therapy

Apoptosis

Combined therapy

A B S T R A C T

The potential use of gene therapy to improve the response of patients with advanced cancer is being

intensively analyzed. We evaluated the cytotoxic impact of the gef gene, a suicide gene, which has a

demonstrated antiproliferative activity in tumor cells, in colon carcinoma cells in order to improve the

antitumour effect of chemotherapeutic drugs used as first line treatment in the management of

advanced colon cancer. We found that the gef gene induced a marked decrease in cell viability (50% in

24 h) in T-84 cells through cell death by apoptosis. Interestingly, when gef gene expression was

combined with drugs of choice in the clinical treatment of colon cancer (5-fluorouracil, oxaliplatin and

irinotecan), a strong synergistic effect was observed with approximately a 15–20% enhancement of the

antiproliferative effect. Our data demonstrate, for the first time, that gef gene expression induces

significant growth arrest in colon cancer cells and that it is able to enhance the effect of some cytotoxic

drugs compared with a single therapeutic approach. These results indicate the potential therapeutic

value of the gef gene in colon cancer combination therapy.

� 2012 Elsevier Masson SAS. All rights reserved.

Available online at

www.sciencedirect.com

1. Introduction

Colorectal cancer is the third most common cancer in men andthe second in women. The annual incidence of colon cancer isestimated to be 1.0 million, and approximately 500,000 people dieeach year as a result of colon cancer worldwide [1]. Recentadvances in chemotherapy and radiotherapy have increased themedian survival of patients. However, advanced or recurrentcolorectal cancer remains incurable by conventional treatments[2]. Therefore, better treatment options need to be explored for thetreatment of advanced colon cancer.

Suicide gene therapy has been proposed as a strategy for thetreatment of patients with advanced colon cancer and has beenassayed alone and in combination with other therapies (radiationor/and drugs) [3]. In colorectal cancer, classical suicide genetherapy with genes encoding enzymes that convert non-toxicprodrugs into cytotoxic compounds has been assayed. However,the two most widely used prodrug systems, the cytosinedeaminase/5-fluorocytosine (CD/5FC) and the herpes simplexvirus thymidine kinase (HSV-tk), have obtained limited results.Therapeutic genes which directly encode cytotoxic proteins could

* Corresponding author. Tel.: +34 958 248819; fax: +34 958 246296.

E-mail address: jcprados@ugr.es (J. Prados).

0753-3322/$ – see front matter � 2012 Elsevier Masson SAS. All rights reserved.

http://dx.doi.org/10.1016/j.biopha.2012.05.004

be an attractive alternative strategy. In contrast to classical suicidegene therapy, these new genes are not dependent on prodrugs,may act by killing both quiescent and rapidly dividing tumor cells,and may induce apoptosis in tumor cells. Interestingly, apoptosisdeficiency is a critical factor in colorectal cancer therapy failure[4,5] and thus, the development of gene therapy strategies thatenhance apoptosis phenomenon may provide a complementarystrategy for its treatment.

Toxic genes from non-eukaryotic organisms such as bacteria,plants, viruses and bacteriophages [6–9], are being widely usedin cancer gene therapy. Our group has developed a new cancergene therapy strategy using a toxic gene from the chromosomeof E. coli (gef) which does not need a prodrug to be effective intumour cells. The gef gene, a member of a gene family withhomologous cell-killing functions, encodes a membrane proteinof 50 amino acids that is anchored in the cytoplasmic membraneby the N-terminal portion. Activation of this protein inducesarrest of cellular respiration and cell death [10]. In humantumour cells, gef gene induces cell cycle arrest and apoptosis[11], and may be a complementary strategy for classic cancertreatments [12].

Based on these observations, we decided to investigate thetherapeutic potential of gef gene to enhance the cytotoxic activityof the classical drug used in the treatment in colorectal cancer.Results obtained suggest that the combination of both treatments

R. Ortiz et al. / Biomedicine & Pharmacotherapy 66 (2012) 563–567564

enhanced their anticancer effect and may be a promising therapyin patients with advanced-stage colorectal cancer.

2. Material and methods

2.1. Cell culture and drugs

The T-84 human carcinoma cell line, selected for its knowntreatment resistance due to an overexpression of labile anti-apoptotic proteins [13], was obtained from American Type CultureCollection (ATCC). Cells were grown in Dulbecco’s Modified Eagle’sMedium (DMEM) (Sigma, St. Louis, MO, USA), supplemented with10% fetal bovine serum (FBS) under air containing 5% CO2 in anincubator at 37 8C. 5-fluorouracil (5-FU), folic acid (FA), irinotecan(IRI) and oxaliplatin (OXA) was provided by the OncologyDepartment, Virgen de las Nieves Hospital, Granada.

2.2. Production of stable inducible cell clones

The pTRE plasmid (Tet-Off gene-expression system) wasobtained from Clontech Laboratories, Inc. (USA). T-84 cells wereinitially transfected with pTet-On (Fugene 6 DNA transfectionreagent, Roche, Spain) and successfully-transfected clones wereselected for geneticin (1 mg/mL) resistance. These clones were thentransfected with pTRE-gef, a pTRE2hyg vector containing the gef

cDNA (provided by Dr J.L. Ramos, Zaidın Experimental Station, CSIC,Granada, Spain), and selected for resistance to geneticin (1 mg/mL)and hygromycin (0.4 mg/mL). All clones were cultured in thepresence of doxycycline (Dox, 0.2 mg/mL, 24 h) to induce gef geneexpression, which was detected by reverse transcriptase (RT)-PCR(Promega Reverse Transcription System, Promega, Spain) using 1 mgof total RNA (Rneasy Mini kit, Qiagen). RNA integrity was assessed byamplification of b-actin. Amplified PCR products were analysedusing a Bio-Rad documentation system (Quantity One AnalysisSoftware). One transfected clone, T-84/S1/gef, presented significantgene gef expression in the presence of Dox with no backgroundexpression, and was used to test the simple and combinedtreatments. To determine the intracellular localization of the gefprotein, a gef-V5 epitope gene fusion was generated usingpcDNA3.1/V5-His-TOPO-TA (Invitrogen, Barcelona) and followingthe manufacturer’s protocol. The resulting plasmid (pcDNA3.1/gef-V5) was amplified in DH5 alpha chemically competent E. coli

(Invitrogene) and confirmed by sequence analysis.

2.3. Microscopic analysis

T-84 cells were transfected with the pcDNA3.1/gef-V5 usingFugene 6 (Roche) as described above. After 24, 48 and 72 h, thecells were washed with PBS, fixed in 100% methanol (roomtemperature) for 5 min, blocked with 1% bovine serum albumin/PBS and incubated with anti-V5-FITC antibody (Invitrogen) (1:500)for 1 h to determine gef/V5 fusion protein expression. The cellswere then rinsed briefly with PBS, mounted and visualized usingfluorescent microscopy analysis (Nikon Eclipse Ti, Nikon Instru-ments Inc. NY, USA). V5 was excited at 488 nm.

2.4. Proliferation assays and apoptosis analysis

T-84/S1/gef cells were seeded in a 96-well plate (6 � 103 cells perwell). After 24, 48, and 72 h Dox induction, 20 mL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution(5 mg/ml) was added to each well and incubated at 37 8C for afurther 4 h. Then, 200 ml of dimethyl sulfoxide (DMSO) was added toeach well after removal of the medium. The optical density wasdetermined using a Titertek multiscan colorimeter (Flow, Irvine,

California) at 570 and 690 nm. On the other hand, T-84/S1/gef cellswere studied by means of an Annexin V-FITC apoptosis detection kit(Pharmingen, San Diego, CA) as describe previously [12] todetermine possible apoptotic cell death resulting from gef genetransfection. Apoptosis was evaluated by fluorescence-activated cellsorter analysis (FACScan). All data were collected and analyzed usingthe Cellfit program with a FACScan flow cytometer (BectonDickinson, San Jose, CA, USA).

2.5. Combined therapy in T-84 cells

In order to determine the effect of the combined therapy (gef

gene/drugs), T-84/S1/gef cells were seeded in 48 well plates (5 � 103

cells per well) and treated with 5-FU (10–100 mM) and FA (5 mM)alone or combined with IRI (10–200 mM) or OXA (1–100 mM)according to Ganten et al. [14] and Rama et al. [15]. The response toeach combined anticancer treatment was evaluated by measuringcell proliferation with MTT as described above.

2.6. Statistical analysis

The SPSS 14 software package (SPSS, Chicago, IL, USA) was usedfor all statistical analyses. Results were compared using Student’s t

test. All data are expressed as means � SD. Differences wereconsidered statistically significant at a P value of less than 0.05.

3. Results

3.1. Gef gene overexpression inhibits T-84 cell growth

The T-84/S1/gef stable clone was cultivated in the presence ofDox. RT-PCR showed a slight time-dependent increase in gef

expression in comparison with the same cells without Dox(Fig. 1A). Analysis of these bands normalized to the correspondingb-actin signal (data not shown) indicated 2- and 2.7-fold higher at48 and 72 h versus cells at 24 h. Immunofluorescence analysisshowed the progressive increase of gef protein in T-84 cell cytoplasm(Fig. 1B). The growth of T-84/S1/gef cells exposed to Doxdemonstrated a significant and time-dependent decrease in cellviability (50.1% at 24 h, 69.7% at 48 h and 85.6% at 72 h). In contrast,the same cells not exposed to Dox had a similar proliferation thanparental T-84 cells or cells transfected with the empty vector(Fig. 1C). Light microscopy observations typically showed T-84 coloncancer cells with polygonal shape and sheet-like pattern in normalmonolayer culture in contrast to cells transfected with gef gene,which showed a progressive loss of monolayer culture uniformitywith the presence of irregular zones without cells (Fig. 1D).

3.2. Gef gene expression induces apoptosis in T-84 cells

To determine whether gef gene expression induced apoptosis,T-84/S1/gef cells were analyzed by FACScan. The apoptosis fractionswere 2.1 � 0.2% for T-84/S1/gef cells not induced with Dox. Similarresults were obtained for the parental cells (1.5 � 0.3%). In contrast, T-84/S1/gef cells induced with Dox at 24 h showed an apoptosis fractionof 61.5 � 3%, which was significantly higher than that of the controlgroups. After 48 and 72 h of transfection, the percentage of apoptoticcells increased to 72.3 � 4% and 85.07 � 3%, respectively.

3.3. Combination of gef gene and cytotoxics produces a synergistic

antitumor effect in T-84 cells

To evaluate the gef gene/drugs combination for the treatment ofcolon cancer, T-84/S1/gef cells (before and after Dox induction)were exposed to a wide range of 5-FU, FA, IRI and OXA drug

Fig. 1. Gef gene expression and growth arrest in T-84 cells. A. RT-PCR detection of gef

gene expression in T-84 transfected cells (T-84/S1/gef) before and after Dox

exposure. Lane 1, Molecular weight; Lane 2, pTRE-gef (positive control); Lanes 3–5,

T-84/S1/gef cells 24, 48 and 72 h after Dox induction, respectively. Lane 6, T-84/S1/

gef cells in absence of Dox (negative control). B. Subcellular localization of the gef/

V5 fusion protein expressed in T-84/S1/gef cells. The fusion, protein detected by

anti-V5-FITC antibody, is shown in green. Twenty-four hours after transfection (a,

�40) the fluorescence pattern was dotted and localized in the cell cytoplasm. The

intensity of staining progressively increased after 48 (b, �40) and 72 h (c, �40) of

transfection. T-84 control cells showed diffuse green staining (d, �40). C. Phase-

contrast photomicrographs showed morphology changes of T-84/S1/gef cells before

and after gef gene expression. In absence of Dox (a) (�20), T-84/S1/gef cells grew in

clumps, were typically polygonal and formed a monolayer culture. In contrast, at 24

(b), 48 (c) and 72 h (d) (�20) after Dox induction, cells formed an irregular

monolayer with progressive presence of zones without cells and lost their

polygonal morphology, appearing rounded. D. MTT assay of T-84/S1/gef cells

induced with Dox showed a clearly increased cell death compared to the T-84/S1/

gef cells in absence of Dox, parental cells and cells transfected with empty vector

(P < 0.05). Values represent means � SD of quadruplicate cultures.

Fig. 2. Combined gef gene/cytotoxic drugs therapy showed a synergistic effect on

growth arrest in the T-84 colon cancer cell line. A. Inhibition of cell proliferation of

T-84/S1/gef cells induced with Dox and treated with 5-FU/FA alone or combined

with IRI at 24 h. All results shown are mean � S.D. of four independent experiments.

B. Inhibition of cell proliferation of T-84/S1/gef cells induced with Dox and treated with

5-FU/FA alone or combined with OXA at 24 h. All results shown are mean � S.D. of four

independent experiments.*Indicates experiments in which combined therapy achieves

100%.

R. Ortiz et al. / Biomedicine & Pharmacotherapy 66 (2012) 563–567 565

concentrations, as described in Materials and methods. In order todetermine the possible synergistic effect and by the highantiproliferative effect of gef gene in T-84 colon cancer cells(50.1% at 24 h), the next experiments were carried out at 24 h. Themost interesting finding was that gef gene/drug therapy (combinedtherapy) produced not simply a sum their individual effects but asignificant enhancement of the antitumor activity. In fact, gef gene/5-FU (10, 50 and 100 m;)/FA induced 74.6 � 3%, 77.3 � 2.5% and88.5 � 2.9% cell growth arrest, respectively. This data represented a15.5 � 0.6%, 12.2 � 0.4% and 16.4 � 1% greater inhibition of prolif-eration than the sum of both gef gene and drug treatments,supporting the synergistic effect of combined therapy (Fig. 2A). Thisenhancement phenomenon was also observed with the use of gef

gene/5-FU/FA/IRI. In fact, the association of gef gene/5-FU (10 m;)/FA/IRI (10, 100 and 200 mM) induced the most significant synergeticeffect with a 17 � 0.4%, 15.2 � 0.8% and 12 � 0.2% greater inhibition

of proliferation respectively, than obtained with the sum of theseparate treatments. This combined therapy was also assayed with 5-FU at 50 and 100 mM although for this last concentration theenhancement effect was slightly less (Fig. 2A). However, the mostrelevant results were found when gef gene was associated to 5-FU/FA/OXA. The T-84 cells expressing gef gene and exposed to 5-FU (10 m;)/FA/OXA (1, 10 and 100 mM) showed a 20.2 � 0.9%, 21.2 � 0.7% and18.5 � 0.9% greater inhibition of proliferation respectively, than thoseobserved with the sum of gef gene and drugs treatment. Similarresults were found with the association 5-FU (50 m;)/FA(5 m;)/OXA(1, 10 and 100 mM) (Fig. 2B).

4. Discussion

Gene therapy strategies are currently being developed for thetreatment of advanced cancer. Up to now, most colon cancer genetherapy assays using the HSV-tk enzyme have had limited results[16–18] and the use of specific promoters, such us Cox-2 or theurokinase plasminogen activator receptor, improves its tissue

R. Ortiz et al. / Biomedicine & Pharmacotherapy 66 (2012) 563–567566

specificity but not its efficiency [19,20]. The bacterial carboxypep-tidase G2 enzyme (CPG2) and the CD enzyme have also been assayedbut without producing definitive results. Zhang et al. [21] describeda decrease in LoVo colon cancer cell viability (23.62%) after CDtransfection using the carcinoembrionary antigen promoter (CEA).Schepelmann et al. [22], using CPG2, detected apoptotic regions andsignificant bystander effects in tumors generated with SW620 coloncancer cells. However, prodrugs continue to be one of the mainlimiting factors of these strategies [23].

In this context, the transfection of cDNA constructs, encodingtoxins with a direct antitumor action, represents an alternativestrategy. The non-systemic administration of a prodrug reduces itsbioavailability limitations, its side effects, and the need for twoconsecutive applications (vector and prodrug). These toxic geneshave been successfully used in pancreas cancer (diphtheria toxingene) [6] and glioma and lung and hepatocellular carcinoma(streptolysin O gene) [24]. Recently, Cheng et al. [25] developed aStaphylococcal enterotoxin C2 (SEC2) mutant gene which inhib-ited the growth of hepatocellular liver carcinoma and lung cancercells (HepG-2 and A549 cell line, respectively). Similar results havebeen obtained with a hybrid toxin obtained from Clostridium

perfringens enterotoxin (CPE) and Pseudomonas aeruginosa exo-toxin A (ETA) in breast, skin, lung, prostate and colon cancer celllines [26]. Bacteriophage lambda-holin protein led to a substantialreduction of cell viability of more than 98% in breast cancer cells[27] and E gene from the fX174 phage was able to induce an intensemelanoma and colon cancer cell destruction [15,28,29]. Recently,we have showed that the suicide gef gene not only inhibitsmelanoma and breast cancer cells proliferation [11,30] but alsoimproves doxorubicin and paclitaxel toxicity against breast andlung cancer cells, respectively [12,31]. However, the gef gene utilityin colon cancer has not been assayed.

Our present results show that gef gene expression in coloncancer cells induced a rapid decrease in cell viability and an intenseapoptosis (61.5% apoptosis at 24 h), which was much greater thanthat observed in others tumor cells derived from melanoma, breastcancer or lung cancer [12,31,32]. This observation was veryinteresting because one of the characteristics implicated in coloncancer development and metastasis formation is the resistance toapoptosis phenomenon [33]. In fact, some antitumor strategiesusing toxic genes, such us Toxin A from Clostridium difficile, haveexplored the possibility of unblocking the apoptosis in this tumor,inducing Bak expression, caspase-3 activation, and finally,apoptosis via p38 [34]. This apoptosis modulation may be apromising new strategy in colon cancer treatment, as shown byFaber et al. [35], using small interfering RNAs (siRNA) to silence theexpression of antiapoptotic proteins and Yamakuchi et al. [36],using MicroRNA 34a which leads to an increase in acetylated p53and the expression of p21 and PUMA.

In spite of the promising results with gene therapy, it is clearthat this therapeutic strategy has limitations in advanced cancer.Interest is growing in the development of combined approachesusing therapeutic genes and local tumor irradiation or chemother-apy. Novel advances have been reported in the use of combinedtherapy for the treatment of bladder cancer [37], pancreatic cancer[38] and hepatocellular carcinoma [39]. In colorectal cancer, Abazaet al. [40] have recently shown that antisense c-myc combinedwith cytotoxic drugs (including 5-FU) increased caspase-3 (amongother apoptotic signals) and exhibited a marked apoptotic effect incomparison with single treatments. With a similar objective, wehave explored the capacity of the gef gene to enhance the cytotoxiceffect of drugs (5-FU/FA, OXA and IRI) whose association hasshown an increase in the survival of patients with colon metastaticdisease [41]. Our results show that the gef gene/5-FU combinedtherapy, either alone or with IRI and OXA, produced a decrease inT-84 cell viability that was greater than that obtained with

individual treatments and around 15–20% greater than the sum ofthe treatments (gef gene/drugs) used separately. This findingsupports the hypothesis that a synergetic effect occurs when bothtreatments are applied together. Interestingly, lower concentra-tions of 5-FU (10 mM) induced a similar enhanced antiproliferativeeffect to that of higher concentrations of this drug, suggesting thatthe gef gene/5-FU combination may reduce the cytotoxicconcentrations needed in colon cancer treatment.

In this study, we demonstrate for the first time, the utility of theE. coli gef gene for inducing growth arrest in colon cancer cells andclearly showed that its antiproliferative effect is significantlyhigher than in other lines assayed. Interestingly, gef gene activity ismediated by the induction of apoptosis which may be deficient inadvanced or metastatic colon cancer. In addition, gef genesignificantly enhanced the cell growth inhibition induced bycytotoxic drugs (either 5-FU alone or combined with IRI and OXA)used in the classical treatment of colon cancer. Although futurestudies will be necessary to determine the utility of combinedtherapy in vivo, at the present time the gef gene may be a candidatefor the development of new colon cancer strategies.

Disclosure of interest

The authors declare that they have no conflicts of interestconcerning this article.

Acknowledgments

This study was supported by the Instituto de Salud Carlos III(FIS) through Project no. PI11/01862, by the Science andInnovation Ministry (Project no. SAF2009-12295) and by theConsejerıa de Salud de la Junta de Andalucıa through Project no.PI-0338.

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