Patent Application
20090264510
The teachings of all of the references, including websites, cited herein are incorporated herein in their entirety by reference. The present invention relates to the application of double-stranded oligonucleotides interfering with the mRNA transcribed from a gene involved in carcinogenesis, particularly the Wnt1, Wnt2 or Her3 gene, as novel anti-tumour agents.
RNA interference is a phenomenon based on the post-transcriptional gene silencing (PTGS) and is an excellent tool for the analysis of their function and role in many processes within an organism. This technique is of great importance in functional genomics, mapping of biochemical pathways, determination of pharmacological treatment directions and in gene therapy. PTGS was first described in plants (Napoli, C., C. Lemieux and R. Jorgensen. Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans. Plant Cell 2:279-289, 1990) In 1998, Andrew Fire and Craig Mello described RNAi for the first time in an animal, C. elegans (Fire, A. et al. 1998 Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-810). Long, double-stranded RNA molecules induced post-transcriptional gene silencing.
However, the application of nucleotides this long also elicited an immune response (increased interferon levels) in mammalian cells and it was T. Tuschl, S M. Elbashir et al. who finally discovered that the application of short, double-stranded nucleotides (19-21 bp) does not induce an immune response (Elbashir, S. M., J Harborth, W. Lendeckel, A. Yalcin, K Weber and T. Tuschl. 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494-498).
Gene silencing is based on double-stranded interfering nucleic, acid (iNA) preferably a double-stranded RNA (dsRNA) molecules, also called siRNA. RNAi is a response to cellular processes induced by dsRNA, which degrades homologous mRNA. Even a few copies of dsRNA may entirely destroy the transcripts for a given gene formed within a cell. The destruction of selected mRNA's through RNAi begins with the activation of RNAse III, which cleaves long hairpin loops of dsRNA or ssRNA fragments into double-stranded small interfering RNA (siRNA) 21-23 nucleotides long. siRNA's prepared earlier may be introduced into cells externally. Next, siRNA molecules bind to a nuclease complex forming a RISC(RNA induced silencing complex). Thanks to the helicase activity which is a part of the RISC, dsRNA is separated into single strands. The ssRNA molecules formed then anneal to complementary mRNA strands. The final stage of PTGS is the degradation of selected mRNA by RISC nucleases. In contrast to traditional methods, such as knockouts, gene silencing is quickly and easily performed, both in animal and in cell line models.
The authors of the present invention have performed intensive research and have determined that the silencing of expression of genes involved in carcinogenesis, e.g. gene Wnt1, using double-stranded oligonucleotides such as an iNA or an siRNA for this gene is an effective strategy for the inhibition of tumour cell proliferation.
Wnt1 is a secretory protein which binds the “frizzled” inter-membrane receptor and transmits a signal to cytoplasmatic phosphoproteins, which in turn downregulate the constitutively high activity of glycogen synthase kinase 3Beta (GSK-3Beta) (Polakis et al., Wnt signaling and cancer, Genes Dev. 2000 Aug. 1; 14(15):1837-51). The result of this is the stabilization and growth of Beta-catenin levels in the cell nucleus.
Wnt-1 overexpression has been noted in many types of tumours, including in cancers of the lung, colon and breast, sarcomas and tumours of the head and neck (Katoh et al. Expression and regulation of WNT1 in human cancer: up-regulation of WNT1 by beta-estradiol in MCF-7, In J Oncol, 2003 January; 22(1):209-12).
Anti-WNT-1 monoclonal antibodies are known. The application of such antibodies resulted in an increase of apoptosis, a decrease in tumour cell proliferation (H460 and MCF-7 lines), as well as inhibition of the take of transplantable murine lung cancer (H460) (Biao He, A Monoclonal Antibody against Wnt-1 Induces Apoptosis in Human Cancer Cells, Neoplasia, Vol. 6, No. 1, January/February 2004, pp. 7-14). In the above report, Biao He et al. also used chemically unmodified siRNA on a breast cancer line (MCF-7), resulting in an increased apoptosis rate in these cells.
Anti-WNT-1 monoclonal antibodies elicited apoptosis in sarcoma cells (A-204) (Iwao Mikami, Efficacy of Wnt-1 monoclonal antibody in sarcoma cells, BMC Cancer 2005, 5:53, 24 May 2005), and in NCI-H1703 and H28 lung cancer cells (Liang You, Inhibition of Wnt-1 Signaling Induces Apoptosis in β-Catenin-Deficient Mesothelioma Cells, Cancer Research 64, 3474-3478, May 15, 2004). This research also made use of chemically unmodified siRNA in MCF-7 breast cancer cells, and NCI-H1703 and H28 lung cancer cells, resulting in an increased apoptosis rate.
You et al. also used unmodified siRNA, which elicited apoptosis to a degree similar to monoclonal antibodies. Similar results of apoptosis induction were obtained in colon cancer cells (SW-480, HCT116) (He et al., Blockade of Wnt-1 signaling induces apoptosis in human colorectal cancer cells containing downstream mutations, Oncogene 2005, 24: 3054-3058). In a recent report, Fukutomi et al. (Hepatology 2005; 41:1096-105) indicated only an indirect effect of the siRNA silencing of WNT-1 on the proliferation of modified liver tumour cells. These experiments made use of liver cancer line cells expressing type C hepatitis virus core protein. Expression of type C hepatitis virus core protein was obtained through the transfection of these cells with vectors coding for said protein. The presence of this protein enhanced WNT-1 expression and cell proliferation. The application of siRNA specific for Wnt-1 in such cells caused the silencing of its expression and inhibited proliferation. This sort of experimental model, however, does not provide evidence which would allow one to hypothesize that a similar effect would be elicited in cells unmodified with the viral protein, upon the application of WNT-1 specific siRNA.
International Patent Application Publication No. WO2004032838 describes a method of inhibiting tumour cell proliferation based on the contact of a cell with a compound which blocks the interaction of WNT with its receptor. As an example of such inhibition, a monoclonal antibody against the WNT-1 protein was used. This patent application also describes the occurrence of apoptosis in the cells of many tumour lines following the application of siRNA for the WNT-1 protein. None of the above publications describes any effect of oligonucleotides which activate the siRNA mechanism in the inhibition of the proliferation of unmodified tumour cells, nor is such an effect known.
The elimination of cells through apoptosis is not a sufficient mechanism for enhancement of anti-tumour activity, because in many tumour types this mechanism is disrupted or inhibited. Among other factors, this is connected with a series of mutations in the p53 gene, which is responsible for regulation of this process. The inefficacy of this process may also be tied in with the absence of proapoptotic proteins such as Bax or Bid in many types of tumours, or the increased expression of apoptosis inhibitors such as Bcl-2. Only the inhibition of tumour take and/or tumour cell proliferation can be evidence of anti-tumour activity.
Experiments on modified cells do not facilitate the prediction of the behaviour of natural, unmodified cells occurring in tumours. The application of monoclonal antibodies as a potential treatment entails a considerable risk of eliciting an immune response in living organisms. Additionally, monoclonal antibodies are very expensive and their production does not guarantee a repeatable response in individual recipients, since genetically modified organisms are used in their manufacture.
Thus, there exists a real need to find new, effective treatments which would exhibit anti-tumour properties but which would not elicit immune responses. Such drugs should be simple and inexpensive to manufacture, preferably using a reproducible technological process.
The present invention relates to the use siRNA against mRNA transcribed by genes involved in carcinogenesis. An example of such an siRNA is one that targets the mRNA of the oncogene Wnt1 gene. An siRNA directed to tumour cell lines expressing the Wnt1 results in a strong inhibition of tumour cell proliferation. This inhibition is dose-dependent. The present invention thus successfully delivers a solution to the problem of tumour treatment through the inhibition of tumour cell growth, using the RNA interference mechanism to degrade the mRNA of the gene involved in carcinogenesis, e.g. gene coding WNT-1.
The present invention provides methods of inducing apoptosis or inhibiting growth of a cancer cell as well the method for obtaining the oligonucleotide useful as an effective anticancer agent. Furthermore, the application of the present invention entails a very limited danger of eliciting an immune response in treated patients. The production of double-helical oligonucleotides is a reproducible process and is simple to perform using standard equipment, the so-called RNA synthesizers. Such oligonucleotides may be designed according to one of many algorithms described to date, such as the one indicated in Example 1.
The sequence of an mRNA gene of interest can be obtained from a database, for example GenBank, and the NCBI Reference Sequence should be chosen. The second structure of the mRNA target sequence can be designed using computer folding algorithm. siRNAs against a chosen mRNA sequence can be generated in silico using known algorithms. There are many algorithms available on-line that are used to design siRNAs against a particular mRNA sequence. These algorithms in general are based on similar equations but there are subtle differences among them. Most of algorithms are based on Tuschl rules of siRNA design but some of them additionally use also Reynolds rules (Reynolds A, Leake D, Boese Q, Scaringe S, Marshall W S, Khvorova A. Rational siRNA design for RNA interference. Nat Biotechnol. 2004 March; 22(3):326-330). It is known that you have to verify siRNAs generated by one of the algorithms with another algorithm. That is why in our method we use different algorithms based on different equations. The Tuschl Rules are:
Select targeted region from a given cDNA sequence beginning 50-100 nt downstream of start condon
2. First search for 23-nt sequence motif AA(N19). If no suitable sequence is found, then,
3. Search for 23-nt sequence motif NA(N21) and convert the 3′ end of the sense siRNA to TT
4. Or search for NAR(N17)YNN
5. Target sequence should have a GC content of around 50%, wherein
Some algorithms also analyze the thermodynamic stability of siRNAs. The distribution of free energy through siRNA molecule is a very important factor in describing the potential of a given sequence. This feature is very important in recognition of the guide strand by RISC because RISC recognizes the 5′ end of a strand that will be incorporated into RISC, and will serve as the guide strand. It is known that the 5′ end of the antisense strand should be less stable than the 3′ end, so the free energy at the 5′ end should be higher than at the 3′ end.
Relying on these rules, there should be a difference in GC content between the 5′ and 3′ ends. More GC pairs are preferred at a 3′ end of antisense strand. Also the total content of GC in an siRNA is important for the thermodynamic stability of siRNA and its potential. In functional siRNA, GC content should be between 30%-60%; this will ensure that a designed duplex will not be too stable to be unwound and will be stable enough to avoid self-unwinding in cytoplasm.
In thermodynamics analysis it is also recommended to design siRNA with a low stability at position 10 of antisense strand. This position is a cleavage site so there should not be formed a strong duplex between guide strand and target mRNA, U base is recommended in this position. Another factor that should be taken into consideration during siRNA designing is to target secondary structure accessibility. This factor describes probability of a single stranded motif in target region in mRNA molecule. In the cytoplasm mRNA never exists as a single strand, its secondary structure is rich in hairpins, loops and other structures which are results of partial paring between bases in given mRNA molecule.
One of the greatest problems in designing siRNA is to avoid potential “off-target” effect. This effect occurs if particular siRNA targets not only desired mRNA but other mRNAs as well. In this case there are also many algorithms like blast or clustal which can predict possible interactions with any known transcript.
1. The sequence of an mRNA gene of interest was obtained from a database, for example GenBank, and the NCBI Reference Sequence was chosen. siRNAs against chosen mRNA sequence were generated in silico using known algorithms.
The designed sequences were ranked according to total filtering score based on following rules:
a) Frequency among algorithms.
a=1*number of algorithms
b) Single stranded region probability
c) Complementary to other mRNA sequences
c=−2*number of molecules
d) Free energy of the antisense strand 5′ end
e) Free energy of the antisense strand 3′ end
f) Free energy at 10 position of the antisense strand
g) GC content
For further analyses the best fifteen siRNAs have been chosen.
Then screenings for inhibition of proliferation, decrease in mRNA and protein levels were performed. The experiments were enforced by transfection efficiency greater or equal to 80%.
The inhibition score for each sequence was evaluated by factor s:
Scores:
Then sequences were ranked by the inhibition score.
The decrease in mRNA level score for each sequence was evaluated by factor r:
Scores:
Then sequences were ranked by the decrease in mRNA level score.
The decrease in protein level score for each sequence was evaluated by factor t:
Scores:
Then sequences were ranked by the decrease in protein level score.
All sequences were characterized by final screening factor z:
Next siRNAs with z factor better or equal to 50% of the best sequence, but not more than 3 were analyzed according to the cell death mechanism. Sequences were ranked by:
number of alive cells
−1*% of necrotic cells
+1*% of early apoptotic cells
+2*% of apoptotic cells
Next dose-response effect was evaluated.
the lowest dose by which over 65% of silencing had been achieved was evaluated,
the longest period of time with effect still observed was evaluated.
Moreover, to limit the immune response, it is preferable that the oligonucleotides be no more than 30 bp long, and preferentially be 21-23 bp long. Sense and antisense oligonucleotides may be symmetrical or not, meaning that i.e. 2 terminal nucleotides may be unhybridized, thus forming sticky ends. In order to enhance their thermal and enzymatic stability, pharmacokinetic, bioavailability and cellular uptake properties the oligonucleotides may be modified chemically. Chemical modifications may pertain to phosphates, ribose or the nucleases themselves. Said chemical modifications may pertain to only selected nucleotides, i.e. terminal or median, or the entire oligonucleotide.
The oligonucleotides may be delivered to tumour cells both by themselves, without vectors, as well as with a vector, both viral and non-viral. Adenoviruses or adeno-like viruses are examples of viral vectors, which facilitate the continual expression of the oligonucleotide following introduction into tumour cells. Non-viral vectors used to introduce oligonucleotides into cells are lipid capsules, lipid complexes or other vectors prolonging their half-lives in a living organism and/or absorption into cells. As a result of use of present invention, considerable inhibition of tumour cell proliferation is achieved.
Although the examples and descriptions presented below illustrate the nature of the present invention and include examples to illustrate it, it is understood that a practical embodiment of the present invention encompasses all normal changes, adaptations, modifications, deletions from or additions to the procedures described, being a part of the below claims and equivalents.
Human breast cancer cell line MCF-7 was obtained from the American Type Culture Collection (Rockville, Md., USA). Cell cultures were maintained in DMEM supplemented with 10% (v/v) fetal calf serum (FCS), 50 μg/ml gentamycin, 2.5 μg/mL fungizone, 50 UI/mL penicillin and 50 μg/ml streptomycin (Invitrogen Carlsbad, Calif. USA) in an atmosphere of 5% CO2/95% humidified air at 37° C., and routinely subcultured every 2 or 3 days.
For proliferation tests MCF-7 cells were plated in Opti-MEM (Invitrogen) at 7×103 cells per well in 96-well plates one day before experiments. The next day the MCF-7 cells were transfected with fifteen siRNAs sequences specific to Wnt1 mRNA and scrambled siRNA sequence (control) in at a concentration of 50 nM for 48 h using Lipofectamine RNAi MAX (Invitrogen) according to manufacturer's protocol. siCONTROL TOX (Dharmacon, Chicago, Ill. USA) was used as a control of transfection efficiency. After 48 h of experiment proliferation inhibition was measured using MTS test (Promega, Madison, Wis. USA).
Western Blot Analysis
Reagents for Western blotting were purchased from BioRad (Hercules, Calif. USA), anti-Wnt1 antibody was from Zymed Invitrogen, anti-actin, anti-phosphor-beta-catenin, anti-c-myc and anti-cyclin D1 were from Santa Cruz Biotechnology (Santa Cruz, Calif. USA), anti-cleaved PARP antibody was from Cell Signaling (Danvers, Mass., USA). Western blotting detection reagents was from Roche Diagnostics (Indianapolis, Ind., USA) and Light Film BioMax was from Kodak (Rochester, N.Y. USA)
On the day before the experiment, 250×103 cells were cultured in Opti-MEM in sterile 25 cm2 conical flasks to 60% confluence. To knock-down the Wnt1 gene, medium was removed and replaced with the transfection medium containing siRNAs, which passed the inhibition score ranking. After 48 h the cultured cells were harvested by trypsinization and centrifuged at 2000 g, for 5 min, at 4° C. and the cells pellet was suspended in ice-cold phosphate buffered saline (PBS). After second centrifugation the supernatant was removed and the cell pellet was re-suspended in 0.5 mL Total Lysis Buffer RIPA (Santa Cruz Biotechnology, Santa Cruz, Calif., USA), and incubated at 4° C. for 30 min. The cells suspended in the buffer were centrifuged at 9000 g, 10 min, at 4° C., then the supernatant (containing the total protein fraction) was carefully removed and passed six times through a 20-gauge syringe needle.
The lysates were mixed 1:2 (v/v) with Laemmli sample buffer (BioRad) containing 2.5% 2-mercaptoethanol and boiled for 3 min. Samples containing identical quantities of proteins were subjected to SDS-PAGE (12% gel) together with a Kaleidoscope Marker (BioRad). The electrophoresis was run for 1 hour at 100 V using a Mini Protean III cell (BioRad,). After electrophoresis the separated proteins were electroblotted on a PVDF membrane (Biorad) for 70 min at 110 V using the Mini Protean III. The membranes were blocked overnight with 5% w/v solution of non-fat powdered milk in TBST (pH 7.5). The following day the membranes were rinsed three times for 10 min in TBST, at room temperature, and then incubated for 1 hour at room temperature with the primary antibodies diluted 1:200. The membranes were then rinsed four times for 10 min in TBST and incubated with diluted 1:2000 secondary antibodies conjugated with horseradish peroxidase (Sigma Aldrich, St. Louis, USA) for another 1 h at room temperature. Finally, the membranes were rinsed three times for 10 min in TBST, and labelled proteins were visualized using the LumiLight (Roche) Western blotting detection reagent on a high performance chemiluminescence BioMAX light film (Kodak). The image on light film was then analyzed with a Kodak Edas System and the integrated optical density (IOD) was measured.
Real Time-PCR
On the day before the experiment, 250×103 cells were cultured in Opti-MEM in sterile 25 cm2 conical flasks to 60% confluence. To knock-down the Wnt1 gene, medium was removed and replaced with the transfection medium with siRNAs, which past the inhibition score ranking. After 48 h the cultured cells were harvested by trypsinization and centrifuged at 2000 g, for 5 min, at 4° C. and the cells pellet was suspended in ice-cold PBS. Then cells were lysed by adding 1 ml of TRIZOL Reagent (Invitrogen) and passed several times through a pipette. After that lysate was incubated for 5 minutes at room temperature. Next 0.2 mL of chloroform per 1 mL of TRIZOL was added and samples were incubated at room temperature for 10 min. Next samples were centrifuged at >12,000 g for 15 min at 4° C. Then the aqueous phase was transferred to a fresh tube and RNA was precipitated from the aqueous phase by mixing with isopropyl alcohol and incubated at room temperature for 10 min and centrifuged at >12,000 g for 10 min at 4° C. The RNA was washed once with 1 mL 75% ice-cold ethanol. Samples were mixed by vortexing and centrifuged at >12,000 g for 5 min at 4° C. At the end of the procedure, the RNA pellet was dried (air-dry for 5-10 min). At the end RNA was dissolved in proper volume of RNase-free water.
Isolated RNA was transcribed to cDNA using ImProm-II Reverse Transcriptase kit (Promega), according to manufacturer's protocol. Changes in mRNA expression of target genes were measured using Rotor-Gene™ 3000 (CORBETT RESEARCH) and calculated as relative expression using Relative Expression Software Tool for Rotor-Gene© (REST-RG©). House-keeping gene was H3F3A (histon H3A). Calibrator sample was from Stratagene, and primers for house-keeping gene were from Eurogenetec and primers specific to target gene (Qiagen, Germany). Samples of cDNA and proper primers were mixed with Fast Start DNA Master SYBR Green I kit (Roche).
Immunofluorescence Staining for Flow Cytometry
On the day before the experiment 250×103 cells were cultured in Opti-MEM in sterile 25 cm2 conical flasks to 60% confluence. To knock-down the Wnt1 gene, medium was removed and replaced with the transfection medium containing the siRNAs, which passed the inhibition score ranking. After 48 h the cultured cells were harvested by trypsinization and centrifuged at 2000 g, for 5 min, at 4° C. and the cells pellet was suspended in ice-cold PBS.
The cells were then fixed in 1% formaldehyde for 15 min, washed twice with PBS, suspended in ice-cold 70% ethanol and stored at −20° C. for 24 h. after this time the cells were washed twice with PBS-1% BSA and incubated for 1 h with either primary antibody anti-Wnt1 (Zymed-Invitrogen) diluted 1:250 with PBS-1% bovine serum albumin (BSA). After primary incubation the cells were washed twice with PBS-1% BSA, and incubated for 1 h with 1:500 secondary antibodies labelled with Alexa Fluor 488 (Molecular Probes, Eugene, Oreg., USA). The cells were then washed twice in PBS-1% BSA and finally incubated with a 10 μg/mL solution propidium iodide with RNase A for 15 min to counterstain the DNA. Then the cells were measured using BD FACS Calibur Flow Cytometry (Becton Dickinson, Franklin Lake, N.J., USA)
Apoptosis Analysis
For caspases 3 and 7 activation, MCF-7 cells were plated in Opti-MEM (Invitrogen) at 7×103 cells per well in 96-well plates one day before experiments. On the next day the MCF-7 cells were transfected with siRNAs sequences which passed the inhibition score ranking at a concentration of 50 nM for 48 h using Lipofectamine RNAi MAX (Invitrogen) according to manufacturer's protocol. After 12 h of siRNA exhibition, activation of caspases 3 and 7 was measured using Caspase-Glo 3/7 assay (Promega) by GloMax™ 96 Microplate Luminometer (Promega) according to manufacturer's protocol.
To analyze apoptosis cells transfected with siRNA which passed the final screening test were harvested by trypsinization and stained using an Annexin V FLUOS Staining Kit (Roche Diagnostics, Indianapolis, Ind., USA), according to the manufacture's protocol. Then stained cells were immediately analyzed by flow cytometry (FACScan; Becton Dickinson, Franklin Lake, N.J.). Early apoptotic cells with exposed phosphatidylserine but intact cell membranes bound to Annexin V-FITC but excluded propidium iodide. Cells in necrotic or late apoptotic stages were labeled with both Annexin V-FITC and propidium iodide.
Design of siRNA Sequences
One of many available algorithms may be used in the design of potent siRNA sequences. Such algorithms are commonly available in literature, such as:
1. http://www.ambion.com/techlib/misc/siRNA_finder.html
2. https://www.genscript.com/ssl-bin/app/rnai
3. http://wwwl.qiagen.com/Products/GeneSilencing/CustomSiRna/SiRnaDesigner.aspx
4. http://sfold.wadsworth.org/sirna.pl
The basis of these algorithms is the introduction of the mRNA or cDNA of the protein which we wish to silence.
mRNA coding the WNT-1 protein or its cDNA is easily accessible and made public (i.e. in the GENMED database: www.ncbi.nlm.nih.gov).
NCBI Reference Sequences (RefSeq) NM005430 (http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=Nucleotide&dopt=GenBank&val=16936523)
The authors of the present invention have designed many potent siRNA sequences for WNT1 mRNA, which are presented in the table below (Tab. 1).
Synthesis of siRNA
RNA synthesis was performed using the solid phase synthesis technique, using typical protocols for the synthesis of nucleic acids using derivatives of β-cyanoethyl phosphamide esters in conjunction the tert-butyldimethyl-silane protection of the 2′-OH group of ribose. Phsosphamide monomers attach to the free 5′-OH group of ribose following activation with 5-benzylmercapto-1H-tetrazole. This reaction proceeds rapidly, efficiently yielding oligomers. The oligomers formed are additionally purified using chromatographic (HPLC) or electrophoretic (PAGE) techniques. The synthesis was performed on an Applied Biosystems 962 RNA synthesizer. siRNA was produced through the gentle agitation of equimolar amounts of complementary RNA strands for 1 hour at −20° C. in 2M acetate buffer in ethanol. Such a solution was centrifuged for 15 min. and dried with 70% ethanol.
Preparation of Individual siRNA Dilutions Using a Lipid Vector
SiRNA (WNT1—16) was diluted using the Hiperfect lipid vector. Hiperfect was purchased from and supplied by Qiagen.
Each siRNA dilution was prepared in a series and then an appropriate amount of HiPerFect was added.
25 nM
3 μl siRNA+1197 μl medium without serum
5 nM
200 μL 25 nM solution+800 μL medium without serum
1 nM
200 μL 5 nM solution+800 μL medium without serum
Transfection of siRNA:
The dilutions prepared in Example 3 were used in transfection.
siRNA transfection was performed at three concentrations: 1, 5 and 25 nm, HiPerfect: constant 0.75 microL/well. Experimental controls consisted of: a) tumour cells, b) a+HiPerfect reagent
Stages:
Reading of the Results
The “Blank” control consisted of a solution from wells containing only culture medium. The positive control consisted of cells suspended in culture medium. The spectrophotometrically determined OD is proportional to the number of living cells in a sample. The results obtained from the measurement of the proliferation rate of individual tumour line cells treated with siRNA were collected in tables (Table 2 and Table 3)
From the results of the experiments on the inhibition of tumour cell proliferation, it is evident that the application of siRNA against the Wnt1 gene entails a significant inhibition of tumour cell proliferation. The values of the inhibition of proliferation are relative to control cells incubated solely in medium. Furthermore, the usage of siRNA against WNT1 resulted in a much stronger inhibitory effect on proliferation when compared to tumour cells treated solely with the Hiperfect lipid vector or the siRNA of other genes, to which anti-tumour properties are ascribed.
Cell proliferation of MCF-7 cells was measured over a 48 h treatment of 50 nM siRNAs sequences specific to Wnt1 gene, using MTS assay for determination of cell growth rates. The growth of cells treated with siRNA was compared to untreated cells (CTRL), cells treated with scrambled (non-coding) siRNA (SC siRNA) and to cells treated with siControl TOX (siTOX) and Docetaxel (DOC). SC siRNA and siTOX were used to determine non-specific inhibition of cell growth caused by nucleic acid chemistry or transfection reagent, and to check efficiency of transfection, respectively. Values shown on
Next, we measured mRNA level after MCF-7 treatment with siRNAs that passed inhibition score ranking. Reduction of mRNA levels is the most direct result of siRNA action. Thus we determined whether MCF-7 cells transfected with siRNA against Wnt1 mRNA would cause a decrease in mRNA level. Analysis was performed 48 h after transfection. Total mRNA isolation, transcription to cDNA and real-time PCR were done as described in Material and Methods. After MCF-7 cells were transfected with an siRNA sequence that targets W15 mRNA, we observed a decrease in mRNA by 61% in comparison to untreated control cells. This experiment was also a control indicating the specificity of our sequence. Additionally we performed a similar experiment with A549 cells, to check if there would be any response. It is known that there is no expression of Wnt1 in A549 cells (He et al. 2004). We observed no changes in proliferation and mRNA level 48 h after A549 cells treatment with W15 siRNA sequence. These data indicate that W15 sequence is specific and potent in decreasing mRNA level, which is a base of siRNA action.
Western blotting analysis of Wnt1 level in MCF-7 cells after transfection with siRNA against Wnt1 was done (
These data indicate that W15 sequence against Wnt1 provides a decrease of Wnt1 level in MCF-7 cells, and it is correlated with a decline of c-myc, cyclin D1 and an increase of phosphorylated beta-catenin level.
Next, the changes in expression of Wnt1 in MCF-7 cells was measured using flow cytometry techniques after the MCF-7 cells were treated with siRNA that targets Wnt1 mRNA (
Analysis of cell cycle of MCF-7 cells treated with siRNA against Wnt1 mRNA was done using flow cytometry techniques (
To verify what kind of cell death is triggered by siRNA treatment we performed caspases activation assay. The results obtained in this assay are presented as inhibition of proliferation in comparison to control. We observed that after treatment of MCF-7 cells with W15 sequence there was at least fivefold increase in activation of caspases 3 and 7, and after W13 sequence treatment it was around fourfold increase while after treatment with cytotoxic docetaxel it was only about twofold increase (
We then determined the number of apoptotic cells, of necrotic cells and of viable cells. Analysis of apoptosis using Annexin V (AV) and propidium iodide (PI) double staining was performed. Double negative are viable cells. AV positive and PI negative are cells in early phase of apoptosis, while AV positive and PI positive are cells in a late phase of apoptosis. Necrotic cells are AV negative and PI positive (
Flow cytometry technique was used to verify if apoptosis was triggered by of the reduction in the level of Wnt1 in MCF-7 cells transfected with siRNA that targets the Wnt1 mRNA. (
The teachings of all of the references including websites cited herein are incorporated in their entirety by reference.
| Number | Date | Country | Kind |
|---|---|---|---|
| P.378857 | Jan 2006 | PL | national |
This is a continuation-in-part of and claims benefit under 35 U.S.C. §120 of International Patent Application No. PCT/PL2007/000006 filed on Jan. 31, 2007, which claims benefit under 35 U.S.C. §119 of Polish Patent Application No. P.378857 filed on Jan. 31, 2006, the teachings of which are all incorporated herein in their entirety by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/PL07/00006 | Jan 2007 | US |
| Child | 12221063 | US |
