Combination Therapy and Noninvasive Imaging With a Dual Therapeutic ...

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Paclitaxel and Doxorubicin Accumulation
Steady-state paclitaxel and doxorubicin accumulation was evaluatedas previously described (8). In brief, parental HCT-15 cells andtransfectants were seeded in 6-well plates and grown for 48 h. Thegrowth medium was aspirated and replaced with I mL of RPMI 1640medium containing 50 nM 3H-paclitaxel (3.7 x 10^sup 11^ Bq/ mmol;Moravek Biochemicals). After incubation for 2 h at 37[degrees]C,the cells were cooled on ice, washed 3 times with ice- cold PBS,and solubilized with 0.2 mL of 1% SDS. The radioactivity in eachsample was determined by scintillation counting. To assess thesteady-state doxorubicin accumulation, HCT-15 or shMDRNIS-expressing cells were incubated with 25 [mu]M doxorubicin for 2 h.At the end of incubation, the cells were washed 3 times with PBSand observed under a confocal microscope with a x 400 lens (Leica).
In Vitro Clonogenlc Assay
Exponentially growing cells were incubated for 7 h with Na131I at18.5-37.0 MBq/10 mL in HBSS supplemented with 10 mM NaI and 10 mMHEPES at pH 7.3. After incubation with 131I, the cells weretrypsinized, seeded at a density of 103 cells per well in 6-wellplates to which a doxorubicin dilution series (25-50 nM) was addedin triplicate, and incubated for 10 d under standard cultureconditions. At the end of incubation, the resulting cell colonieswere stained with a Diff Quick staining kit (IMEB Inc.), andcolonies containing more than 10 cells were counted. Parallelexperiments were performed with HBSS without Na131I or doxorubicin,and all values were adjusted for plating efficiency. Cell survivalwas expressed as the percentage of colonies relative to that in theuntreated control.
Small-Animal PET
Small-animal PET was performed with a Concorde microPET R4 rodent-model scanner (Concorde Microsystems Inc.) (77). An animal bearingtumor xenografts was injected with 18.5 MBq of 124I via a tailvein. At 1, 3, and 15 h after injection, the animal was placed in aprone position on the bed of the small-animal PET scanner.Anesthesia was performed with l%-2% isoflurane in 100% O2 duringinjection and imaging. The images were reconstructed with a2-dimensional ordered- subsets expectation maximum algorithm.Corrections were not required for attenuation or scattering.Activity was quantified by viewing the regions of interest in thetumors and averaging the activity concentrations over the containedvoxels.
Statistical Analysis
All numeric data were expressed as mean +- SD. The statisticalsignificance of differences was assessed by analysis of variance(ANOVA). P < 0.05 was considered to be statisticallysignificant.
RESULTS
Stable Expression of MDR1 shRNA and NIS Gene in HCT-15 Cells
As shown in Figure 1, hNIS cDNA was cloned into an expressionvector under the control of the CMV promoter, whereas the MDRlshRNA sequence was recombined under the control of the U6 promoter.For characterization of the dual expression vector, the HCT-15colon cancer cell! line, which has high levels of endogenous MDRlexpression, was stably transfected with the shMDR-NIS dualexpression construct. Two stable cell lines, MN-61 and MN-62, wereobtained by selection with G418, and the levels of expression ofMDR1 mRNA and P-glycoprotein were determined by RT-PCR and Westernblotting, respectively. As shown in Figure 2A, the levels ofexpression of endogenous MDRl mRNA were markedly decreased in MN-61and MN-62 cells relative to those in parental HCT-15 and HCT/Mockcells. In contrast, no significant differences were observed Jnglyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA expression inany of the cell lines. The expression of P-glycoprotein was alsosignificantly decreased in MN-61 and MN-62 cells, in agreement withthe results of the RT-PCR analysis (Fig. 2B). The expression ofactin was not affected by the expression of MDRl shRNA. hNISprotein expression was further confirmed by Western blotting andimmunohistochemical analysis. Western blotting of cell lysatesderived from MN-61 and MN-62 cells with a monoclonal antibody thatrecognizes hNIS protein revealed a band with a molecular mass ofapproximately 90 kDa, which was not detected in parental HCT-15 orHCT/Mock cells (Fig. 3A). In addition, NISspecific immunoreactivityin MN-61 cells was revealed by immunofluorescence staining. Incontrast, HCT/Mock cells did not show NIS-specificimmunoreactivity. Control slides stained with isotype-matchednonimmune immunoglobulin were consistently negative (Fig. 3B).
Drug Accumulation and Sensitivity in shMDR-NIS-Expressing Cells
To clarify the functional activity of MDRI shRNA, we measured thedegree of drug accumulation in MN-61 and MN-62 cells. Theaccumulation of paclitaxel, a P-glycoprotein-transportablecompound, was increased by about 3.8- and 2.8-fold in MN-61 andMN-62 cells, respectively, compared with that in parental HCT-15cells (Fig. 4A). To assess whether shRNA-directed suppression ofP-glycoprotein sensitized HCT-15 cells to the anticancer drug, wecompared the drug sensitivity of shMDR-NIS-expressing cells withthose of parental HCT- 15 and HCT/Mock cells by using a cellproliferation assay. The level of accumulation of doxorubicin,another drug that is transported by P-glycoprotein, was also higherin shMDR-NIS-expressing cells (MN- 61 ) than in HCT-15 cells (Fig.4B). As shown in Figure 4C, sensitivity to doxorubicin wassignificantly higher in MN-61 and MN- 62 cells than in parentalHCT-15 cells. Functional hNIS Activities in shMDR-NIS-ExpressingCells
The functional activity of hNIS protein was clearly shown by themeasurement of cellular iodide uptake. MN-61 and MN-62 cells showedup to 28.6- and 24,8-fold increases in iodide accumulation,respectively, compared with that in cells coincubated withperchlorate, a competitive NIS inhibitor (Fig. 5A). Parental HCT-15and HCT/ Mock cells did not show an increase in iodideaccumulation. Iodide accumulation reached approximately 80% within10 min and became saturated at 20 min (Fig. 5B), but almost 80%-90%of the accumulated ^sup 125^I was released into the medium duringthe initial 10 min (Fig. 5C).
FIGURE 2. Suppression of MDR1 mRNA and P-glycoprotein in shMDR-NlS-expressing cells. (A) Total RNA was isolated from parental HCT-15, HCT/ Mock, MN-61, and MN-62 cells, and MDR1 gene was amplifiedby PCR. GAPDH was used as internal control. PCR products wereelectrophoresed on agarose gels and then visualized by ethidiumbromide staining (top). Amplified DNA levels obtained bydensitometry were normalized to GAPDH signals, and relativeintensities were expressed as percentage of that observed incontrol HCT-15 cells (bottom). Results are expressed as mean +- SDof 3 independent experiments. "P < 0.01, as determined by ANOVA.(B) Total cell lysates were prepared from parental HCT-15, HCT/Mock, MN- 61, and MN-62 cells. Equal amounts (50 [mu]g of proteins)of cell lysates were separated by 7% SDS-PAGE and then transferredto nitrocellulose membranes. Membranes were immunoblotted withanti-P- glycoprotein or antiactin antibody (top). Protein levelsobtained by densitometry were normalized to actin signals, andrelative intensities were expressed as percentage of that obtainedin control HCT-15 cells (bottom). Results are expressed as mean +-SD of 3 independent experiments. *P < 0.01, as determined byANOVA.
FIGURE 3. Expression of NIS protein. (A) Total cell lysates wereprepared from HCT/ Mock, MN-61, and MN-62 cells. Equal amounts (50[mu]g of proteins) of cell lysates were separated by 7% SDS-PAGEand then transferred to nitrocellulose membranes. Membranes wereimmunoblotted with anti-NIS (clone C-2) or anti-actin antibody.Results shown are representative of 3 independent experiments. (B)Immunohistochemical analysis of NfS expression in MN-61 cells. HCT/Mock and MN-61 cells were stained with anti-NIS antibody (clone 44)or isotype-matched control IgG (Ab). Scale bar = 50 [mu]m. DAPI =4',6'diamidino-2-phenylindole.
Next, we investigated the therapeutic effectiveness of ^sup 131^Iin MN-61 and HCT/Mock cells by using a clonogenic assay. Theviability of MN-61 cells decreased in correlation with an increasein the ^sup 131^I concentration (Fig. 5D). In contrast, thepercentage of viable HCT/Mock cells remained at almost 100% evenwhen these cells were incubated with various concentrations of ^sup131^I. These results indicated that transduction with the dualexpression vector could induce the expression of a functional hNISgene and that, although the iodide efflux rate was rapid, theamount of accumulated ^sup 131^I was sufficiently high toselectively kill MN-61 cells.
Combination Therapy with ^sup 131^I and Doxorubicin
The effects of combination therapy with 131I and doxorubicin invitro were estimated from the survival of HCT/ Mock and MN-61 cellsin a clonogenic assay. ^sup 131^I at 18.5-37.0 MBq and 25-50 nMdoxorubicin were administrated at concentrations that resulted in20%-40% cell survival when either drug was given alone. When MN-61cells were treated with ^sup 131^I at 18.5 MBq and 25 nMdoxorubicin for 10 d, cell survival decreased to 4.9% +- 1.9%compared with that obtained with ^sup 131^I treatment (24.7% +-3.2%) or doxorubicin treatment (36.3% +- 4.4%) alone (Fig. 6B). Incontrast, HCT/Mock cells did not show a therapeutic effect of thecombination of ^sup 131^I and doxorubicin (Fig. 6A). To determinewhether this combination had a synergistic or additive effect, wecalculated a combination index as previously described (18,19). Thecombination of ^sup 131^I at 18.5 MBq and 25 nM doxorubicin had a95% killing effect, with a combination index of 0.662, suggestingthat this combination was synergistic.
FIGURE 4. Accumulation of paclitaxel and sensitivity to doxorubicinin shMDR-NlS-expressing cell lines. (A) shMDR-NlS- expressing cellswere seeded in 6-well plates, grown for 48 h, and incubated with 50nM 3H-paclitaxel for 2 h at 37[degrees]C. At end of incubation,cells were cooled on ice, washed 3 times with ice- cold PBS, andsolubilized with 0.2 ml. of 1% SDS. Radioactivity in each samplewas determined by scintillation counting. Results are expressed asmean +- SD of 3 independent experiments. 'P < 0.01, asdetermined by ANOVA. (B) HCT-15 or shMDR-NIS-expressing celts (MN-61) were treated with 25 [mu]M doxorubicin for 2 h. At end ofincubation, cells were washed 3 times with PBS and then observed byconfocal microscopy. Results are representative of 2 similarexperiments. Scale bar = 50 [mu]m. (C) Cells (103 per well) wereseeded in 96-well plates, doxorubicin dilution series was added intriplicate, and cells were incubated for 4 d under standard cultureconditions. Cell vtabilitv was assessed with CellTiter 96 AqueousOne solution cell proliferation assay. Results are expressed asmean +- SD of 3 independent experiments.
FIGURE 5. Iodide uptake in shMDR-NlS-expressing cells. (A) Iodideaccumulation was measured in HCT-15, HCT/ Mock, and shMDR-NIS-expressing cells (MN-61 cells and MN-62 cells). shMDRNlS-transfected HCT-15 cells showed 25-fold increase in iodideaccumulation compared with shMDR-NIS-expressing cells coincubatedwith perchlorate. Parental HCT-15 or vector-transfected HCT-15cells did not show perchloratesensitive iodide accumulation.Results are expressed as mean +- SD of 3 independent experiments."P < 0.01, as determined by ANOVA. (B) MN-61 cells wereincubated with Na^sup 125^I at 37[degrees]C for 10-60 min. Atvarious time points, cells were quickly washed with PBS, and theirradioactivity was counted with gamma-counter. Results are expressedas mean +- SD of 3 independent experiments. (C) MN-61 cells wereincubated with Na^sup 125^I at 37[degrees]C for 30 min, medium wasremoved at various time points, and its radioactivity wasdetermined. Results are expressed as mean +- SD of 3 independentexperiments. (D) Cytotoxicity of ^sup 131^I. HCT/Mock or MN-61cells were incubated for 7 h with Na^sup 131^I at 18.5-37.0MBq/10mL in HBSS supplemented with 1OmM NaI. After incubation withradioiodine, cells were trypsinized, seeded in 6-well plates(10^sup 3^ cells per well), and incubated for 10 d. After cellcolony development, cells were fixed with methanol and stained withDiff Quick staining kit. Colonies containing more than 10 cellswere counted, and cell survival was expressed as percentage ofcolonies relative to that in untreated control. Results areexpressed as mean (remaining percentage of ^sup 125^I) +- SD of 3independent experiments.
^sup 124^I Imaging of Tumor Xenogratts In Vivo and Quantification
At 8-12 wk after the inoculation of HCT/Mock and MN61 cells, tumordiameter reached up to -10 mm, and whole-body imaging was performedby small-animal PET after the intraperitoneal injection of ^sup124^I at 18.5 MBq. In contrast to control HCT/Mock tumors, whichshowed negligible uptake of ^sup 124^I, MN-61 tumors showedprominent uptake, indicating functional NIS expression (Figs. 7Aand 7B). Physiologic uptake was also observed at the sites of thethyroid and stomach, in which NIS is normally expressed (Fig. 7B).Regions of interest were drawn on tumors expressing the shMDR-NISconstruct, and ^sup 124^I activity in tumors was quantified atselected time points (Fig. 7C). The accumulation of ^sup 124^I intumors was maintained for up to 3 h; the percentage injected doseper gram (%ID/g) at l h was 7.2, and that at 3 h was 7.3, However,a higher signal -to-noi se ratio was achieved at 3 h than at 1 hbecause of decreased nonspecific background activities. By 15 h,the accumulation had decreased to 1.2 %ID/g.
FIGURE 6. Effect of combination of ^sup 131^I and doxorubicin onsurvival of MN-61 celts. MN-61 (B) or HCT/Mock (A) cells wereincubated for 7 h with Na^sup 131^I at 18.5 MBq/10 mL in HBSSsupplemented with 10 mM NaI. After incubation with radioiodine,cells were trypsinized and seeded in 6-well plates (10^sup 3^ cellsper well), 25 [mu]M doxorubicin was added, and cells were incubatedfor 10 d. After cell colony development, cells were fixed withmethanol and stained with Diff Quick staining kit. Coloniescontaining more than 10 cells were counted, and cell survival wasexpressed as percentage of colonies relative to that in untreatedcontrol. Results are expressed as mean +- SD of 3 independentexperiments.
FIGURE 7. In vivo tumor images of tumor-bearing nude mouse at 3 hafter injection of ^sup 124^I. (A and B) Mouse was subcutaneouslytransplanted with shMDR-NIS-expressing cells (MN-61) in right flankand control cells (HCT/Mock) in contralateral left flank.Transverse (A) and coronal (B) small-animal PET images clearlyvisualized shMDR- NIS-expressing tumor in right flank. (C) ^sup124^I accumulation in shMDR-NIS-expressing cells (MN-61) plottedover time. Uptake of ^sup 124^I in tumor lesions was maintained forup to 3 h after injection but, by 15 h, had decreased to 1.2 %ID/g.
DISCUSSION
NIS expression could lead to a novel gene strategy for radioiodinetherapy in thyroid diseases (20). Transfer of the NIS gene into avariety of tumors, including melanoma, colon carcinoma, ovarianadenocarcinoma, and lung and prostate cancers, confers radioiodideuptake capacity (21-23), Although NIS-based radiotherapy iseffective for a wide range of cancers, several problems remain,such as rapid washout of radioiodide from cells and limitedretention of radioiodide within cells (24). Many strategies havebeen proposed to enhance the antitumor effects of NIS-basedradioiodide therapy; these include combination with variousreagents or gene therapy. Several antineoplastic drugs, such ascisplatin, doxorubicin, and 5-fluorouracil, are already known tohave radiosensitizing effects and are used widely in the field ofexternal radiotherapy for various cancers (25-27).Chemoradiotherapy has the advantage of enhancing the sensitivity oftumor cells to ionized radiation at a concentration lower than theoptimal cytotoxic range (28). It has the potential disadvantage,however, of increased toxicity because chemotherapy may inhibit therepair of radiotherapy-induced sublethal damage in normal tissue.In the present study, to minimize the extratumoral side effects ofchemotherapy, we developed a dual expression vector system in whichNIS gene expression is combined with MDRl shRNA expression. Weinvestigated the feasibility of the combination of NIS-basedradioiodide therapy and RNAi-based gene therapy with our novelvector system in vitro and performed ^sup 124^I small-animal PET ofnude mice bearing tumor xenografts expressing both the NIS gene andMDRl shRNA. RNAi relies on the sequence-specific interactionbetween siRNA and mRNA. The degradation of long double-stranded RNAto siRNA is mediated by a double-stranded RNA-specific enzyme,RNase III dicer. siRNA is incorporated into a nuclease complexknown as the RNA-induced silencing complex (RISC), in whichunwinding of the duplex siRNA takes place. The antisense strandbinds in a highly sequencespecific manner to target mRNA, which isthen endonucleolytically cleaved and degraded (29). Many studieshave used siRNA as an experimental tool to dissect the cellularpathways that lead to uncontrolled cell proliferation and cancer.To develop siRNA for cancer therapy, several researchers haveinvestigated the effects of siRNA in cancer models (30-32). In thepresent study, we developed a novel dual expression vector systemcombining the NIS gene with MDRl shRNA to allow an effectivecombination of ^sup 131^I with doxorubicin, which is aP-glycoprotein-transportable compound. Four MDR1-specific targetsequences were tested according to published recommendations(8-10,33), and the most effective sequence was chosen. Theintroduction of this vector increased sensitivity to the anticancerdrug in HCT-15 colon cancer cells, thereby allowing the effectiveuse of low-dose doxorubicin and resulting in synergisticcytotoxicity in combination with ^sup 131^I.
The combination of doxorubicin and radioiodine could be a usefulstrategy for cancer therapy, but the molecular mechanism of thissynergy is presently unknown. Doxorubicin acts as an intercalatingagent by binding to DNA and inhibiting chain elongation but alsointerferes with topoisomerases and induces breaks in DNA (34,35).In addition, doxorubicin is also known to be an activator of thep53 pathway (36), which acts as a transcriptional factor andinduces the expression of several proteins related to cell cyclearrest and apoptosis. Indeed, previous studies showed thattreatment with adriamycin induced the expression and activation ofp53 (37), Thus, it is possible that the sequential addition ofdoxorubicin may accelerate radiotherapyinduced apoptosis throughthe increased activation or expression of p53. Furthermore, aprevious study showed that the expression of p53 was positivelycorrelated with hNIS activity in a dual expression vectorexpressing hNIS and the p53 gene (11). More recently, it wasreported that doxorubicin enhanced the expression of a transgeneunder the control of the CMV promoter in anaplastic thyroidcarcinoma cells (38). Thus, treatment with doxorubicin may enhancehNIS activity through p53 upregulation as well as activation of theCMV promoter. Although our results showed that treatment with ^sup131^I and then doxorubicin was effective in cancer therapy,doxorubicin pretreatment before radioiodide therapy may be alsoeffective in treating cancers that are resistant to conventionalchemotherapy or radiotherapy. Our dual expression vector could be auseful therapeutic tool in a combination of NIS-based radiotherapyand chemotherapy.
For safe and efficient gene therapy in clinical applications,technology that allows noninvasive monitoring of the level anddistribution of vector-mediated gene expression in vivo isrequired. The NIS gene is well known for its many advantages as anideal reporter gene, the well-understood biodistribution of itsligands, and its nonimmunogenic properties in humans. Ligands ofthis reporter gene include ^sup 123^I, ^sup 131^I, and ^sup 99^mTc-pertechnetate as gamma-tracers and ^sup 124^I for PET. In thepresent study, small-animal PET with ^sup 124^I revealed a clearimage of shMDR-NIS-expressing tumors in an in vivo tumor xenograftmodel. The uptake of ^sup 124^I in tumors could also be quantifiedat selected time points successfully. Therefore, radiologic imagingof NIS derived from our dual expression vector could be a highlyeffective method for monitoring targeted sites in a noninvasivemanner.
The ultimate success of gene therapies will depend on gene transfervectors that facilitate the expression of a specific gene attherapeutic levels in cancer cells without eliciting cytotoxicity.Although the data presented here appear to be promising, the lackof specificity conferred by nonspecific promoters may beproblematic. MDR1 gene products are present not only in cancercells but also in various normal tissues, such as the endotheliumof blood vessels in the brain (39) and hematopoietic progenitorsfound in normal bone marrow (40). Although the level of MDR1expression in these tissues is relatively low, functional P-glycoprotein expression may be important in limiting potentialtoxicity after exposure to anticancer drugs. Thus, a targeteddelivery system in which shRNA is selectively expressed in cancercells should be developed. Furthermore, our results were obtainedin stable transfected cells with a high transduction efficiency andeasy drug administration. Our findings will need to be confirmedwith clinically relevant models in vivo.
CONCLUSION
We have developed a dual expression vector system containing theNIS gene and MDRl shRNA and tested its therapeutic effects in coloncancer cells. A combination of ^sup 131^I and low-dose doxorubicinwas more effective in killing colon cancer cells than ^sup 131^I ordoxorubicin alone. Moreover, ^sup 124^I small-animal PET imagingrevealed a clear image of the in vivo tumor xenografts, andaccumulated ^sup 124^I activity could be quantified over time.Although several problems need to be resolved for furtherapplications, the present study suggests the possibility of a newstrategy of RNAi-based gene therapy accompanied by NIS-basedradioiodide therapy and imaging in colon cancer cells.
ACKNOWLEDGMENTS
We thank Dr. Je-Yeol Cho (Kyungpook National University, Daegu,Republic of Korea) for the kind donation of the hNIS expressionconstruct. The production of ^sup 124^I was supported partially bythe QURI project of MOST (Ministry of Science and Technology) andby a project titled "Development of Diagnosis and Therapy forIntractable Disease Using Radioisotopes" of KOSEF (Korea Scienceand Engineering Foundation). This study was supported by grantRTI040 - 010 -01 from the Regional Technology Innovation Program ofThe Ministry of Knowledge Economy (Advanced Medical TechnologyCluster for Diagnosis and Prediction at Kyungpook NationalUniversity); by grant 0720550-2 from the National R&D Program forCancer Control, Ministry of Health & Welfare, Republic of Korea;and by the Brain Korea 21 Project in 2008.
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Seung-Yoon Park1, Wonjung Kwak2, Narendra Tapha3, Mi-Yeon Jung3,Ju-Ock Nam3, In-Seop So3, So-Youn Kim3,
Jeongsoo Yoo2,4, Jaetae Lee4, and In-San Kim3
1 Department of Biochemistry, School of Medicine, DonggukUniversity, Kyungju, Republic of Korea; 2Department of Molecular
Medicine, School of Medicine, Kyungpook National University, Daegu,Republic of Korea; 3 Department of Biochemistry and Cell
Biology, Cell and Matrix Research Institute, School of Medicine,Kyungpook National University, Daegu, Republic of Korea; and
4 Department of Nuclear Medicine, School of Medicine, KyungpookNational University, Daegu, Republic of Korea
Received Jan. 22, 2008; revision accepted May 7, 2008.
For correspondence or reprints contact: In-San Kim, School ofMedicine, Kyungpook National University, 101 Dongin-dong, Jung-gu,Daegu 700-422, Republic of Korea.
E-mail: iskim@knu.ac.kr
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Copyright Society of Nuclear Medicine Sep 2008
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