DNA triplex-mediated inhibition of MET leads to cell death ... - Nature

Cancer Gene Therapy (2011) 18, 520–530 r

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ORIGINAL ARTICLE

DNA triplex-mediated inhibition of MET leads to cell death and tumor regression in hepatoma G Singhal1, MZ Akhter1, DF Stern2, SD Gupta3, A Ahuja3, U Sharma4, NR Jagannathan4 and MR Rajeswari1 1

Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India; 2Department of Pathology, School of Medicine, Yale University, New Haven, CT, USA; 3Department of Pathology, All India Institute of Medical Sciences, New Delhi, India and 4Department of Nuclear Magnetic Resonance, All India Institute of Medical Sciences, New Delhi, India

Mesenchymal epithelial transition factor (MET) is one of the critical cell signaling molecules whose aberrant expression is reported in several human cancers. The aim of the study is to investigate the antigene and antiproliferative effect of short triplex forming oligonucleotides, TFO-1 (part of the positive regulatory element) and TFO-2 (away from the transcription start site) on MET expression. HepG2 cells transfected only with TFO-1 (but not with TFO-2 and non-specific TFO) significantly decreased MET levels, which is accompanied by decrease in antiapoptotic proteins and increase in pro-apoptotic proteins. Phosphoproteome-array analysis of 46 intracellular kinases revealed hypophosphorylation of about 15 kinases including ERK, AKT, Src and MEK, suggesting the growth inhibitory effect of TFO-1. Further, the efficacy of TFO-1 was tested on diethylnitrosamine-induced liver tumors in wistar rats. T2-weighted magnetic resonance imaging showed decrease in liver tumor volume up to 90% after treatment with TFO-1. Decreased MET expression and elevated apoptotic activity further indicate that TFO-1 targeted to c-met leads to cell death and tumor regression in hepatoma. Formation of stable DNA triplex between TFO-1 and targeted gene sequence was confirmed by circular dichroic spectroscopy and gel retardation assay. Therefore, it can be concluded that DNA triplex-based therapeutic approaches hold promise in the treatment of malignancies associated with MET overexpression. Cancer Gene Therapy (2011) 18, 520–530; doi:10.1038/cgt.2011.21; published online 10 June 2011 Keywords: c-met; DNA triplex; tyrosine kinase; triplex forming oligonucleotide; hepatoma

Introduction

The c-met gene encodes a receptor tyrosine kinase, called hepatocyte growth factor receptor, also commonly referred as mesenchymal epithelial transition factor (MET).1 MET/hepatocyte growth factor signaling has a crucial role in a variety of cellular functions during normal cell growth.2 However, aberrant signaling of MET is frequently associated with cell scattering, angiogenesis, proliferation, enhanced motility, invasion and eventually metastasis.3 Several clinical studies have shown the relationship between overexpression of MET with the aggressive nature of tumors and poor therapeutic response. The constitutive activation of MET in cancers has been ascribed to (i) primarily overexpression of MET being the most frequent mechanism in various solid tumors such as hepatocellular carcinoma (HCC),4 biliary tract carcinomas5 and lung cancer6, (ii) the formation of Correspondence: Dr MR Rajeswari, Department of Biochemistry, All India Institute of Medical Sciences, New Delhi 110029, India. E-mail: [email protected] Received 2 September 2010; revised 24 January 2011; accepted 18 March 2011; published online 10 June 2011

autocrine loops between MET and its ligand, hepatocyte growth factor causing uncontrolled cell signaling like osteosarcoma7 and breast cancer8 and (iii) the activation of point mutations in the tyrosine kinase region of MET which is although less frequent as reported in sporadic papillary renal cancer,9 gastric cancer10 and so on. Engelman et al.,6 showed that MET amplification led to the gefitinib (inhibitor of epidermal growth factor receptor) resistance in non-small cell lung cancer cell line via activation of PI3K signaling. This clearly generated a lot of interest in MET and therapies.11,12 Therapies targeted against MET have been widely anticipated as a potential antitumor strategy. Therefore, keeping in view of the crucial role of MET in cancer, the primary interest in developing therapeutics is either to downregulate the (over) expression of MET or to inhibit the functional activity of MET. A number of small molecules which are competitors of ATP binding site and target intracellular kinase domain of the MET receptor thereby block the kinase activity like PHA-665752,13 K252a,14 SU11274,15 and PF 2341066.16 Petrelli et al.,17 used the antibody-based approach and targeted the extracellular domain of MET. Majority of above-mentioned attempts, targeting MET at the protein level, are found to

Downregulation of MET by triplex-based antigene therapy G Singhal et al

50 -GGGGCAGAGGCGGGAGGAAACGCG-30 , 50 -CG CGTTTCCTCCCGCCTCTGCCCC-30 and TFO-1, 50 - AG GAGGGGGGAGAGG-30 . The molar extinction coefficients were calculated using nearest neighbor analysis of the unfolded species.25 It is possible that an oligonucleotide in solution can alone form self-structures like hairpins or triplexes or tetraplexes. To rule out the possibility of existence of such intramolecular or interstranded ordered structures in solution, UV melting experiments were carried out. No hyperchromicity was observed for any of the oligonucleotides and this is an indication of the presence of a linear unfolded oligonucleotide entity in solution.

be partially toxic and non-specific in nature. Therefore, antigene- or antisense-based technologies pose themselves as most specific approach in comparison with them. There are several ways by which antigene therapy have been attempted in context to c-met in variety of cancers in cell lines18,19 and in animal models.20 However, to the best of our knowledge, there are no reports on targeting c-met by triplex forming oligonucleotide (TFO)-based antigene therapy. However, DNA triplex-based antigene approach on various proto-oncogenes like c-myc,21 ki-ras,22 bcl-2,23 and her-224 is being investigated. The focus of the present study is to investigate the antitumorigenic activity of specific TFOs targeted against the promoter part of c-met in hepatoma both in vitro in HepG2 cell line and in vivo using rat model of HCC. Results provide relevant direction toward designing of TFOs as a potential therapeutic alternative in malignancies with MET involvement.

Reagents and antibodies Dulbecco’s Modified Eagle’s medium, fetal calf serum, trypsin-EDTA, penicillin G, streptomycin and amphotericin B were from GIBCO BRL (Gaithers-burg, MD). Lipofectamine LTX Reagent (Cat. No. 15338-100) along with PLUS Reagent (Cat. No. 11514-015) used for the transfection procedure were procured from Invitrogen, Carlsbad, CA. Antibodies, Met 25H2, Y1234/1235 Phospho-Met, Myc, Phospho-Myc, p53, Ser-392 Phospho-p53, Bcl-xl and Bax were from Cell Signaling Technology (Irvine, CA), while GAPDH antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The g-32P [ATP] was obtained from Bhabha Atomic Research Center, India, while T4 polynucleotide kinase was procured from New England Biolabs (Hertfordshire, UK).

Materials and methods

Designing of TFOs The schematic structure of c-met promoter and the sequences of the triplex target sites are shown in Figure 1a. The sequences of the used oligonucleotides were TFO-1 (50 -AGGAGGGGGGAGAGG-30 ) and TFO-2 (50 -AAGAAAAAAAGAAAAAAAAAAAG-30 ), respectively. TFO-3 (50 -AGAAGAGAGGGGGGG-30 ), the control had the same length with five mismatched nucleotides (one base per codon) at different positions of TFO-1. All the oligonucleotides were purchased from Integrated DNA Technology (Coralville, IA). The oligonucleotides were dissolved in DNase- and RNase-free water and aliquots were kept at 20 1C until use. For the circular dichroic spectroscopy and gel retardation assay (GRA), the concentrations of the oligonucleotides were estimated using molar extinction coefficient at e260 of 254 680, 193 860 and 173190 M1 cm1, respectively, for

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Cell growth measurement and protein expression The HepG2 cells were maintained in Dulbecco’s Modified Eagle’s Medium supplemented with 10% (v/v) fetal calf serum and incubated in 5% CO2 humidified incubator at 37 1C. Transfection was performed according to the manufacturer’s instructions for HepG2 cells. Cytotoxicity assay and the western blot analysis were carried out as described previously.25

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Figure 1 (a) Map of the human c-met promoter indicating the positive ( þ ve), strongly positive ( þ þ þ ve) and negative (ve) regulatory elements. Location of 24 bp purine rich triplex target sequences (TTSs) (1) TTS-1, 142 to 119 and (2) TTS-2, 644 to 620 targeted by triplex forming oligonucleotide (TFO)-1 and TFO-2 and the corresponding DNA triplex (1) and DNA triplex (2), respectively, are indicated. Watson–Crick base pairing is shown by a dot (K) while Hoogsteen bonds between the duplex and TFO is shown by star (*). (b) MTT (3-[4, 5-dimethylthiazol-2yl]-2, 5-diphenyltetrazolium bromide) assay for the growth proliferation of HepG2 cells in presence of TFO-1, TFO-2 and TFO-3 is shown as ( ), ( ) and ( ), respectively. Absorbance at 570 nm was measured, and the results are expressed as percentage of absorbance in untreated cells.

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Human phospho-intracellular kinase array The phospho-kinase array was used to detect the relative phosphorylation of 46 different intracellular signaling molecules, captured on nitrocellulose membrane. Array (Array 003) was obtained from R&D Systems (Foster City, CA) and was used according to the manufacturer’s instructions. Briefly, arrays were incubated overnight with a total 1000 mg of cell lysates followed by washing. Phosphorylation status of cell proteome was evaluated by incubating the membranes in a pool of provided conjugated antibodies and then by chemiluminescence. Intensity of each spot detected with phospho-kinase array was measured by Image J software (National Institutes of Health, Bethesda, MD) and calculated for the relative intensity of the average signal of the pair of duplicated spots to the negative control spots. Background intensity was also determined and subtracted from each average signal. Schedule of liver tumor induction and TFO-1 treatment in wistar rats Random bred 6–8-week-old male wistar rats (100–150 g) were obtained and maintained in the experimental animal facility of All India Institute of Medical Sciences with food and water ad libitum. Animals were handled according to Animal Ethical Committee guidelines for the care of laboratory animals (Project clearance no. 373/ Institutional Animal Ethical Committee/06). Animals were divided into three groups, that is, normal, control and test groups. Liver tumors were induced in the control and test groups by oral administration of diethylnitrosamine (DEN) (Sigma, St Louis, MO) in drinking water (40 p.p.m.) up to 8 weeks. Water bottles were protected from light and fresh solutions of DEN were prepared every day. Control group was given the free liposomes, that is, without encapsulation of TFO. Test group was subdivided into two groups, group I (2 weeks) and group II (5 weeks) and treated with liposomes encapsulated with TFO-1 (4 mg kg1 body weight, i.p.). Formation of liposomes was confirmed by Transmission Electron Microscopic imaging (data not shown). Monitoring of tumor volume by magnetic resonance imaging analysis Magnetic resonance (MR) experiments were carried out using 4.7 T scanner (BIOSPEC Bruker BioSpin MRI GmbH, Ettlingen, Germany) using a 72-mm resonator as transmitter/receiver coil. The animals were placed with the upper abdomen area centered on the surface coil of the equipment. Animals were anesthetized using chloral hydrate (400 mg kg1 body weight, i.p.) and their body temperature was constantly maintained using a circulating warm water bath attached to the animal bed. Following the scout scan in all three planes, T2-weighted (T2-W) images were acquired using RARE sequence in axial plane using the following parameters: retention time/echo delay time ¼ 3000/56 ms, RARE factor ¼ 8, slice thickness ¼ 2 mm, interslice distance ¼ 2 mm, averages ¼ 4 and matrix size ¼ 256 256.

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Histopathological and immunohistochemical analysis After evaluation of tumor by magnetic resonance imaging (MRI), animals were sacrificed by approved euthanasia techniques according to Institutional Animal Ethical Committee guidelines under aseptic conditions. Histopathological analysis and immunohistochemistry were performed as described previously.26 In negative controls, the primary antibody was replaced by non-immune IgG of the same isotype to ensure specificity. Image analysis was done using Nikon microscope (Otawara, Tochigi, Japan). Protein extraction and western blotting Total cell protein was extracted from the liver tissue using a lysis buffer (50 mmol l1 Tris–HCl (pH 8.0), 1% Triton X-100, 0.1% sodium dodecyl sulfate, 250 mmol l1 NaCl and 5 mmol l1 phenylmethanesulfonylfluoride, 5 mg ml1 pepstatin and 5 mg ml1 leupeptin). Tissue sample was cut and minsed in lysis buffer followed by homogenization and centrifugation at 12 000 r.p.m. for 30 min. All the procedures were performed at 4 1C. Western blot for the MET protein was performed according to the above-described protocol.25 Measurement of apoptosis Apoptosis was determined by TUNEL assay using Fluorescein FragEL DNA fragmentation detection kit from Calbiochem (San Diego, CA) (Catalog no. QIA39) according to the manufacturer’s instructions. Circular dichroism spectroscopy Circular dichroism (CD) experiments were carried out using a JASCO-810 spectropolarimeter (Jasco International, Tokyo, Japan) equipped with in-built peltier controlled thermostat cell holder (PTC-423S) and data processor. The melting profile and isothermal CD spectrum were acquired as described previously.25 Gel retardation assay For GRA, the pyrimidine strand of DNA-1 duplex and TFO-1 was 50 end labeled with g-32P [ATP] by T4 polynucleotide kinase using standard protocol.27 The triplex was obtained by mixing 50 nM each of the DNA-1 (with labeled pyrimidine strand) and cold TFO-1 in 10 mM sodium cacodylate buffer containing 10 mM NaCl, pH 7.4. The assay was performed as per our earlier method.28 Statistical analysis Body weight, apoptotic index, immunostaining scores and quantitative densitometry values were analyzed by the unpaired Student’s t-test (SPSS statistical analysis software, version 10.0, IBM SPSS, Chicago, IL). Po0.05 was considered statistically significant. Values were expressed as mean values±s.d. as indicated.


Results

presence of TFO-1. Three dosages 10, 100 nM and 1 mM for the TFO-1 were selected depending upon its inhibitory concentrations. Basal levels of MET protein were found to be reduced by 25% and phosphorylation was almost undetectable after 72 h treatment of TFO-1 at 10 nM. At concentrations 100 nM and 1 mM of TFO-1, the basal levels of MET were reduced 450%. Figure 2c shows the fold intensity change in the MET levels. In contrast to TFO-1, other two TFOs did not show any inhibition on MET protein expression (data not shown).

The positive regulatory element of c-met gene contains a 155-bp long (–223 to 68) sequence which is proven to be important for transcriptional regulation.29 Promoter sequence analysis have a very high degree of sequence homology (B90%) between human and rat c-met gene.30 Based on our experience on c-myc, hmg, frataxin and so on, two possible triplex targeted sequences (TTSs) were selected for the present study after thorough screening of c-met promoter, and by considering all the prerequisites for the DNA triplex formation (Figure 1a).31 TTS-1 is located at 144 to 119 (within the positive regulatory element) while TTS-2 is situated at 644 to 620, which is slightly away from transcription start site. TFO-3 has been included in the study for checking the non-specific interaction and inhibiton of MET. Sequence specificity of TFOs was also confirmed by BLAST.

Effect of TFO-1 on pro-apoptotic and antiapoptotic proteins Western blot analysis of some pro-apoptotic and antiapoptotic proteins is shown in Figure 2b. Hyperphosphorylation of p53 under TFO-1 treatment was observed, while endogenous levels of p53 remian unaffected. Similary, basal levels of c-myc were also unchanged while the phosphorylated status was reduced. Bax protein was found to be increased while Bcl-xl was decrased after initial treatment of TFO-1. Figure 2c shows the fold change in the intensities of the western blots.

TFO mediated cell growth inhibition of HepG2 cells To increase the resistance toward exogenous and endogenous nucleases, the triplex forming oligonucleotides were phosphorothioated in their sugar-phosphate backbones. Figure 1b shows the cell viability of HepG2 cells in the presence of different TFOs in a dose-dependent manner. It can be seen that TFO-1 was the most effective inhibitor of cell proliferation as the number of viable cells reduced by 50% even at a very low concentration of 10 nM while the growth was inhibited by 85% at 1 mM. No detectable cell inhibition was observed in the cells treated with TFO-2 and TFO-3.

Intracellular signaling kinase array To determine the effect of MET knockdown on key signaling pathways, an intracellular signaling kinase array was performed. A representative map of kinase array is providing the alignment of the kinases on the membrane and is shown in Figure 3c. The HepG2 cells were grown separately under normal conditions and with the TFO-1 for 72 h at 100 nM concentration and the kinase arrays are shown in Figures 3a and b. Normalized pixel density for all the included kinases was measured and have shown in Figures 3d and e. Loss of phosphorylation was observed in several kinases like p38a, ERK1/2, MEK1/2, MSK1/2, AKT, mTOR, b-catenin, Src, Fyn, Yes, FAK, PLCy-1,

Reduction of endogenous MET expression in response to TFO-1 Figure 2a shows the protein expression pattern of phospho and basal levels of MET in HepG2 cells in the TFO1 C

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Figure 2 Western blot analysis of the tyrosine phosphorylated MET, total MET (a) and of phosho-myc, c-Myc, phospho-p53, p53, Bax and Bclxl and GAPDH (b). Positive control for MET, ‘C’ is the total cell lysate from HepG2 cells which were induced with hepatocyte growth factor. Lanes 0, 1, 2 and 3 represent HepG2 cell lysate without triplex forming oligonucleotide (TFO)-1, and in presence of 10, 100 nM and 1 mM, respectively. ), ( ), ( ) and ( ), respectively. (c) Fold change in the intensity of proteins at 0, 10, 100 nM and 1 mM of TFO-1 as shown by ( GAPDH has been used as an internal control.

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Figure 3 Intracellular signaling kinase array of HepG2 cells under no treatment (a) and treatment with triplex forming oligonucleotide (TFO)-1 (100 nM) for 72 h (b). (c) Alignment of all the kinases as per the manufacturer. Numbers in & represent the kinases which are downregulated while numbers in J represent the kinases which are activated in response to the TFO-1. The three positive controls are indicated by ‘ þ ’ sign at the corners of the membrane. Three sets of duplicates represent p-53. Similarly for AKT, two sets of duplicates have been placed. Each duplicate indicates a different phosphorylation site of the protein. Spots represent the respective kinase positions. 1: p38; 2: ERK1/2; 3: JNK pan; 4: MEK1/ 2; 5:MSK1/2; 6: AMPKa1; 7a & 7b: AKT; 8: mTOR; 9: CREB; 10: AMPKa2; 11: b-catenin; 12: Src; 13: Fyn; 14: Yes; 15: Fgr; 16: STAT 3; 17: STAT 5b; 18: Chk2; 19: FAK; 20: Stat 6; 21: Stat 5a/b; 22(a, b & c): p53; 23: p70S6; 24: Stat1; 25: RSK; 26: PLCy-1. Bar diagram indicates the normalized pixel density of hypophosphorylated (d) and hyperphosphorylated (e) kinases. Normal and TFO-1-treated HepG2 cells are shown as ( ) and ( ), respectively.

CREB, RSK1/2/3 and c-jun (indicated by numbers in &, Figure 3d). In contrast to these molecules, phosphorylation status of some kinases was elevated like p53, Chk-2 and Stats (indicated by numbers in J, Figure 3e). Kinases whose phosphorylation levels were undetectable in both the conditions were excluded. The biological activity of the TFO-1 on the growth of human HCC cells was also accompanied by evaluating them in vivo. Oral administration of DEN induced HCC in wistar rats and animals were measured for body and liver weight during the complete course of the study. Tumorigenesis caused increase in the liver weight of animals in comparison with the normal conditions as shown in Figure 4c. When animals were subjected to TFO-1, a decrease in the liver weight was observed while body weight was not grossly effected. Overall appearance of the animals was also improved which was accompanied

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with 100% survival rate in the test group in comparison with the control group.

MRI analysis of animals MRI examination proves to be a useful technique not only for detection of rat liver tumors, but also for the study of their dynamic growth. Figure 4a shows T2weighted MR images of rat liver from normal, control and TFO-1-treated test groups. MR examination of normal group of rats showed no visible changes in the liver with time (Figure 4a, upper panel i–iii). Middle panel in Figure 4a (iv–vi) shows control group where both tumor size and intensity increase with time, that is, 16 (group 0), 18 (16 þ 2, group I) and 21 (16 þ 5, group II) weeks of tumor progression, respectively. On contrary, the lower panel in Figure 4a (vii–ix) which is of the test group shows the decrease in both the tumor size as well as


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Figure 4 (a) T2-weighted trans-axial magnetic resonance (MR) images of rat abdomen highlight the normal liver (dotted line). MR image of normal (upper panel, i–iii), control (middle panel, iv–vi) and test (treated with triplex forming oligonucleotide (TFO)-1) (lower panel, vii–ix) wistar rat at 16 weeks (left panel), 16 þ 2 weeks (middle panel) and at 16 þ 5 weeks (right panel) time points are shown. Liver tumors are encircled. (b) Liver tumor volumes were calculated for a 2-mm slice thickness (width) and multiplied by the tumor area and presented as of ±s.d. (Po0.005). Bars represent the tumor volume corresponding to the respective images of (a). (c) The whole liver weight after magnetic resonance imaging (MRI) of normal, control and test groups is represented as g per 100 g of the body weight.

the intensity with respect to the above-mentioned time points. Figure 4b represents the calculated liver tumor volumes at all the mentioned stages. MRI unveiled the most interesting finding that in test group tumor volumes decreased by 80% after 2 weeks and 490% after 5 weeks of TFO-1 treatment.

Histopathological analysis and TUNEL assay Further, the observations made by MRI were correlated with the histological findings. The same animals were sacrificed and histopathological examination was done (Figure 5a). The biopsy of the normal rat showed hepatocytes that are within the normal histological limits (Figure 5ai). The rat liver from the control group showed histological features of poorly differentiated HCCs (Figure 5aii) like tumor cells show marked nuclear polymorphism with prominent nucleoli, atypical mitotic figures and moderate amount of cytoplasm. The histological features of the TFO-1-treated test group I showed a group of few apoptotic bodies along with individual cell necrosis (Figure 5aiii). As the TFO-1 treatment continued till 5 weeks, in test group II, increase in apoptotic bodies was observed on microscopy. The characteristic features of apoptosis like cell shrinkage, nuclear fragmentation, chromatin condensation and chromosomal DNA fragmentation were seen (Figure 5aiv).

TUNEL assay is a standard method to confirm the presence of apoptosis in the cells and the same has been performed for all the groups, that is, normal, control and TFO-1-treated test groups. Figure 5aix–x shows the normal and the control groups liver cells with low amount of apoptotic bodies. Significant increase in the number of fragmented nuclei was observed in the test I group which further increased in test II group (Figure 5axi–xii). Figure 5b shows the apoptotic indices calculated for all these above-described groups.

Expression of MET protein Figure 5av–viii shows the immunohistochemical analysis (IHC) of the rat livers from different treatment groups. The MET expression in normal rats was almost undetectable (immunoscore 0); however, strong cytoplasmic and membranous positivity of MET protein was found in the DEN-treated samples of liver tumor (immunoscore þ 7). Tumor samples of TFO-1-treated test group I showed a moderate amount of immune-reactivity for MET (immunoscore þ 4). Test group II showed very few faint and focal MET-positive cells (immunoscore þ 2). Western blotting was performed for the same liver samples and is shown in Figure 5c and the fold change in the intensities is given in Figure 5d. Normal rat livers have moderate amount of MET expression which

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Figure 5 (a) Clinicopathological analysis (i–iv) of DEN induced liver tumor. As described in ‘Materials and methods’, paraffin-embedded section of liver from rats of normal, control, test group I and test group II rats was analyzed by hematoxylin-eosin staining. In histological analysis, apoptotic bodies are indicated by arrows. Magnification: 400. Photographs of immunohistochemical detection of MET protein expression form normal (v), control (vi) and triplex forming oligonucleotide (TFO)-1-treated test I (vii) and test II (viii) show immunoreactivity scope 0, þ 7, þ 4 and þ 2, respectively, and TUNEL assay reveals the fragmented nuclei in the respective groups. Magnification: 200. TUNEL assay reveals the fragmented nuclei (ix–xii) of the rats from different groups. Magnification: 1000. (b) Apoptotic Index calculated by TUNEL assay represent as mean±s.d. (Po0.005). (c) Upper panel shows the western blot analysis of MET in the different groups as mentioned in (a). Lower panel represents the respective coomassie blue gel staining pattern for the equal protein loading. (d) Normalized fold change in the protein expression. The number of animals in various groups is as follows: normal group (n ¼ 6), control groups (n ¼ 8) and test groups (n ¼ 8).

considerably increase with DEN treatment. Similar to the IHC results, MET protein was found to be downregulated with TFO-1 treatment. Coomassie blue gel have been shown to indicate the same loading for the protein.

Circular dichroic spectroscopy CD is one of the simplest yet confirmatory techniques to determine the conformation of different forms of DNA like B-DNA, A-DNA and Z-DNA and other unusual DNA structural elements like hairpins,32 triplex28 and quadruplex. Therefore, CD was exploited to analyze the structural aspects of the DNA triplex formation. Figure 6a shows the CD spectra of duplex DNA-1, TFO-1 and of the mixture of duplex and TFO-1. The spectrum of duplex corresponds to the usual B-DNA with one positive ellipticity at 270 nm and two negative

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ellipticities at 242 and 216 nm. The CD spectrum of complex has additive effect of both duplex and TFO-1. Upon complexation, shift in the positive peak toward 260 nm, decrease in the negative ellipticity of 216 nm and increase in the 242 nm peak was observed. Since the CD spectra of the mixture differ substantially from that of the duplex and the third strand, temperature-induced changes in the CD spectra of the complex were monitored which represents the two transitions in the melting profile (Figure 6b). First transition covers relatively small portion of the melting and centered around 47 1C, which corresponds to the dissociation of third strand while second transition is centered around 69 1C. The second transition is accompanied by a large ellipticity change and corresponds to dissociation of the duplex to single strand. CD spectroscopy confirms the presence of a DNA triple helical structure in the solution.


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Figure 6 (a) DNA duplex formed by triplex targeted sequence (TTS)-1 and its complementary strand, and its corresponding DNA triplex with triplex forming oligonucleotide (TFO)-1 (see Figure 1). The concentration of TFO-1, duplex (1:1) and triplex is 1.0 mM. The circular dichroic spectra were recorded in 10 mM sodium cacodylate, 10 mM NaCl, 10 mM MgCl2 at pH 7.0 and 20 1C. (b) The CD thermal melting curve of triplex followed by the ellipticity at 270 nm as a function of temperature. The values of Tm1 and Tm2 are 471 and 69 1C, which correspond to the melting temperature of duplex and triplex, respectively.

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Figure 7 Polyacrylamide gel retardation assay of duplex (DNA-1) alone and the mixture of DNA-1 and triplex forming oligonucleotide (TFO)-1 on 15% polyacrylamide gel in Tris-borate buffer containing 10 mM NaCl, pH 7.4 at 4 1C. The pyrimidine strand of DNA-1 was 50 end labeled with g-32P [ATP]. The total concentration of oligonucleotide was kept constant at 50 nM. Lane 1 represents DNA-1 alone while lanes 2–4 correspond to the mixtures of DNA-1 and TFO-1 in mole ratios 2:1, 1:1 and 1:2, respectively.

Gel retardation assay Figure 7 shows the autoradiogram of GRA of 50 nM duplex, DNA-1 (lane 1) and mixtures of DNA-1 and TFO-1 in different mole ratios 2:1 (lane 2), 1:1 (lane 3) and 1:2 (lane 4). Gel result shows two bands, one the faster moving (lower band) corresponding to the DNA-1 and the retarded one (upper band) which corresponding to the DNA triplex. It is evident that as the TFO-1 ratio in the mixture is increased, the intensity of the DNA triplex band is increased which clearly proposes the triplex formation (lanes 3 and 4, Figure 7). Simultaneously, the band intensity corresponding to DNA-1 gets diminished and later disappears, as the DNA triplex formation is maximum. Discussion

The hepatocyte growth factor/MET pathway is one of the most frequently deregulated pathways in human cancers, which activate several downstream pathways including

RAS-MAPK (mitogen-activated protein kinase), PI3K (phosphotidylinositol 3 kinase)-AKT, STATs (signal transducer and activator of transcription), PLCg (phospholipase Cg) and c-Src. The activated MET kinase elicits different cellular responses by triggering these distinct pathways for example activation of RAS-MAPK pathway is mainly required for oncogenic transformation and cell proliferation, while the PI3K-AKT pathway primarily regulates both cell survival and motility. In addition, the recruitment of the protein phosphatase SHP2 (a Src homology 2-containing tyrosine phosphatase) by Gab1 is essential for branching morphogenesis. Therefore, phosphorylation profiles of multiple kinases shed ample light on our understanding of how cells recognize and respond to changes in their environment. Present results of phospho-array of HepG2 cell line after treatment with TFO-1 reveal loss of phosphorylation status of molecules ERK, MEK, AKT, Src, FAK, MSK and p38a. This clearly indicate the decreased activity of signaling pathways leading to proliferation inhibition. A similar observation of association of MET knockdown and decreased levels of phosphorylation of intracellular signaling molecules has been reported in lung cancer cell lines.33 MET also has dual role with having antiapoptotic and pro-apoptotic properties and is involved in the differential regulation of the survival/apoptosis balance. There are studies which have shown the survival responses induced by hepatocyte growth factor/scatter factor were correlated with an increased expression of the antiapoptotic Bcl-xl and Bcl-2 proteins, which inhibit mitochondrial-dependent apoptosis.34 In the view of the present results, the antiproliferative and apoptotic effect of TFO-1 can be suggested, based on the hyperphosphorylation of p53, Chk-2, the DNA damage indicators, the increased activity of Bax and decreased levels of Bcl-xl. Overexpression of p53 in HCC has been shown to induce apoptosis35 and diminish the growth of tumor cells.36 It may be recalled that the TTS-1 is located within positive regulatory element and contains the Sp1 binding

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site (GGCGGG). Sp1 is an essential transcription factor and any mutation in Sp1 binding site leads to drastic reduction in c-met transcription.37 Therefore, it is obvious that binding of TFO-1 to TTS-1 is a primary cause for the observed inhibition of MET expression. TFO-2 could not generate any transcription inhibition as the targeted region TTS-2 is away from the transcription start site. Similarly, TFO-3 which is a non-specific sequence containing five mismatch base pairs (of TFO-1) could not again influenced the MET expression. These two findings further strongly support the sequence-specific antiproliferative effect of TFO-1 on c-met. However, a small insignificant inhibitory effect shown by TFO-2 and TFO-3 could be a possibility of exposure of cells to high concentrations of phosphorothioated oligonucleotides. As the inhibition of MET by TFO-1 is primarily due to the triple helical DNA, it was important to confirm the formation and stability of DNA triplex between the TFO1 and TTS-1 and the same was performed by CD spectroscopy and GRA. Differential mobilities of DNA1 duplex alone and in the presence of the TFO-1 clearly suggest DNA triplex formation (Figure 7). DNA triplex-based antigene strategy provide additional advantage over other strategies, for example, small molecule kinase inhibitors, antibodies or decoy molecules are non-specific in nature while antisense stratgey can block mRNA translation but does not prevent the corresponding gene from being transcribed resulting in repopulating the RNA pool. In contrast, DNA triplex formation prevents gene transcription in a long-lasting way and specificially targeting overexpressed c-met oncogene without effecting the tumor micro-enviornment. Further, for a more complete assessment, it is mandatory to use a comprehensive experimental model that mimics, as completely as possible, a large variety of pathological changes in humans. Therefore, the effect of TFO-1 was also assessed on the DEN induced HCC in rats as DEN causes liver lesions which closely mimic the different types of benign and malignant tumors in humans. Our laboratory has demonstrated the role of MRI in the extent of tumor invasion in skin tumor previously.38 In the present study, MRI was used as the first step to monitor the development and the dynamic growth of the liver tumors non-invasively and non-destructively. MR image of the normal rat shows homogeneous appearance of liver lobules (encircled) as shown in Figure 4ai, while liver tumor in control rat appears as hyperintense nodules in the T2-weighted images (Figure 4aiv). It may be mentioned here that MRI is based on the differences in the environment of protons within and surrounding the tissue. The intensity of the tumor was found to be further increased with the tumor progression. Neoplasms have more free water than benign tissues and thus have longer relaxation times with progression of tumors;39 therefore, tumor appears hyperintense in imaging. In TFO-1-treated groups, tumor intensity was less that may be due to the lesser cell mass accumulation. Tumor inhibition, apparently, is a result of decreased cellular proliferation and induction of cellular apoptosis as

Cancer Gene Therapy

increased number of DNA frangmentation was observed in TUNEL assay. It is interesting to see that TFO-1 cause downregulation of MET by B50% in both in vitro and in vivo. From this observation, it can be said that TFO-1 has high anticancer property. The western blot results show almost 50% downregulation in the protein expression after 5 weeks TFO treatment, which seems to be sufficient to check the tumor growth as the reduction in tumor growth is significantly higher at this stage. It has been reported earlier that even 30% downregulation in MET stopped the tumor growth in nude mice as reported by Herynk et al.40 This can be understood by the phenomenon of ‘Oncogene Addiction’. As the sustained activity of a particular oncogene has found to be required for maintenance of tumors and attenuation of pro-survival signals in oncogene-dependent cells contribute to cell death following acute oncogene inactivation.41,42 The present study suggests that the apoptotic response to MET inhibition by TFO-1 reflects the cellular ‘addiction’ for MET. Oncogene inactivation leads to a transient imbalance in survival and apoptotic signals because survival signals are relatively short-lived in comparison with the apoptotic signals. However, there are at least three possible results when the oncogenes are inactivated: first, malignant cells might differentiate into normal cells; second, their inactivation leads to proliferative arrest; and finally, tumor cells undergo apoptosis. The in vitro and in vivo experiments in the present study show that proliferation of HCC cells was significantly inhibited after knockdown of c-met by TFO-1 followed by apoptosis. Therefore, the present study establish the role of TFO-1 as a potential tumor suppressor agent. MET in the intial stages of invasion and metastasis43,44 and to sustain the metastasis20 has been very well established in different experimental systems, suggesting that MET targeting could be fruitful therapeutic approach. From the present results, we conclude that c-metspecific DNA triplex-based therapeutic approach is a promising tool to modulate gene expression and for the inhibition of cancer cell proliferation. TTS in the genome is either directly important for gene functionality or they act as spacing fragment to help the correct positioning of transcription factors. Therefore, this strategy can be useful to target a number of oncogenes. Conflict of interest

The authors declare no conflict of interest. Acknowledgements

We sincerely thank Yale Liver Centre, Yale University, USA for providing HepG2 cells. We also would like to thank Dr Ashutosh Haldar, Department of Reproductive Biology, A.I.I.M.S. for allowing us to use Fluorescence Microscope and Electron Microscopy Facility, A.I.I.M.S. for the help in acquiring TEM images. This work is


funded by the Department of Science and Technology, India (Ref No: SR/SO/HS-118/2008). We wish to thank Professor Sudha Bhattacharya, Dean, School of Environmental Sciences, Jawaharlal Nehru University, India for providing us g-32P [ATP] and Professor Shyamal K Goswami, School of Life Sciences, Jawaharlal Nehru University, India for allowing us to carry out the gel retardation assay using radioactive material.

References 1 Cooper CS, Park M, Blair DG, Tainsky MA, Huebner K, Croce CM et al. Molecular cloning of a new transforming gene from a chemically transformed human cell line. Nature 1984; 311: 29–33. 2 Weidner KM, Sachs M, Birchmeier W. The Met receptor tyrosine kinase transduces motility, proliferation, and morphogenic signals of scatter factor/hepatocyte growth factor in epithelial cells. J Cell Biol 1993; 121: 145–154. 3 Jiang WG, Martin TA, Parr C, Davies G, Matsumoto K, Nakamura T. Hepatocyte growth factor, its receptor, and their potential value in cancer therapies. Crit Rev Oncol Hematol 2005; 53: 35–69. 4 Varnholt H, Asayama Y, Aishima S, Taguchi K, Sugimachi K, Tsuneyoshi M. C-met and hepatocyte growth factor expression in combined hepatocellular and cholangiocarcinoma. Oncol Rep 2002; 9: 35–41. 5 Nakazawa K, Dobashi Y, Suzuki S, Fujii H, Takeda Y, Ooi A. Amplification and overexpression of c-erbB-2, epidermal growth factor receptor, and c-met in biliary tract cancers. J Pathol 2005; 206: 356–365. 6 Engelman JA, Zejnullahu K, Mitsudomi T, Song Y, Hyland C, Park JO et al. MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science 2007; 316: 1039–1043. 7 Ferracini R, Di Renzo MF, Scotlandi K, Baldini N, Olivero M, Lollini P et al. The Met/HGF receptor is over-expressed in human osteosarcomas and is activated by either a paracrine or an autocrine circuit. Oncogene 1995; 10: 739–749. 8 Tuck AB, Park M, Sterns EE, Boag A, Elliott BE. Coexpression of hepatocyte growth factor and receptor (Met) in human breast carcinoma. Am J Pathol 1996; 148: 225–232. 9 Schmidt L, Duh FM, Chen F, Kishida T, Glenn G, Choyke P et al. Germline and somatic mutations in the tyrosine kinase domain of the MET proto-oncogene in papillary renal carcinomas. Nat Genet 1997; 16: 68–73. 10 Lee JH, Han SU, Cho H, Jennings B, Gerrard B, Dean M et al. A novel germ line juxtamembrane Met mutation in human gastric cancer. Oncogene 2000; 19: 4947–4953. 11 Ma WW, Adjei AA. Novel agents on the horizon for cancer therapy. CA Cancer J Clin 2009; 59: 111–137. 12 Eder JP, Vande Woude GF, Boerner SA, LoRusso PM. Novel therapeutic inhibitors of the c-Met signaling pathway in cancer. Clin Cancer Res 2009; 15: 2207–2214. 13 Puri N, Khramtsov A, Ahmed S, Hetzel JT, Jagadeeswaran R, Karczmar G et al. A selective small molecule inhibitor of MET, PHA665752, inhibits tumorigenicity and angiogenesis in mouse lung cancer xenografts. Cancer Res 2007; 67: 3529–3534. 14 Morotti A, Mila S, Accornero P, Tagliabue E, Ponzetto C. K252a inhibits the oncogenic properties of Met, the HGF receptor. Oncogene 2002; 21: 4885–4893.

15 Berthou S, Aebersold DM, Schmidt LS, Stroka D, Heigl C, Streit B et al. The Met kinase inhibitor SU11274 exhibits a selective inhibition pattern toward different receptor mutated variants. Oncogene 2004; 23: 5387–5393. 16 Christensen JG, Zou HY, Arango ME, Li Q, Lee JH, McDonnell SR et al. Cytoreductive antitumor activity of PF-2341066, a novel inhibitor of anaplastic lymphoma kinase and MET, in experimental models of anaplastic large-cell lymphoma. Mol Cancer Ther 2007; 6: 3314–3322. 17 Petrelli A, Circosta P, Granziero L, Mazzone M, Pisacane A, Fenoglio S et al. Ab-induced ectodomain shedding mediates hepatocyte growth factor receptor down-regulation and hampers biological activity. Proc Natl Acad Sci USA 2006; 103: 5090–5095. 18 Kaji M, Yonemura Y, Harada S, Liu X, Terada I, Yamamoto H. Participation of c-met in the progression of human gastric cancers: anti-c-met oligonucleotides inhibit proliferation or invasiveness of gastric cancer cells. Cancer Gene Ther 1996; 3: 393–404. 19 Stabile LP, Lyker JS, Huang L, Siegfried JM. Inhibition of human non-small cell lung tumors by a c-Met antisense/U6 expression plasmid strategy. Gene Ther 2005; 11: 325–335. 20 Corso S, Migliore C, Ghiso E, De Rosa G, Comoglio PM, Giordano S. Silencing the MET oncogene leads to regression of experimental tumors and metastasis. Oncogene 2008; 27: 684–693. 21 McGuffie EM, Pacheco D, Carbone GM, Catapano CV. Antigene and antiproliferative effects of a c-myc-targeting phosphorothioate triple helix-forming oligonucleotide in human leukemia cells. Cancer Res 2000; 60: 3790–3799. 22 Cogoi S, Ballico M, Bonora GM, Xodo LE. Antiproliferative activity of a triplex-forming oligonucleotide recognizing a Ki-ras polypurine/polypyrimidine motif correlates with protein binding. Cancer Gene Ther 2004; 11: 465–476. 23 Shen C, Rattat D, Buck A, Mehrke G, Polat B, Ribbert H. Targeting bcl-2 by triplex-forming oligonucleotide—a promising carrier for gene-radiotherapy. Cancer Biother Radiopharm 2003; 18: 17–26. 24 Ebbinghaus SW, Gee JE, Rodu B, Mayfield CA, Sanders G, Miller DM. Triplex formation inhibits HER-2/neu transcription in vitro. J Clin Invest 1993; 92: 2433–2439. 25 Singhal G, Rajeswari MR. Interaction of actinomycin D with promoter element of c-met and its inhibitory effect on the expression of MET. J Biomol Struct Dyn 2009; 26: 625–636. 26 Varnholt H, Asayama Y, Aishima S, Taguchi K, Sugimachi K, Tsuneyoshi M. C-met and hepatocyte growth factor expression in combined hepatocellular and cholangiocarcinoma. Oncol Rep 2002; 9: 35–41. 27 Sambrook J, Fritch EF, Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbour Press: NY, 1989. 28 Jain A, Rajeswari MR, Ahmed F. Formation and thermodynamic stability of intermolecular (R*R*Y) DNA triplex in GAA/TTC repeats associated with Freidreich’s ataxia. J Biomol Struct Dyn 2002; 19: 691–699. 29 Liu Y. The human hepatocyte growth factor receptor gene: complete structural organization and promoter characterization. Gene 1998; 215: 159–169. 30 Liu Y, Tolbert EM, Sun AM, Dworkin LD. Primary structure of rat HGF receptor and induced expression in glomerular mesangial cells. Am J Physiol 1996; 271: F679–F688. 31 Chan PP, Glazer PM. Triplex DNA: fundamentals, advances, and potential applications for gene therapy. J Mol Med 1997; 75: 267–282.

Cancer Gene Therapy

529


530

32 Rajeswari MR, Bose HS, Kukreti S, Gupta A, Chauhan VS, Roy KB. Binding of oligopeptides to d-AGATCTAGATCT and d-AAGCTTAAGCTT: can tryptophan intercalate in DNA hairpins? Biochemistry 1992; 31: 6237–6241. 33 Lutterbach B, Zeng Q, Davis LJ, Hatch H, Hang G, Kohl NE et al. Lung cancer cell lines harboring MET gene amplification are dependent on Met for growth and survival. Cancer Res 2007; 67: 2081–2088. 34 Fan S, Wang JA, Yuan RQ, Rockwell S, Andres J, Zlatapolskiy A et al. Scatter factor protects epithelial and carcinoma cells against apoptosis induced by DNA-damaging agents. Oncogene 1998; 17: 131–141. 35 Mitry RR, Sarraf CE, Havlik R, Habib NA. Detection of adenovirus and initiation of apoptosis in hepatocellular carcinoma cells after Ad-p53 treatment. Hepatology 2000; 31: 885–889. 36 Xu GW, Sun ZT, Forrester K, Wang XW, Coursen J, Harris CC. Tissue-specific growth suppression and chemosensitivity promotion in human hepatocellular carcinoma cells by retroviral-mediated transfer of the wild-type p53 gene. Hepatology 1996; 24: 1264–1268. 37 Koritschoner NP, Bocco JL, Panzetta-Dutari GM, Dumur CI, Flury A, Patrito LC. A novel human zinc finger protein that interacts with the core promoter element of a TATA box-less gene. J Biol Chem 1997; 272: 9573–9580.

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38 Rajeswari MR, Jain A, Sharma A, Singh D, Jagannathan NR, Sharma U et al. Evaluation of skin tumors by magnetic resonance imaging. Lab Invest 2003; 83: 1279–1283. 39 Peemoeller H, Shenoy RK, Pintar MM, Kydon DW, Inch WR. Improved characterization of healthy and malignant tissue by NMR line-shape relaxation correlations. Biophys J 1982; 38: 271–276. 40 Herynk MH, Stoeltzing O, Reinmuth N, Parikh NU, Abounader R, Laterra J et al. Down-regulation of c-Met inhibits growth in the liver of human colorectal carcinoma cells. Cancer Res 2003; 63: 2990–2996. 41 Druker BJ, Talpaz M, Resta DJ, Peng B, Buchdunger E, Ford JM et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med 2001; 344: 1031–1037. 42 Felsher DW, Bishop JM. Reversible tumorogenesis by MYC in hematopoietic lineages. Mol Cell 1999; 4: 199–207. 43 Patane` S, Avnet S, Coltella N, Costa B, Sponza S, Olivero M et al. MET overexpression turns human primary osteoblasts into osteosarcomas. Cancer Res 2006; 66: 4750–4757. 44 Taulli R, Accornero P, Follenzi A, Mangano T, Morotti A, Scuoppo C et al. RNAi technology and lentiviral delivery as a powerful tool to suppress Tpr-Met-mediated tumorigenesis. Cancer Gene Ther 2005; 12: 456–463.