ANTICANCER THERAPEUTIC INTERVENTION
20170362598 · 2017-12-21
Inventors
Cpc classification
C12N15/1138
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
C12N15/113
CHEMISTRY; METALLURGY
Abstract
The present invention is directed to a method of treating cancer using interfering RNA duplexes to mediate gene silencing. The present invention is also directed to interfering RNA duplexes and vectors encoding such interfering RNA duplexes.
Claims
1. An siNA (short interfering nucleic acid) molecule, wherein said molecule targets at least one sequence selected from SEQ ID NO: 1-SEQ ID NO: 104 and SEQ ID NO: 209-SEQ ID NO: 297 or a variant thereof or at least one sequence complementary to a sequence selected from SEQ ID NO: 1-SEQ ID NO: 104 and SEQ ID NO: 209-SEQ ID NO: 297 or a variant thereof for use as a medicament.
2. An siNA molecule according to claim 1, wherein the siNA specifically targets at least one sequence selected from SEQ ID No 4, 6, 10, 13, 22, 34, 58, 61, 81, 83, 87 and 95 to 104 or a variant thereof, and wherein said molecule reduces expression of the LAT1 gene.
3. An siNA molecule according to claim 1, wherein the siNA specifically targets at least one sequence selected from SEQ ID NO: 209, 216, 225, 226, 228, 235 to 238, 245, 260, 264, 267, 271, 272, 278, 279 and 281 to 297 or a variant thereof, and wherein said molecule reduces expression of the ASCT2 gene.
4. An siNA molecule of claim 1, wherein said molecule is between 19 and 25 base pairs in length.
5. An siNA molecule of claim 1, wherein the siNA is selected from dsRNA, siRNA or shRNA.
6. An siNA molecule of claim 5, wherein the siNA is siRNA.
7. An siNA molecule of claim 1, wherein the siNA comprises 5′ and/or 3′ overhangs.
8. An siNA of claim 1, wherein the siNA comprises at least one chemical modification.
9. An siNA molecule of claim 1, wherein the siNA molecule comprises a sense strand, and preferably an antisense strand, wherein the sense strand comprises a sequence selected from SEQ ID NO: 105 to 208 and 298 to 386 or a variant thereof.
10. An siNA molecule of claim 9, wherein preferably the sense strand comprises at least one sequence selected from 108, 110, 114, 117, 126, 138, 162, 165, 185, 187, 191 and 199 to 208 or a variant thereof, and wherein preferably, said molecule reduces expression of the LAT1 gene.
11. An siNA molecule of claim 1, wherein preferable the sense strand comprises at least one sequence selected from 298, 305, 314, 315, 317, 324-327, 334, 349, 353, 356, 360, 361, 367, 368 and 370 to 386 or a variant thereof, and wherein preferably, said molecule reduces expression of the ASCT 2 gene.
12. An siNA molecule of claim 1, wherein the sense strand comprises a sequence selected from SEQ ID NO: 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407 and 409 or a variant thereof and the antisense strand comprises a sequence selected from SEQ ID NO: 388, 390, 392, 394, 306, 398, 400, 402, 404, 406, 408 and 410 respectively or a variant thereof.
13. A pharmaceutical composition comprising at least one siNA of claim 1 and a pharmaceutically acceptable carrier.
14. A method for the treatment of cancer, the method comprising administering the siNA of claim 1.
15. A method for the treatment of cancer, the method comprising administering the pharmaceutical composition of claim 13 to a patient in need thereof.
16. The method of claim 14, wherein the cancer is selected from bladder, blood, brain, colon, head and neck, kidney, liver, lung, lymph node, mammary gland, metastatic, muscle, ovary, pancreas, prostate, skin, stomach and uterus cancer.
17. The method of claim 15, wherein the cancer is selected from bladder, blood, brain, colon, head and neck, kidney, liver, lung, lymph node, mammary gland, metastatic, muscle, ovary, pancreas, prostate, skin, stomach and uterus cancer.
18. A method of treating cancer comprising administrating to a patient in need thereof, the siNA of claim 1 in combination with one or more anti-cancer agents, preferably wherein the anti-cancer agent comprises an anti-antineoplastic agent.
19. A method of reducing cell proliferation, the method comprising contacting the cell with the siNA of claim 1.
20. A nucleic acid construct or vector comprising a nucleic acid sequence encoding an siNA of claim 1.
Description
DESCRIPTION OF THE DRAWINGS
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[0179] MATERIALS AND METHODS
[0180] Cell Culture
[0181] SK-HEP-1, T24, HT-1080 and HCT-116 cell lines were maintained in a humidified atmosphere of 5% CO.sub.2 at 37° C. SK-HEP-1 cells were grown in RPMI-1640 (Sigma, St. Louis, Mo.) supplemented with 20% fetal bovine serum (FBS) (Gibco, UK), 100 U/mL penicillin G, 0.25 μg/mL amphotericin B, 100 μg/mL streptomycin (Gibco, UK), 25 mM sodium bicarbonate (Merck, Germany) and 25 mM N-2-hydroxyethylpiperazine-N′-2-ethanosulfonic acid (HEPES) (Sigma, St. Louis, Mo.). T24 and HT-1080 cells were grown, respectively, in Dulbecco's Modified Eagle's Medium (DMEM)—high glucose (Sigma, St. Louis, Mo.) and DMEM—low glucose (Sigma, St. Louis, Mo.), supplemented with 10% FBS (Gibco, UK), 100 U/mL penicillin G, 0.25 μg/mL amphotericin B, 100 μg/mL streptomycin (Gibco, UK), 25 mM sodium bicarbonate (Merck, Germany) and 25 mM HEPES (Sigma, St. Louis, Mo.). HCT-116 cells were grown in McCoy's 5A (Sigma, St. Louis, Mo.) supplemented with 10% FBS (Gibco, UK), 100 U/mL penicillin G, 0.25 μg/mL amphotericin B, 100 μg/mL streptomycin (Gibco, UK), 25 mM sodium bicarbonate (Merck, Germany) and 25 mM HEPES (Sigma, St. Louis, Mo.). For all cell lines the medium was changed every 2 days, and cells reached confluence 3-4 days after initial seeding. For subculturing, cells were dissociated with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) (Sigma, St. Louis, Mo.), split 1:15 or 1:20 and subcultured in a 21-cm.sup.2 growth area (Sarstedt, Germany).
[0182] LAT1 and ASCT2 Protein Expression
[0183] Cells were rinsed twice with cold phosphate-buffered saline (PBS) and incubated with 100 μL RIPA lysis buffer (154 mM NaCl, 65.2 mM TRIZMA base, 1 mM EDTA, 1% NP-40 (IGEPAL), 6 mM sodium deoxycholate) containing protease inhibitors: 1 mM PMSF, 1 μg/mL leupeptine and 1 μg/mL aprotinin; and phosphatase inhibitors: 1 mM Na.sub.3VO.sub.4 and 1 mM NaF. Cells were scraped and briefly sonicated. Equal amounts of total protein (30 μg) were separated on a 10% SDS-polyacrylamide gel and electrotransferred to a nitrocellulose membrane in Tris-Glycine transfer buffer containing 20% methanol. The transblot sheets were blocked in 5% non-fat dry milk in Tris-buffered saline (TBS) for 60 min and then incubated overnight, at 4° C., with the following antibodies: rabbit anti-LAT1 (1:1000; Cell Signalling); rabbit anti-ASCT2 (1:1000; Cell Signalling); or mouse monoclonal anti-GAPDH (1:20,000; Santa Cruz Biotechnology Inc.), diluted in 2.5% non-fat dry milk in TBS-Tween 20 (0.1% vol/vol). The immunoblots were subsequently washed and incubated with fluorescently-labelled goat anti-rabbit (1:20,000; IRDye™ 800, Rockland) or fluorescently-labelled goat anti-mouse secondary antibody (1:20,000; AlexaFluor 680, Molecular Probes) for 60 min at room temperature (RT) and protected from light. Membranes were washed and imaged by scanning at both 700 nm and 800 nm with an Odyssey Infrared Imaging System (LI-COR Biosciences).
[0184] LAT1 and ASCT2 Gene Expression
[0185] Total RNA was isolated and purified using the SV Total RNA Isolation System (Promega, USA) according to manufacturer's instructions. RNA quality and concentration were verified in the NanoDrop ND1000 Spectrophotometer (Thermo Scientific, USA), and RNA integrity and genomic DNA contamination were evaluated by agarose gel electrophoresis. Total RNA (1 μg) was converted into cDNA using the Maxima Scientific First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Scientific, USA), according to instructions. The following protocol was used: 1.sup.st step, 10 min at 25° C.; 2.sup.nd step, 15 min at 50° C.; 3.sup.rd step, 5 min at 85° C. cDNA was used for qPCR analysis using Maxima SYBR Green qPCR Master Mix (Thermo Scientific, USA) in the StepOnePlus instrument (Applied Biosystems, USA). QuantiTect Primer Assay for LAT1 and ASCT2 and for the endogenous control gene GAPDH (Quiagen, Germany) were used. The qPCR reaction was performed in 96-well PCR plates (Sarstedt, Germany) as follows: one cycle of 10 min at 95° C., followed by 40 PCR cycles at 95° C. 15 s and 60° C. 60 s. A melting curve was made immediately after the qPCR, to demonstrate the specificity of the amplification. No template controls were always evaluated for each target gene. Quantification cycle (Cq) values were generated automatically by the StepOnePlus 2.3 Software and the ratio of the target gene was expressed in comparison to the endogenous control gene GAPDH. Real-time PCR efficiencies were found to be between 90% and 110%.
[0186] LAT1 Activity
[0187] Cells were plated in 24-well plates (Sarstedt, Germany) and grown until confluence was reached. On the day of the experiment, cell culture medium was aspirated and cells were preincubated for 15 min in Hanks' medium (NaCl 140 mM. KCl 5 mM, MgSO.sub.4.7H.sub.2O 0.8 mM, K.sub.2HPO.sub.4 0.33 mM, KH.sub.2PO.sub.4 0.44 mM, MgCl.sub.2.6H.sub.2O 1 mM, CaCl.sub.2 0.025 mM, Tris-HCl 9.75 mM, pH 7.4). Uptake was initiated by addition of Hanks' medium with 0.25 μM [.sup.14C]-L-leucine in the absence and in the presence of 3 mM unlabeled L-leucine. During preincubation and incubation cells were continuously shaken and maintained at 37° C. Uptake was terminated after 1 min by rapid removal of uptake solution by means of a vacuum pump connected to a Pasteur pipette, followed by a rapid wash with Hanks' medium. Subsequently, cells were solubilized in 0.1% vol/vol Triton X-100 (dissolved in 5 mM Tris-HCl, pH 7.4), and radioactivity was measured by liquid scintillation counting.
[0188] ASCT2 Activity
[0189] Cells were plated in 24-well plates (Sarstedt, Germany) and grown until confluence was reached. On the day of the experiment, Cell culture medium was aspirated and cells were preincubated for 15 min in Hanks' medium (ChCl 140 mM, KCl 5 mM, MgSO.sub.4.7H.sub.2O 0.8 mM, K.sub.2HPO.sub.4 0.33 mM, KH.sub.2PO.sub.4 0.44 mM, MgCl.sub.2.6H.sub.2O 1 mM, CaCl.sub.2 0.025 mM, Tris-HCl 9.75 mM, pH 7.4). Uptake was initiated by the addition of Hanks' medium with 0.25 μM [.sup.14C]-L-alanine in the absence and in the presence of 3 mM unlabeled L-alanin. During preincubation and incubation cells were continuously shaken and maintained at 37° C. Uptake was terminated after 1 min by rapid removal of uptake solution by means of a vacuum pump connected to a Pasteur pipette, followed by a rapid wash with Hanks' medium. Subsequently, cells were solubilized in 0.1% vol/vol Triton X-100 (dissolved in 5 mM Tris-HCl, pH 7.4), and radioactivity was measured by liquid scintillation counting.
[0190] LAT1 Gene Silencing
[0191] Cells were plated in 24-well (Sarstedt, Germany) or 6-well plates (Sarstedt, Germany) or 96-well plates with black walls clear bottom (BD Biosciences, USA) and incubated 24 h under normal growth conditions. siRNAs against LAT1 and transfection agent were diluted at desired concentrations and mixed according to transfection agent manufacturer's instructions. The mixture was incubated 20 min at RT for siRNA-complex formation, after which it was added to the cells and incubated at 37° C., 5% CO.sub.2. After the incubation period, serum and antibiotic was restored and cells were further incubated at normal conditions for the desired time points until evaluation of LAT1 activity or LAT1 expression (immunoblotting and RT-qPCR).
[0192] ASCT2 Gene Silencing
[0193] Cells were plated in 24-well (Sarstedt. Germany) or 6-well plates (Sarstedt, Germany) or 96-well plates with black walls clear bottom (BD Biosciences, USA) and incubated 24 h under normal growth conditions. siRNAs against ASCT2 and transfection agent were diluted at desired concentrations and mixed according to transfection agent manufacturers instructions. The mixture was incubated 20 min at RT for siRNA-complex formation, after which it was added to the cells and incubated at 37° C., 5% CO.sub.2. After the incubation period, serum and antibiotic was restored and cells were further incubated at normal conditions for the desired time points until evaluation of ASCT2 activity or ASCT2 expression (immunoblotting and RT-qPCR).
[0194] Cell Proliferation Assay
[0195] Cell proliferation was measured using calcein-AM (Thermo Fisher Scientific, USA). The membrane permeant calcein-AM, a nonfluorescent dye, is taken up and converted by intracellular esterases to membrane impermeant calcein, which emits green fluorescence. Cells were plated in 96-well plates with black walls clear bottom (BD Biosciences, USA) and incubated 24 h under normal growth conditions. Cells were incubated with test items at 37° C., 5% CO.sub.2. After the incubation period, serum and antibiotic was restored and cells were further incubated at normal conditions during 72 h. After treatment with test substances or vehicle, cells were washed twice with Hanks' medium and loaded with 2 μM calcein-AM in Hanks' medium, at at 37° C. for 30 min. Fluorescence was measured at 485 nm excitation and 530 nm emission wavelengths in a microplate spectrofluorometer (Gemini EM, Molecular Devices). Nine consecutive fluorescence measurements are performed per well, to allow fluorescence readings in the whole area of the well, which was then considered for the calculation of mean fluorescence per well. To determine minimum staining for calcein (calcein.sub.min), eight wells were treated with ethanol 30 min before calcein-AM addition. The percent cell number is calculated as [(calcein.sub.sample)/(calcein.sub.control)]×100.
[0196] Animals and Tumour Implantation
[0197] Human colon cancer HTC-116 cells grown in tissue culture and 10.sup.7 cells per mouse were injected into the hind flank of female NMRI nu/nu mice. Once tumours have developed and tumour volumes reached randomisation criteria, therapy will commence by every other day daily, intra-tumoral injections. A vehicle treated group was included in the study as control. Female immunodeficient NMRI nu/nu mice from Charles River were used. The animals were delivered at the age of 4-6 weeks and are used for implantation after at least 1 week of quarantine. All animals interventions were performed in accordance with the European Directive number 86/609, and the rules of the “Guide for the Care and Use of Laboratory Animals”, 7th edition, 1996, Institute for Laboratory Animal Research (ILAR), Washington. D.C. Only animals with unobjectionable health were selected to enter testing procedures. During the experiments, animals were monitored at least daily. Each cage was labelled with a record card indicating animal source, gender, and the delivery date. Animals were numbered during tumour implantation or at the initiation of a dose finding experiment.
[0198] The tumour volume was determined by a two-dimensional measurement with callipers on the day of randomization (Day 0) and then twice weekly. Tumour volumes were calculated according to the following equation:
Tumour Vol[mm.sup.3]=a[mm]×b.sup.2[mm.sup.2]×0.5
where “a” is the largest diameter and “b” is the perpendicular diameter of the tumour representing an idealized ellipsoid.
[0199] The relative volume of an Individual tumour on day X (RTV.sub.x) was calculated by dividing the absolute volume [mm.sup.3] of the respective tumour on day X (T.sub.x) by the absolute volume of the same tumour on the day of randomization, i. e. on day 0 (T.sub.0), multiplied by 100, as shown by the following equation:
[0200] RTVs were used for growth characterization and compound activity rating as follows:
TABLE-US-00006 Rating RTV.sub.x [%] CR Complete remission ≦10 PR Partial remission >10; ≦50 MR Minor remission >50; ≦75 NC No change >75; ≦125 P Progression >125
[0201] Group median and range (alternatively geometric mean+/−SEM) of RTVs were calculated, considering only the tumours of animals that were alive on the day in question (for median). Group median (geometric mean) RTVs were used for drawing tumour growth curves and for treatment evaluation.
[0202] Tumour inhibition on a particular day (T/C.sub.x) was calculated from the median RTV of a test group and the median RTV of a control group multiplied by 100, as shown by the following equation:
[0203] The optimum/minimum/best T/C [%] value recorded for a particular group during an experiment represents the maximum anti-tumour activity for the respective treatment and is rated as follows:
TABLE-US-00007 Rating T/C [%] − Inactive ≧65 +/− Borderline activity ≧50; ≦65 + Moderate activity ≧25; ≦50 ++ High activity ≧10; ≦25 +++ Very high activity ≧5; ≦10 ++++ Complete remission <5
[0204] Tumour volume doubling/quadrupling time (DT/QT) is defined as the time interval (in days) required for a group to reach a median RTV of 200%/400% of the initial tumour volume. Growth delay is defined as the difference in days between the tumour volume doubling and quadrupling times of a test group and the respective control group.
[0205] Non-Viral Delivery siRNA Systems
[0206] 1. Liposomes carrying therapeutic siRNA-LAT1 agents are capable of passing through the membrane of the target cell to deliver cargo. A large number of lipids can be used for the synthesis of liposomes used for the delivery of siRNAs. Neutral lipids that can be complexed with siRNA-LAT1 include DOPE (1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine), egg PC (phosphatidylchone), DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine and DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamlne).
[0207] 2. Cationic lipids that can be complexed with siRNA-LAT1 include DOTAP (1,2-dioley-3-trimetylammonium propane), CDAN (N(1)-cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine)/DOPE, DC-Chol (3β-[N—(N′,N′-dimethylaminoethane)carbamoyl] cholesterol)/DOPE. DOTAP/DOPE, cationic lipid RPR209120 (2-(3-[Bis-(3-amino-propyl)-amino]-propylamino)-N-ditetradecylcarbamoylme-thyl-acetamide). Galactosylated (Gal-C4-Chol/DOPE) liposomes/siRNA-LAT1 complex can also induce gene silencing.
[0208] 3. Cationic polymers can also be used in siRNA-LAT1 or siRNA-ASCT2 delivery. These materials combine with anionic siRNA-LAT1 or siRNA-ASCT2 to form a siRNA-LAT1-polymer complex or siRNA-ASCT2-polymer complex that can interact with the negatively charged cell surfaces through the cationic portion of the complex. Among the available polymers, polyethyleneimine (PEI) has the ability to bind strongly to negatively charged siRNA-LAT1 or siRNA-ASCT2. Biodegradable polymers such as poly(L-lysine) (PLL) are known for their lower toxicity and higher biocompatibility than PEI. A derivative of PLL, poly[α-(4-aminobutyl)-L-glycolic acid] exhibits higher transfection efficiency and lower immunogenicity and cytotoxicity than the original PLL polymer and can be used with siRNA-LAT1 or siRNA-ASCT2.
[0209] 4. Cationized gelatin microspheres can be prepared by chemically cross-linking gelatin in the water-in-oil emulsion state. To impregnate siRNA-LAT1 or siRNA-ASCT2 expression plasmid DNA into cationized gelatin microspheres, PBS containing siRNA-LAT1 expression plasmid DNA can be dropped onto freeze-dried cationized gelatin microspheres and then kept for 24 h at 4° C.
[0210] 5. Nanoparticles can be produced based on modified ionic gelation of tripolyphosphate (TPP) with chitosan. Two different types of chitosan (chitosan hydrochloride and glutamate) and each type with two different molecular weights can be used. Nanoparticles can be spontaneously obtained upon the addition of a TPP aqueous solution to chitosan solution under constant magnetic stirring at room temperature. The particles can then be incubated at room temperature for before use or further analysis. Nanoparticles are collected by centrifugation. The supernatants are discarded and nanoparticles are resuspended in filtered distilled water. For the association of siRNA-LAT1 or siRNA-ASCT2 with the chitosan-TPP nanoparticles (chitosan-TPP-siRNA-LAT1 or chitosan-TPP-siRNA-ASCT2), siRNA-LAT1 or siRNA-ASCT2 in double distilled water is added to the TPP solution before adding this drop-wise to the chitosan solution under constant magnetic stirring at room temperature. The particles are then incubated at room temperature before use or further analysis.
[0211] 6. Chitosan (114 kDa) was dissolved in sodium acetate buffer to obtain a 0.2-1 mg/ml working solution range. Twenty microliters of siRNA-LAT1 or siRNA-ASCT2 (20-250 μm range) was added to 1 ml of filtered chitosan while stirring and left for 1 h. To calculate specific N:P ratios (defined as the molar ratio of chitosan amino groups/RNA phosphate groups) a mass per phosphate of 325 Da was used for RNA and mass per charge of 167.88 for chitosan (84% deacetylation).
[0212] 7. The siRNA-LAT1 or siRNA-ASCT2 can be encapsulated in stable nucleic acid lipid particles (SNALP) and administered by intravenous injection. The SNALP formulation contained the lipids 3-N-[(ω-methoxypoly(ethylene glycol).sub.2000)carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-DMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-qminopropanone (DLinDMA), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol.
[0213] 8. The siRNA-LAT1 or siRNA-ASCT2 can be encapsulated in Injectin In Vivo SiRNa Delivery Reagent (BioCelChallenge SAS, Toulon, France) and administered by intravenous injection. The Injection formulation contained the following mixture: 10 μg of siRNA in 10 pIL of glucose containing buffer, 40 μL with a sterile RNase-free water, 10 μL of Injedin reagent. The mixture should be mix by pipetting up and down and incubated 15 minutes at room temperature before injection.
Example 1
[0214] LAT1 and ASCT2 Immunoblotting in Human Cancer Cells
[0215] The presence of LAT1 protein and ASCT2 was studied by means of immunoblotting using an antibodies raised against LAT1 and ASCT2. As shown in
Example 2
[0216] LAT1 and ASCT2 Gene Expression in Human Cancer Cells
[0217] The presence of LAT1 and ASCT2 mRNA was studied by means of Real-time PCR using primers against ASCT2. As shown in
Example 3
[0218] [.sup.14C]-L-Leucine and [.sup.14C]-L-Alanine Uptake
[0219] Sodium-independent [.sup.14C]-L-leucine (0.25 μM) uptake at initial rate of uptake (1 min) in epithelial carcinoma cells was significantly (P<0.001) reduced by 3 mM unlabelled L-leucine as shown in
Example 4
[0220] LAT1 and ASCT2 Gene Expression in Human Cancer Cells
[0221] As shown in
Example 5
[0222] [.sup.14C]-L-Leucine and [.sup.14C]-L-Alanine Uptake
[0223] As shown in
Example 6
[0224] Modifications of siRNA-LAT1 and siRNA-ASCT2
[0225] siRNA-LAT1 against nucleotide sequences No 58 and No 61 (for example, an siRNA comprising or consisting of SEQ ID NO: 162 and 165 respectively) are shown in
Example 7
[0226] [.sup.14C]-L-Leucine and [.sup.14C]-L-Alanine Uptake
[0227] As shown in
Example 8
[0228] LAT1 and ASCT2 Gene Expression in Human Cancer Cells
[0229] As shown in
Example 9
[0230] LAT1 Gene Expression in Human Cancer Cells
[0231] As shown in
Example 10
[0232] Cell Proliferation of Human Colon Cancer HTC-116 Cells, Transfection Agents and Negative Controls
[0233] As shown in
Example 11
[0234] Cell Proliferation of Human Colon Cancer HTC-116 Cells
[0235] As shown in
[0236] Treatment for 6 h of cells with the siRNA-LAT1 against nucleotide sequence No 58t (for example, an siRNA comprising or consisting of SEQ ID NO: 387 as the sense strand and SEQ ID NO: 388 as the antisense strand), as shown in
Example 12
[0237] Cell Proliferation of Human Colon Cancer HTC-116 Cells in the Presence of Cytotoxic Antineoplastic Agents
[0238] As shown in
Example 13
[0239] Cell Proliferation of Human Colon Cancer HTC-116 Cells in the Presence Anti-LA T1 and Anti-ASCT2 siRNAs
[0240] As shown in
Example 14
[0241] LAT1 and ASCT2 Gene Expression and Cell Proliferation of Human Colon Cancer HTC-116 Cells in the Presence Anti-LA T1 and Anti-ASCT2 siRNAs
[0242] As shown in
[0243] As shown in
Example 1
[0244] LAT1 and ASCT2 Immunoblotting in the Xenograft Tumour Model
[0245] The presence of LAT1 protein and ASCT2 was studied by means of immunoblotting using an antibodies raised against LAT1 and ASCT2. As shown in
Example 16
[0246] LAT1 and ASCT2 Gene Expression in Human Cancer Cells
[0247] The presence of LAT1 and ASCT2 mRNA was studied by means of Real-time PCR using primers against LAT1 and ASCT2. As shown in
Example 17
[0248] Tumour Growth
[0249] As shown in
CONCLUSION
[0250] The treatment of cancer cells expressing LAT1 and/or ASCT2 transporter with siRNA-LAT1 and/or siRNA-ASCT2 leads to a decrease in LAT1 and/or ASCT2 protein and a decrease in [.sup.14C]-L-leucine uptake and [.sup.14C]-L-alanine uptake, which is accompanied by a decrease in cell proliferation. The decrease in cell viability and proliferation of cancer cells induced by the siRNA-LAT1 and/or the siRNA-ASCT2 is accompanied by apoptosis and a decrease in tumour growth and metastasis potential, as evidenced in nude mice subcutaneous tumours of human colon cancer HTC-116 cells.
[0251] Additional aspects of the invention will be apparent to those skilled in the art, or may be learned from the practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.
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