ORIGINAL ARTICLE—ALIMENTARY TRACT
Conditionally replicative adenovirus-based mda -7/IL-24expression enhances sensitivity of colon cancer cells to 5-?uorouracil and doxorubicin
Jing Xu ?Yiwo Mo ?Xiaoyun Wang ?
Jun Liu ?Xinjin Zhang ?Junfeng Wang ?
Lei Hu ?Chao Yang ?Lei Chen ?Yankun Wang
Received:25October 2011/Accepted:28May 2012óSpringer 2012
Abstract
Background Multiple drug resistance (MDR)greatly lim-its the ef?cacy of chemotherapy for colon cancer.An ade-novirus armed with Melanoma differentiation associated gene -7/interleukin-24(mda-7/IL-24;abbreviated to ‘IL-24’here)was shown to reverse the MDR of colon cancer cells to oxaliplatin and doxorubicin.However,the relatively low expression level of IL-24mediated by a replication-de?cient adenoviral vector hindered its clinical application.
Methods To enhance IL-24-dependentreversion of the MDR phenotype,we utilized a conditionally replicative adenoviral vector,AdBB-IL24,to express IL-24at a high level for more ef?cient MDR reversion.
Results An enzyme-linked immunosorbent assay (ELISA)suggested conditionally replicative adenoviral vector-medi-ated IL-24expression was elevated in comparison with that of a replication-de?cient adenoviral vector,Ad-IL24.AdBB-IL24was shown to reverse MDR in colon cancer cells more potently than Ad-IL24.The AdBB-IL24-induced MDR reversion was linked to reduced P-glycoprotein (Pgp)and breast cancer resistance protein 1(BCRP1)expression.Consistently,5-?uorouracil and doxorubicin induced more apoptosis in AdBB-IL24-infected colon cancer cells com-pared with that in the Ad-IL24-infected cells.A cell viability assay showed that AdBB-IL24could enhance the growth-inhibitory effect of 5-?uorouracil and doxorubicin on colon cancer cells more effectively than Ad-IL24in vitro.In a
mouse model,we also found that the combination of 5-?u-orouracil and doxorubicin with AdBB-IL24completely inhibited the growth of colon cancer cells.
Conclusion We here provide evidence supporting condi-tionally replicative adenoviral vector-based gene therapy as a powerful strategy to enhance mda7/IL-24-dependent MDR reversion of colon cancer cells.
Keywords Colon cancer áGene therapy ámda-7/IL-24áCRAd áMDR
Introduction
Colon cancer is the third most common cancer type in the world.In spite of progressive improvements in diagnosis and therapeutics,40%of colon cancer patients still fail to survive for 5years [1].Chemotherapy is one of the stan-dard treatment regimens for colon cancer patients [1].However,its clinical outcome is shown to be usually impeded by multiple drug resistance (MDR)in colon cancer cells.In some cases,patients even display little response to high doses of cytotoxic agents.The ATP-binding cassette (ABC)transport family plays a central role in MDR induction and maintenance in cancer cells [2].The transmembrane ABC transporters,mainly including P-glycoprotein (Pgp),multidrug resistance-associated pro-tein 1(MRP1),and breast cancer resistance protein (BCRP1),reduce the intracellular concentrations of cyto-toxic drugs through actively pumping the drugs out of cells,thereby protecting cancer cells from the chemotherapeutic agent-induced anti-tumor effect.
To reverse the MDR phenotype of colon cancer cells,researchers have developed strategies targeting some ABC transporters,such as Pgp [3,4].A speci?c inhibitor [5],a
J.Xu and Y.Mo contributed equally to this work.J.Xu áY.Mo áX.Wang áJ.Liu áX.Zhang áJ.Wang áL.Hu áC.Yang áL.Chen áY.Wang (&)
Department of Hepatobiliary Surgery,The First People’s Hospital of Yunnan Province,Kunhua Hospital Af?liated to Kunming Medical College,Kunming,China e-mail:yankunwang@https://www.doczj.com/doc/a818014298.html,
J Gastroenterol
DOI 10.1007/s00535-012-0623-y
function-blocking monoclonal antibody[4,6],and Pgp-targeting short-interfering RNA(siRNA)[7]have been demonstrated to sensitize colon cancer cells to chemo-therapeutic agents through abrogating Pgp function.How-ever,the inef?ciency of survival-bene?ting effect of speci?c inhibitors in clinical trials does not provide a promising perspective[8].Therefore,effective alternative strategies are greatly required to reverse MDR[9].
Recently,Melanoma differentiation associated gene-7/ interleukin-24(mda-7/IL-24;abbreviated here to IL-24)was demonstrated to reverse the MDR phenotype of colon cancer cells.Fisher’s research team(Emdad et al.[10])found that IL-24was able to increase the intracellular accumulation of doxorubicin in colon cancer cells,and eventually enhance the cell sensitivity to doxorubicin-induced apoptosis10. Another study,by Reardon et al.[11],also showed that an IL-24-armed adenovirus(AdIL-24)could restore the apop-tosis-inducing activity of oxaliplatin to oxaliplatin-resistant HT-29colorectal cancer cells(HT-29/oxa)11.The IL-24-dependent MDR reversion in colon cancer cells seems to be caused by the downregulation of Pgp expression[10]. Compared with the Pgp inhibitors currently being examined in clinical trials,IL-24has high selectivity for tumor cells, and is almost without cytotoxicity to normal tissues[12]. The safety and ef?ciency of IL-24have attracted the attention of researchers who have been searching for novel chemosensitizers.
It is worth noting,however,that both of the above research groups(Emdad et al.[10]and Reardon et al.[11]) used replication-de?cient adenoviral vectors to transfer IL-24-encoding DNA into colon cancer cells.In this system of gene transfer,the copy number of the DNA sequence encoding IL-24cannot multiply,only allowing low or moderate IL-24expression.The low expression level of IL-24probably severely limits its clinical application as a chemosensitizer.The same problem is also seen during the utilization of adenovirus-based gene therapy for the p53-mediated sensitizing of hepatocellular carcinoma(HCC) cells to radiation therapy[13].
Conditionally replicative adenovirus(CRAd)is a pow-erful vehicle with which to express an inserted gene at a high level.To increase the expression level of adenoviral vector-mediated IL-24,we thus constructed a CRAd, AdBB-IL24.As a potent expression vector for IL-24, AdBB-IL24replication is almost preserved in cancer cells. Unlike the replication-de?cient adenoviral vectors used in previous studies,CRAd can duplicate its genome selec-tively in neoplastic cells,rather than in normal cells.Also, exogenous genes inserted into CRAd would be copied at an almost exponential level.With this CRAd system,trans-ferred IL-24was shown to be expressed at a signi?cantly higher level than in systems using a replication-de?cient adenoviral vector.More importantly,we con?rmed that a CRAd system would potentiate the chemosensitizing effect of IL-24on colon cancer cells by enhancing MDR rever-sion.The widely used cytotoxic chemicals,5-?uorouracil and doxorubicin,were employed in our experiments of IL-24-dependent reversion of MDR.
Methods
Cell lines and cell culture
Human colorectal adenocarcinoma cell lines(HT-29, SW480,and SW620),a human normal liver cell line(L-02), and a human normal lung?broblast cell line(MRC-5)were purchased from the Shanghai Cell Collection(Shanghai, China).A human embryonic kidney cell line(HEK-293) was obtained from Microbix Biosystems(Toronto,Canada). The cells were cultured in Dulbecco’s modi?ed Eagle’s medium(DMEM)supplemented with10%of fetal bovine serum(FBS)(Gibco-BRL,Gaithersburg,MD,USA)and 4mM of L-glutamine.The incubators we used contained 5%CO2in a humidi?ed37°C atmosphere.
Adenoviruses and chemicals
Replication-de?cient adenoviruses,Ad-enhanced green ?uorescent protein(EGFP)and Ad-IL24,were preserved in our laboratory.The production of AdBB-IL24and AdBB-EGFP was done as follows:pBB-EGFP and pBB-IL-24 were generated according to the standard molecular cloning protocols.Homologous recombination between pBB-EGFP and pBB-IL-24plasmids and the pAd-bone plasmid-carrying oncolytic adenoviral backbone was per-formed in the BJ5183Escherichia coli strain to generate pAdBB-EGFP and pAdBB-IL-24,respectively.Viral parti-cles were produced in HEK-293cells by transfection with PacI-digested pAdBB-EGFP and pAdBB-IL-24to generate recombinant AdBB-EGFP and AdBB-IL-24.The construc-tion of adenoviral vectors involved in this work is summarized schematically in Fig.1.The adenoviruses were then concentrated and puri?ed by a CsCl gradient ultracen-trifugation method.The titers of the adenoviruses involved in our work were quanti?ed using the TCID50assay.
5-Fluorouracil and doxorubicin(Sigma-Aldrich,St.Louis, MO,USA)were used and stored following the manufac-turer’s instructions.
Virus progeny determination
Cells were plated in6-well plates at19105/well.Over-night,cells were infected with adenoviruses(multiplicity of infection[MOI]10).Two hours later,adenoviruse-containing cell media were removed,and then the cells
J Gastroenterol
were cultured in 5-?uorouracil-or doxorubicin-containing media.At that moment (0h)or 48h later,cells were lysed through cycles of freezing and thawing.The titers of viral progenies were quanti?ed in HEK-293cells with the TCID 50(50%tissue culture infective dose)assay and were expressed as plaque-forming units (pfu).ELISA
A two-antibody sandwich enzyme-linked immunosorbent assay (ELISA)was developed for the detection of human IL-24.The antibodies used were monoclonal mouse anti-human IL-24antibody (R&D Systems,Minneapolis,MN,USA),goat anti-human IL-24antibody (R&D Systems),and
peroxidase-conjugated rabbit anti-goat IgG (H&L)(R&D Systems).The absorbance was read at a 450-nm wavelength.The concentration of IL-24was evaluated with a standard curve.
Cell viability assay
Cells were plated on 96-well plates at 19104/well.Overnight,adenoviruses and/or the cytotoxic chemicals were added to the media.At various time points,cells were treated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT,1mg/ml)followed by cell viability measurement on a microplate reader (Tecan,Mannedorf,Switzerland).
HT-29
SW480
SW620
1.E+03
1.E+041.E+051.E+061.E+071.E+081.E+09HT-29SW-480SW-620L-02MRC-5 1.E+03
1.E+041.E+051.E+061.E+071.E+081.E+09HT-29SW-480SW-620L-02MRC-5
1.E+03
1.E+041.E+051.E+061.E+071.E+081.E+09HT-29
SW-480SW-620L-02MRC-5******
**
**
******
**
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**
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****5-fluorouracil (2.5μg/mL)no drug treatment doxorubicin (400 nM)
a J Gastroenterol
Cell apoptosis detection
Cells were plated in6-well plates at 3.59105/well. Overnight,adenoviruses and/or the cytotoxic chemicals were added to the media.Ninety-six hours later,the cells were harvested,followed by staining with?uorescein iso-thiocyanate(FITC)-conjugated anti-Annexin V antibody and propidium iodide(PI).Apoptotic cells(Annexin V-positive/PI-negative cells)were quanti?ed by?uores-cence activated cell sorting(FACS)analysis.
Detection of Pgp,MRP1,and BCRP1
Cells were plated on6-well plates at3.59105/well.Over-night,adenoviruses and/or the cytotoxic chemicals were added to the media.Ninety-six hours later,the cells were harvested,followed by staining with monoclonal mouse ?uorescein-conjugated anti-human P-glycoprotein(Abcam, Cambridge,UK),monoclonal mouse?uorescein-conjugated anti-human MRP1,and monoclonal mouse?uorescein-con-jugated anti-human BCRP1(R&D Systems).Pgp-,MRP1-, and BCRP1-positive cells were detected by FACS analysis.
Doxorubicin accumulation assay
The intracellular amount of doxorubicin was assessed when HT-29,SW480,and SW620cells were infected with dif-ferent adenoviruses(10MOI).After24h,400nM of doxorubicin was added to cell cultures and the cultures were then incubated at37°C for1h.The cells were washed with cold phosphate-buffered saline(PBS), re-suspended in PBS,and analyzed using?ow cytometry. Animal experiments
All procedures for animal experiments were approved by the Committee on the Use and Care of Animals(The Third Military Medical University,Chongqing,China)and per-formed in accordance with the institution guidelines. SW620tumor xenografts were established by subcutane-ously inoculating59106cells into the right?anks of4-to 6-week-old BALB/c nude mice(Institute of Animal Cen-ter,Daping Hospital,Third Military Medical University, Chongqing,China).When the tumors reached between6 and8mm in diameter,34mice were randomly assigned to a PBS-treated group(n=6),a5-?uorouracil-or doxoru-bicin-treated group(n=6),an Ad-IL24plus5-?uorouracil or doxorubicin-treated group(n=7),an AdBB-EGFP plus 5-?uorouracil or doxorubicin-treated group(n=7),and an AdBB-IL24plus5-?uorouracil or doxorubicin-treated group(n=8).The established tumors were injected with 100l L of PBS with or without19109pfu of Ad-IL24, AdBB-EGFP,or AdBB-IL24.5-Fluorouracil(5mg/kg)or doxorubicin(4mg/kg)was injected intraperitoneally (4mg/kg)at the same time.Tumor growth was monitored by periodic measurements with calipers and tumor volume was calculated using the following formula:tumor volume (mm3)=maximal length(mm)9(perpendicular width) (cm)2/2.In our experiments,no mice were observed to have died of tumor loading.
Statistical analysis
All the statistical tests involved in our work were two-tailed. Data were considered statistically signi?cant when P\0.05 and highly statistically signi?cant when P\0.01. Results
AdBB-IL24replicated selectively and ef?ciently
in colon cancer cells in the presence of5-?uorouracil
or doxorubicin
CRAd retains the ability to infect cancer cells and replicate within them,thereby allowing the ampli?cation of gene-encoding open reading frames(ORFs)that are inserted into CRAd for high expression of these genes.However,it is not clear whether CRAds also replicate ef?ciently in colon cancer cells in the presence of chemotherapeutic agents.
We investigated the infectivity and replication ef?-ciency of AdBB-IL24in5-?uorouracil-or doxorubicin-treated colon cancer cells HT-29,SW480,and SW620. AdBB-EGFP,a CRAd almost identical to AdBB-IL24 except for EGFP in place of IL-24,was used to investigate the infectivity of the CRAd.HT-29,SW480,and SW620 cells were all treated with10MOI of AdBB-EGFP as well as5-?uorouracil(2.5l g/mL)or doxorubicin(400nM). Twelve hours later,the cells were observed under a?uo-rescence microscope.EGFP-positive cells were extensively seen in the infected cells(Fig.1),showing that this CRAd had high infectivity in colon cancer cells.
Subsequently,we infected HT-29,SW480,and SW620 cells with Ad-EGFP(10MOI),Ad-IL24(10MOI),AdBB-EGFP(10MOI),or AdBB-IL24(10MOI)with or without 5-?uorouracil(2.5l g/mL)or doxorubicin(400nM). Forty-eight hours later,the titers of adenoviral progenies were determined by the TCID50method.AdBB-EGFP and AdBB-IL24were both shown to ef?ciently replicate in colon cancer cells,rather than in normal cells(L-02and MRC-5),regardless of whether drug treatment was per-formed.However,the replication-competent adenoviral vectors,Ad-EGFP and Ad-IL24,failed to replicate in cancer cells and normal cells(Fig.1).
Collectively,we con?rmed that colon cancer cells were sensitive to infection with AdBB-EGFP,and we found that
J Gastroenterol
AdBB-EGFP and AdBB-IL24ef?ciently replicated in the presence of 5-?uorouracil and doxorubicin.AdBB-IL24-infected colon cancer cells had a higher level of IL-24expression than the Ad-IL24-infected cells
Associated with the replication of CRAd,the inserted ORFs were also effectively propagated in cancer cells,thereby elevating the expression of the inserted genes.We next compared the IL-24expression levels of AdBB-IL24and Ad-IL24in colon cancer cells with or without 5-?uorouracil or doxorubicin.HT-29,SW480,and SW620were infected with 10MOI of AdBB-IL24or Ad-IL24followed by the administration of 5-?uorouracil (2.5l g/mL)or doxorubicin (400nM)or no administration.Forty-eight hours later,IL-24expression was quanti?ed using an ELISA assay.AdBB-IL24was shown to have a higher expression of IL-24than the replication-de?cient vector,Ad-IL24(Fig.2),in colon cancer cells with or without 5-?uorouracil or doxorubicin.Furthermore,there was no difference between normal cell lines infected with AdBB-IL24and Ad-IL24(Fig.2).
These data suggested that CRAd was a more potent vector for exogenous IL-24expression in colon cancer cells than the replication-de?cient adenovirus.
AdBB-IL24reversed MDR of colon cancer cells more ef?ciently than Ad-IL24
IL-24was reported to reverse the MDR of colon cancer cells,and therefore we investigated the expression levels of the MDR-related proteins,Pgp,BCRP1,and MRP1,in AdBB-IL24-or Ad-IL24-infected colon cancer cells in the presence of 5-?uorouracil (2.5l g/mL)or doxorubicin (400nM).FACS analysis showed that both 5-?uorouracil and doxorubicin induced the expression of Pgp and BCRP1,but not MRP1,in the colon cancer cells (Fig.3a–c).AdBB-IL24and Ad-IL24suppressed the cytotoxic chemi-cal-induced Pgp and BCRP1expression,and AdBB-IL24suppressed the levels of these two proteins relatively more ef?ciently than Ad-IL24(Fig.3a–c).
We also performed a doxorubicin accumulation assay to further con?rm the improved MDR-reversing activity of AdBB-IL24in colon cancer cells.The results had revealed that Ad-IL24infection led to the enhanced accumulation of doxorubicin in colon cancer cells.However,increased intracellular doxorubicin accumulation was observed in AdBB-IL24-infected HT-29,SW480,and SW-620cells (Fig.3d).
Conclusively,AdBB-IL24was shown to have a stronger MDR-reversing activity than Ad-IL24in colon cancer cells.
HT-29
SW480SW620L-02MRC-5
AdBB-IL24
100200300400500600700800HT-29
SW480SW620L-02MRC-5HT-29SW480SW620L-02MRC-5
**
**
**
5-fluorouracil (2.5μg/mL)
mediated by AdBB-IL24in colon cancer with AdBB-IL24or Ad-IL24at 10MOI 5-?uorouracil (2.5l g/mL)or doxorubicin (400nM).Forty-eight hours later,IL-24levels were quanti?ed Columns average values of three independent mean ±SD.**P \0.01
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AdBB-IL24infection enhanced drug-induced apoptosis of colon cancer cells
5-Fluorouracil and doxorubicin inhibited the growth of cancer cells by the induction of apoptosis.Therefore,we identi?ed whether the stronger reversion of MDR caused by AdBB-IL24could lead to enhanced 5-?uorouracil-and doxorubicin-induced apoptosis in the colon cancer cells.AdBB-IL24-or Ad-IL24-infected HT-29,SW480,and SW620cells were exposed to 5-?uorouracil (2.5l g/mL)or doxorubicin (400nM).Ninety-six hours later,we quanti-?ed the apoptosis rates of the cancer cells with the Annexin-V/PI staining assay.FACS analysis data sug-gested that the percentage of Annexin-V ?/PI -cells in the presence of 5-?uorouracil (2.5l g/mL)or doxorubicin
(400nM)was much higher in the AdBB-IL24-infected group than in the Ad-IL24-infected group (Fig.4).
Thus,we showed that AdBB-IL24conferred higher vulnerability to 5-?uorouracil-and doxorubicin-induced apoptosis than Ad-IL24in colon cancer cells.AdBB-IL24potentiated the growth-inhibiting effect of 5-?uorouracil and doxorubicin in colon cancer cells Cancer cell survival was also determined when AdBB-IL24and cytotoxic drugs were administered together.AdBB-IL24or Ad-IL24(10MOI)was used to infect HT-29,SW480,and SW620colon cancer cells,as well as L-02and MRC-5normal cells,and 5-?uorouracil (2.5l g/mL)or doxorubicin (400nM)was added to the media.
H T
-29 SW480SW620
H T
-29 SW480SW620
H T -29 SW480
SW620
H T -29 SW480
SW620
H T -29 SW480
SW620
H T
-29 SW480SW620
5-fluorouracil (2.5μg/mL)
doxorubicin (400nM)
H T -29 SW480
SW620
Ad-EGFP Ad-IL24AdBB-EGFP AdBB-IL24
H T -29 SW480SW620H T -29 SW480SW620
H T -29 SW480SW620
cells.HT-29,
Ad-IL24,of 5-?uo-hours later,cells was percentages of the
Pgp-,MRP1-,and BCRP1-positive population in each group.average values of three independent experiments are shown with SDs.d A doxorubicin accumulation assay was performed to investigate MDR reversion cancer cells after adenoviruses were administered
J Gastroenterol
The survival was quanti?ed with the MTT assay at various time points.Although Ad-IL24displayed moderate MDR-reversing activity in SW480and SW620cells,we observed an improved sensitizing effect of AdBB-IL24to5-?uoro-uracil or doxorubicin in all colon cancer cells(Fig.5a,b). Furthermore,neither AdBB-IL24nor Ad-IL24showed any additional toxicity to the normal cells,L-02and MRC-5 (Fig.5a,b).In addition,only limited anti-tumor capacity was observed when AdBB-IL24or Ad-IL24was applied alone(Fig.5c).
These data demonstrate that,in the presence of che-motherapeutic agents,AdBB-IL24inhibited the growth of colon cancer cells more effectively than Ad-IL24,almost without cytotoxicity to normal cells.
AdBB-IL24-enhanced MDR reversion induced
5-?uorouracil and doxorubicin to inhibit the growth
of colon cancer in an animal model more ef?ciently than Ad-IL24
Finally,we investigated whether AdBB-IL24enhanced the anti-tumor effect of5-?uorouracil and doxorubicin in colon cancer cells in nude mice.SW620was used to establish tumors in BALB/c nude mice.Cytotoxic chemicals,cyto-toxic chemicals plus Ad-IL24,cytotoxic chemicals plus AdBB-EGFP,or cytotoxic chemicals plus AdBB-IL24 were administered to mice after the tumors had formed.Our data suggested that the growth of cytotoxic chemical-treated tumors was almost completely inhibited when AdBB-IL24was used simultaneously.No signi?cant dif-ference in tumor growth was observed between the group treated with cytotoxic chemicals plus Ad-IL24and that treated with the cytotoxic chemicals alone(5-?uorouracil: P=0.2031;doxorubicin:P=0.1774)(Fig.6a,b).Com-pared with Ad-IL24,AdBB-IL24potentiated the growth-suppressing effect of both5-?uorouracil and doxorubicin on SW620xenografts much more ef?ciently(5-?uoroura-cil:P=0.0001,doxorubicin:P=0.0002)(Fig.6a,b,d). We also investigated the growth-suppressing activity of the adenoviruses,Ad-IL24or AdBB-IL24,on SW620tumor xenografts,when the adenoviruses were applied alone.The two adenoviruses showed moderate anti-tumor capacity, but the effect was not signi?cant(P=0.3061)(Fig.6c).
The animal model experiments con?rmed that AdBB-IL24-mediated MDR reversion enhanced the anti-tumor activity of5-?uorouracil and doxorubicin in colon cancer cells in vivo.
Discussion
We utilized a human telomerase reverse transcriptase (hTERT)promoter to drive the expression of the E1A protein of adenoviral vectors in our study.hTERT has been
HT-29SW480SW620Ad-EGFP AdBB-EGFP
HT-29SW480SW620 HT-29SW480SW620
induced by5-?uorouracil or doxorubicin when were infected with AdBB-IL24.Ad-EGFP, or AdBB-IL24at10MOI was added
in the presence or absence of5-?uorouracil
g/mL)or doxorubicin(400nM).Ninety-six
rates of cells were quanti?ed using Annexin-V-FITC/PI
FACS analysis.Columns average values experiments;bars means±SD.*P\0.05.**
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well documented to be overexpressed in a variety of cancer types,including colon cancers[14,15],ensuring the can-cer-speci?c expression of introduced exogenous genes with the hTERT promoter.In spite of the hTERT promoter having lower activity than the original E1A promoter, replacement of the promoters did not greatly affect the replication of adenoviruses,either in vitro or in vivo[16]. More importantly,hTERT-regulated oncolytic adenovi-ruses showed very low cytotoxicity to normal tissue-derived cells[16].Therefore,hTERT-modulated oncolytic adenoviruses were shown to be promising agents that have the potential for clinical application.
hTERT has been well documented to be highly expressed in cancer cells,rather than normal cells. Therefore,the differential activity of the hTERT promoter ensured the selective replication of adenovirus particles in cancer cells when it was used to drive E1A expression.Although CRAds possess the ability to replicate selec-tively and,to a great extent,more effectively in cancer cells rather than normal cells[17,18],their capability for replication in the presence of chemotherapeutic agents has been poorly understood.A research team has reported that lovastatin can promote the proliferation of a well-designed CRAd,dl1520,both in vitro and in vivo[19].In our present study,AdBB-IL24was also con?rmed to replicate ef?ciently in colon cancer cells treated with a cytotoxic agent.Within48h,the amount of CRAd vec-tors was increased by up to more than1000fold,whereas the replication-de?cient adenoviral vector,Ad-IL24, showed no change in its copy number(Fig.1).Thus,we here provide evidence that CRAds are able to replicate in colon cancer in the presence of chemotherapeutic agents, ensuring the further application of CRAds for high IL-24 expression in chemotherapy.
J Gastroenterol
A previous study has revealed that doxorubicin impairs the replication of adenoviruses in some osteosarcoma cells [20],and this impaired replication could probably decrease the concomitant duplication of a transgene inserted into the adenovirus genome and its high expression.Therefore,in light of this?nding,we investigated combinatorially trea-ted colon cancer cells for their IL-24level.Our data show that a high level of IL-24can be obtained from5-?uoro-uracil-or doxorubicin-treated colon cancer cells(Fig.2), although we have not yet detected the effect of these two chemotherapeutic agents on AdBB-IL24replication.
Intracellularly accumulated doxorubicin can be contin-uously exported from the cytoplasm,as a substrate,by the membrane-located ABC transporters Pgp and MRP1,but not by other family members[3].Overexpression of these two proteins in cancer cells reduces the intracellular doxorubicin concentration to under the lethal threshold, thereby conferring MDR.Because IL-24reverses the MDR of colon cancer cells through reducing the Pgp level[9],we studied whether a CRAd-mediated high level of IL-24was able to more ef?ciently reverse MDR.An analysis of the side population,which represents cells with high resistance to small molecules such as cisplatin and5-?uorouracil[21], was used to quantify MDR phenotype-possessing cells.In accordance with the downregulation of Pgp and BCRP1 expression,we also observed a marked decline in the side population in AdBB-IL-24-infected SW620cells(data not shown).
We also observed that all the tested colon cancer cell lines showed enhanced sensitivity to5-?uorouracil and doxorubicin when infected with AdBB-IL24,in compari-son to?ndings with Ad-IL24(Fig.5).The fact that the 5-fluorouracil5-fluorouracil
+Ad-IL24
5-fluorouracil
+AdBB-IL24
doxorubicin doxorubicin
+Ad-IL24
doxorubicin
+AdBB-IL24
kg)plus AdBB-IL24(19109pfu)(n=8).c The growth-inhibiting effect was also investigated when adenoviruses were administered
J Gastroenterol
difference in cell viability was not observed until almost 48h may be explained by the time required for adenoviral DNA replication.Similarly,the data obtained from our animal experiment also showed the superiority of AdBB-IL24over Ad-IL24in increasing the sensitivity of colon cancer cells to doxorubicin(Fig.6).
Side effects of chemotherapeutic agents severely impede the application of chemotherapy.The use of anticancer chemicals can damage normal somatic tissue,especially that of the liver[22].Unfortunately,in many cases,higher doses of cytotoxic agents have to be administered to patients because of an MDR phenotype,leading to more damage to normal tissue.Similarly,the Pgp inhibitors currently used for MDR reversion also damage normal tissue,due to their lack of speci?city[3].Therefore,safety is highly required for clinical tumor therapy.Here,we examined the cytotoxicity of combinatorial treatment in normal cell lines,MRC-5and L-02.Encouragingly,no additional cytotoxicity was observed in the AdBB-IL24-infected groups(Fig.5),suggesting the safety of the CRAd-based strategy of combinational chemotherapy. AdBB-IL24was well designed to replicate selectively in cancer cells,but not in normal cells(Fig.1),ensuring its speci?c cytotoxicity to cancer cells.To further investigate the cytotoxicity of combinations of AdBB-IL24and che-motherapeutic agents to normal hepatic tissues,a precision-cut human liver slice assay is required[23].
In addition to tumor-suppressing proteins,miRNAs have also been used to reverse the MDR phenotype of cancer cells.For example,miR-122has been documented to not only inhibit the growth of cancer cells,but also to sensitize hepatocellular carcinoma(HCC)cells to doxorubicin [24,25].Modi?cation of the?ber region of an adenoviral vector has also been demonstrated to improve the anti-tumor ef?cacy of adenovirus-based gene therapy[26], suggesting the need to engineer the?ber protein of AdBB-IL24for further enhancing its tumor-suppressing activity.
In our present study,CRAd was shown to be a potent vector for IL-24expression to reverse the MDR of colon cancer cells,providing an effective strategy for enhancing IL24-mediated MDR reversion in colon cancer cells.The safety of this strategy suggests that it is suitable for a clinical trial.We hope our work will offer valuable infor-mation for further research.
Con?ict of interest None declared
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