US20090048195A1

US20090048195A1 - Compositions and methods of sphingosine kinase inhibitors for use thereof in cancer therapy

Info

Publication number: US20090048195A1
Application number: US11/948,998
Authority: US
Inventors: Uttam K. Sinha; Rizwan Masood
Original assignee: University of Southern California USC
Current assignee: University of Southern California USC
Priority date: 2006-11-30
Filing date: 2007-11-30
Publication date: 2009-02-19
Also published as: US20080261907A1; US20120259150A1; US8084438B2; WO2008067526A2; WO2008067526A3; WO2008067560A9; US20100331393A1; WO2008067560A2; WO2008067560A3

Abstract

The present invention relates to Sphingosine kinase inhibitors that are useful for treating various cancers. The invention specifically relates to compositions and methods of SPK inhibitors, including siRNAs, which specifically block gene expression of SPK and induce apoptosis in a variety of cancers.

Description

The present application claims the benefit of the filing date of U.S. Provisional Application No. 60/868,046, filed Nov. 30, 2006, the disclosure of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates in general to treatment of diseases. More specifically, the invention provides compositions comprising sphingosine kinase inhibitors and methods of using the compositions for treating various cancers.

BACKGROUND OF THE INVENTION

Cancer is a major health problem in the world. It is the second leading cause of human death next to coronary disease. As reported by the American Cancer Society, cancer causes the death of well over a half-million people annually, with over 1.2 million new cases diagnosed per year. Although significant progress has been made in the detection and treatment of specific types of cancers, no universal treatment method for the treatment of all types cancer exists. Management of the disease involves the combination of as early as possible diagnosis and aggressive treatment including a variety of elements such as surgery, radiotherapy, chemotherapy or also hormone therapy.
Sphingolipids such as ceramide and sphingosine (SPH) are a class of apoptosis regulators in cancer cells. Ceramide inhibits proliferation and promotes apoptosis (10), while sphingosine-1-phosphate (S1P) is a key tumor-promoting lipid, responsible for tumor cell proliferation, migration and invasion (1). S1P is synthesized by phosphorylation of SPH. However, the formation of S1P antagonizes the formation of ceramide. The opposing directions between the formation of ceramide and S1P is referred to as the “sphingolipid rheostat” and plays a pivotal role in regulating tumor growth (10) (FIG. 1). The levels of ceramide and SIP are regulated by sphingosine kinase-1 (SPK-1), whose overexpression has been shown to inhibit apoptosis (10). Studies have also shown that elevated levels of SIP and increased SPK-1 activity in cancers is due to the overexpression of SPH (2,3), while the reduction of SPK1 levels in cancer cells results in apoptosis of cancer cells (10, 15).
SPK-1 activity has been shown to be upregulated in many types of cancers including various squamous cell carcinomas (SCC) such as head and neck, lung, bladder, ovary, prostate, and skin cancers. Likewise, cell lines from various types of cancers including the human breast cancer (MCF-7) (6-8); intestinal tumors (9); prostate adenocarcinomas (10); colon cancers (11); lung cancers (12); erytholeukemias (13); and bladder tumors (14) also exhibit SPK-1 overexpression.
French et. al. used various cancer cells and cell lines including human breast cells and breast, colon, lung, ovary, stomach, uterus, kidney and rectum tumors from patients, to demonstrate that SPK mRNA is over-expressed in cancer cells as compared to normal tissue in the surrounding area of the same organ (15). French used a chemical library of 16,000 compounds to screen for compounds that inhibited SPK overexpression. Four compounds were discovered, but not all of these compounds were specific for SPK inhibition, as some compounds inhibited other human kinases.
Other pharmacological agents that have been shown to reduce the levels of SPK in tumor cells include phenoxodiol (16); dimethylsphingosine (DMS) (18); docetaxel and camptothecin (10); agents derived from marine bacterium B-5354 (19) and fungus (20); and sphingoside analogs (17). However, these pharmacological agents exhibit moderate levels of antitumor activity or toxicity that make the use of pharmacological inhibitors undesirable.
Small interfering RNAs (siRNAs) have been shown to specifically “knock out” or “silence” the gene of specific proteins and enzymes and are effective at inhibiting the overexpression of SPK without the toxicities and other undesirable side effects associated with the use of pharmacological inhibitors. Various siRNAs have been shown to reduce SPK-1 activity and induce apoptosis in MCF-7 cells (21) and decrease cell viability in PC-3 cells (22).

SUMMARY OF THE INVENTION

In one embodiment, the invention relates to SPK inhibitors that specifically block SPK overexpression in a variety of cancer cells.
In another embodiment, the invention relates to SPK inhibitors that silences or knocks out the gene responsible for SPK over-expression and induces apoptosis in a variety of cancer cells.
In a related embodiment, the invention relates to compositions comprising SPK inhibitors that specifically blocks SPK over-expression in a variety of cancer cells.
In yet another embodiment, the invention relates to compositions comprising SPK inhibitors that silences or knocks out the gene responsible for SPK over-expression and induces apoptosis in a variety of cancer cells.
In accordance with another embodiment, the invention relates to methods using SPK inhibitors that knock out or silence the gene responsible for SPK over-expression and induce apoptosis in a variety cancer cells. The method comprises transfecting cancer cells with SPK inhibitors and determining the amount of SPK expression. If SPK expression in cancer cells that have been transfected with SPK inhibitor decreases more than the SPK expression of cancer cells that have not been transfected with SPK inhibitor, then the SPK inhibitor has silenced or knocked out SPK gene expression and apoptosis has been induced.
In a closely related embodiment, the invention relates to a method of using SPK inhibitors to specifically block gene expression of SPK and induce apoptosis in vivo. The method comprises injecting a mammal with cancer cells that have been transfected with and without SPK inhibitors. If the tumor volume of the mammal treated with a SPK inhibitor decreases more than the tumor volume of the mammal treated with no SPK inhibitor, then the SPK inhibitor has blocked SPK gene expression and induced apoptosis.
Within one aspect, the present invention provides SPK inhibitors comprising siRNA. More specifically the siRNA comprises 19-25 oligonucleotide bases.
Within one further aspect, the present invention provides SPK inhibitors comprising double stranded siRNA (HNB-001) with a sense of 5′-GGG CAA GGC UCU GCA GCU CTT-3′ (SEQ ID NO: 1) and antisense sequence of 5′-GAG CUG CAG AGC CUU GCC CTT-3′ (SEQ ID NO: 2).
Within another aspect, the present invention provides SPK inhibitors comprising double stranded siRNA (HNB-002) with a sense sequence of 5′-GGG CAA GGC CUU GCA GCU CTT-3′ (SEQ ID NO: 3) and antisense sequence of 5′-GAG CUG CAA GGC CUU GCC CTT-3′ (SEQ ID NO: 4).
The above-mentioned and other features of this invention and the manner of obtaining and using them will become more apparent, and will be best understood, by reference to the following description, taken in conjunction with the accompanying drawings. The drawings depict only typical embodiments of the invention and do not therefore limit its scope.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic representation of the synthesis of Ceramide, Sphingosine (SPH) and Sphingosine-1-Phosphate (S-1-P).

FIG. 2. Representative Immunohistochemistry (IHC) staining of head and neck tumor tissue for SPK expression. The left panel shows an absence of SPK staining in normal tissue. The arrows in the right panel show positive staining for SPK in tumor regions.

FIG. 3. Representative western blot for SPK-1 expression in HNSCC primary tissues and metastases. Western blots of HNSCC tissue lysates consisting of lymph node biopsies (LN), tumor (T), and normal tissue (N) were prepared using a SPK specific antibody. The tumor and lymph node tissue show positive SPK staining, while the normal tissue shows minimal SPK staining.

FIG. 4. Representative western blot for SPK-1 expression in siRNA treated cells. (Left) SCC cells treated with siRNA (SPK1 lane) and without siRNA (control lane). (Right) Schematic showing a decrease in the concentration of SIP and increase the concentration of ceramide when SPK mRNA is inhibited by siRNA.

FIG. 5. Graph of viable SCC-71 cells as a function of siRNA concentration.

FIG. 6. Graph of viable SCC-71 and SCC-15 cells as a function of siRNA concentration.

FIG. 7. Graph of viable MCF-7 cells as a function of siRNA concentration.

FIG. 8. Graph of viable PC-3 cells as a function of siRNA concentration.

FIG. 9. Graph of viable HT-29 cells as a function of siRNA concentration.

FIG. 10. Graph of tumor size as function of siRNA concentration.

FIG. 11. Photograph of tumor size in mice treated with siRNA.

DETAILED DESCRIPTION OF THE INVENTION

It is well known that various types of cancer cells (i.e., head and neck squamous cell carcinoma, leukemia, breast, colon, lung, ovary, stomach, uterus, kidney, rectum, prostate, bladder, skin, ovary, brain, etc.) overexpress SPK (10-15), and that apoptosis of these cancer cells may be induced by inhibiting SPK overexpression (15).
The prior art has shown siRNAs that were used to reduce SPK-1 activity and induce apoptosis or cell viability specifically in the MCF-7 cells or PC-3 cells (21,22). However, there are no siRNAs that have been shown to reduce SPK-1 activity and induce apoptosis in various types of cancers.
The present invention relates to siRNAs which are unique in characteristic as they are capable of inducing apoptosis in various cancer cells. These siRNAs specifically bind and knock out SPK expression and induce apoptosis in various types of cancers.
As used herein the terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include breast cancer, brain cancer, bladder cancer, prostate cancer, colon cancer, intestinal cancer, squamous cell cancer, lung cancer, stomach cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, skin cancer, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, thyroid cancer, various types of head and neck cancer, and the like.
The term “overexpression,” as used herein refers to overexpression of a gene and/or its encoded protein in a cell, such as a cancer cell. A cancer cell that “overexpresses” a protein is one that has significantly higher levels of that protein compared to a noncancerous cell of the same tissue type.
The phrase “induces apoptosis” refers to the ability of a SPK inhibitor to induce programmed cell death by inhibiting SPK overexpression by silencing or knocking out SPK gene expression.
“Therapeutic agent” is an agent that may directly decrease the pathology of tumor cells, render the tumor cells more susceptible to treatment by other therapeutic agents, e.g., radiation and/or chemotherapy, or reduces the percentage of cells overexpressing SPK.
“Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, mice, primates, rabbits, rats, cats, dogs, and the like.
To practice the methods relating to silencing or knocking out SPK gene expression, a cancer cell line or cancer cells are obtained from a tissue containing cancer. The cancer cells are transfected with a SPK inhibitor and the expression level of the SPK gene in the cells are determined and compared to a control level. A control level may be the expression level of the SPK gene in a cancer cell that has not been transfected with a SPK inhibitor, may be transfected with green fluorescent protein siRNA (GFP siRNA), or a normal cell from a tissue of the same body location. If the expression level of the SPK gene in the test sample is lower than the control level, the SPK inhibitor has knocked out or silenced the gene responsible for SPK over-expression.
To select SPK specific inhibitors, several mouse and human SPK-1 phosphorothioate antisense oligonucleotides were tested in head and neck squamous cell lines. Phosphothionate-modified oligonucleotides were synthesized and purified (Core facilities at the USC/Norris Comprehensive Cancer Center, Microchemical Core Facility Los Angeles, Calif.). SPK-1 antisense oligonucleotides from different regions of the mouse SPK-1 and human SPK-1 coding region were synthesized. The sequence of each oligonucleotide are as follows:

hSPK-1-1:
5′-GAG CTG CAA GGC CTT GCC CTT-3′	(SEQ ID NO: 5)

hSPK-1-2:
5′-AGG CCG CTC CAT GAG CCC GTT-3′	(SEQ ID NO: 6)

hSPK-1-3:
5′-GTT GGT CAG GAG GTC TTC ATT-3′	(SEQ ID NO: 7)

hSPK-1-4:
5′-GGT GTC TTG GAA CCC ACT CTT-3′	(SEQ ID NO: 8)

hSPK-1-5:
5′-ATA CTC CAT ATG CCT GCC CTT-3′	(SEQ ID NO: 9)

hSPK-1-6:
5′-CGG CCT CGC TAA CCA TCA ATT-3′	(SEQ ID NO: 10)

mSPK-1-1:
5′-GAG CTG CAG AGC CTT GCC CTT-3′	(SEQ ID NO: 11)

mSPK-1-2:
5′-TCC GTT CGG TGA GTA TCA GTT-3′	(SEQ ID NO: 12)

mSPK-1-3:
5′-CAC CAG CTC CCT GGC ATG GTT-3′	(SEQ ID NO: 13)

mSPK-1-4:
5′-GTT GAT GAG CAG GTC TTC ATT-3′	(SEQ ID NO: 14)

mSPK-1-5
5′-GCA CAA CAG CAG TGT GCA GTT-3′	(SEQ ID NO: 15)

mSPK-1-6
5′-CCA GGT ATG GAC AGT CAA GTT-3′	(SEQ ID NO: 16)

Six of each, mouse and human antisense oligonucleotides (1-6) corresponding to different regions of SPK-1 cDNAs were tested for activity against HNSCC cell growth. Only one of the six antisense oligonucleotides (hSPK-1-2) showed potent inhibitory activity. The hSPK-1-1 oligonucleotide had minimal inhibitory effect on HNSCC cells and the hSPK-1-3, hSPK-1-4, and hSPK-1-5 antisense oligonucleotides also showed some inhibitory activity in HNSCC cells.
The SPK inhibitor HNB-001 showed potent inhibitory activity against HNSCC cell growth. It corresponds to a SPK-1 coding region in mouse and comprises the sense sequence: 5′-GGG CAA GGC UCU GCA GCU CTT-3′ (SEQ ID NO: 1) and antisense sequence: 5′-GAG CUG CAG AGC CUU GCC CTT-3′ (SEQ ID NO: 2). Likewise, HNB-002 also showed potent inhibitory activity against HNSCC cell growth. It corresponds to a SPK-1 coding region in humans, and comprises the sense sequence: 5′-GGG CAA GGC CUU GCA GCU CTT-3′ (SEQ ID NO: 3) and antisense sequence: 5′-GAG CUG CAA GGC CUU GCC CTT-3′ (SEQ ID NO: 4).
SPK inhibitors of the present invention may comprise siRNA. More specifically the siRNA may be 19-25 oligonucleotide bases long.
Control siRNA may be green fluorescence protein siRNA (GFP siRNA) comprising the sense sequence: 5′-CGG CCA CAA GUU CAG CGU GUC dTdT-3′ (SEQ ID NO: 17) and antisense sequence: 5′-GAC ACG CUG AAC UUG UGG CCG dTdT-3′ (SEQ ID NO: 18).
Gene expression levels may be detected and quantified at the mRNA or protein level using a number of means well known in the art. To detect mRNAs or measure mRNA levels, cells in biological samples (e.g., tissues and body fluids) may be lysed and the mRNA in the lysates or in RNA purified or semi-purified from the lysates detected or quantified by any of a variety of methods familiar to those in the art. Such methods include, without limitation, hybridization assays using detectably labeled gene-specific DNA or RNA probes and quantitative or semi-quantitative RT-PCR (e.g., real-time PCR) methodologies using appropriate gene-specific oligonucleotide primers. Alternatively, quantitative or semi-quantitative in situ hybridization assays can be carried out using, for example, unlysed tissues or cell suspensions, and detectably (e.g., fluorescently or enzyme-) labeled DNA or RNA probes. Additional methods for quantifying mRNA levels include RNA protection assay (RPA), cDNA and oligonucleotide microarrays, and colorimetric probe based assays.
Methods for detecting proteins or measuring protein levels in biological samples are also known in the art. Many such methods employ antibodies (e.g., monoclonal or polyclonal antibodies) that bind specifically to target proteins. In such assays, an antibody itself or a secondary antibody that binds to it can be detectably labeled. Alternatively, the antibody can be conjugated with biotin, and detectably labeled avidin (a polypeptide that binds to biotin) can be used to detect the presence of the biotinylated antibody. Combinations of these approaches (including “multi-layer sandwich” assays) familiar to those in the art can be used to enhance the sensitivity of the methodologies. Some of these protein measuring assays (e.g., ELISA or Western blot) can be applied to body fluids or to lysates of test cells, and others (e.g., immunohistological methods or fluorescence flow cytometry) applied to unlysed tissues or cell suspensions. Methods of measuring the amount of a label depend on the nature of the label and are known in the art. Appropriate labels include, without limitation, radionuclides (e.g., 125I, 131I, 35S, 3H, or 32P), enzymes (e.g., alkaline phosphatase, horseradish peroxidase, luciferase, or 6-glactosidase), fluorescent moieties or proteins (e.g., fluorescein, rhodamine, phycoerythrin, GFP, or BFP), or luminescent moieties (e.g., Qdot™ nanoparticles supplied by the Quantum Dot Corporation, Palo Alto, Calif.). Other applicable assays include quantitative immunoprecipitation or complement fixation assays.
Therapeutic agents are formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.
In one embodiment, the therapeutic agents are prepared with carriers that will protect the compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Therapeutic agents, may also comprise siRNAs conjugated to cationic polypeptides, amphipathic compounds, polycations, liposomes or PEGlyated liposomes. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. “Dosage unit form,” as used herein, refers to physically discrete units suited as unitary dosages for the subject to be treated, each unit containing a predetermined quantity of an active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
The dosage required for treating a subject depends on the choice of the route of administration, the nature of the formulation, the nature of the subject's illness, the subject's size, weight, surface area, age, and sex, other drugs being administered, and the judgment of the attending physician. Wide variations in the needed dosage are to be expected in view of the variety of compounds available and the different efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art. Encapsulation of the compound in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) may increase the efficiency of delivery, particularly for oral delivery.
The following examples are intended to illustrate, but not to limit, the scope of the invention. While such examples are typical of those that might be used, other procedures known to those skilled in the art may alternatively be utilized. Indeed, those of ordinary skill in the art can readily envision and produce further embodiments, based on the teachings herein, without undue experimentation.

EXAMPLE 1

Immunohistochemistry Staining for SPK Expression

Head and neck tumor biopsy samples and adjacent normal tissue were paraffin-embedded according to the methods of Atkins et. al., who demonstrated a preferred method for detection of protein with specific antibody (23). Sections were studied by immunohistochemistry within one week of cutting the sections. Cut sections were placed on slides, rinsed twice in PBS and preincubated with blocking buffer (0.2% Triton-X100, 1% BSA in PBS) for 20 min, followed by incubation at 4° C. for 16 hr with SPK as a primary antibody that was added to the blocking buffer. After washing three times in PBS, sections were incubated with HRP-conjugated secondary antibody for 1 hr at 25° C. Peroxidase activity was revealed by the diaminobenzidine (Sigma) cytochemical reaction. Sections were counterstained with 0.12% methylene blue or H&E and mounted in IMMU-MOUNT (Shandon, Astmoor UK). SPK was expressed in the tumor regions only, as there was no SPK staining in the stroma and normal tissues (FIG. 2). Likewise, a metastatic tumor site in the lymph node showed positive staining while the normal lymph node was negative (data not shown).

EXAMPLE 2

Western Blotting for SPK Expression in Tumor Tissue

Western blots of tissue from primary tumor, lymph node metastases, and normal tissue were carried out to determine the relative levels of SPK expression in those sites. Normal tissues adjacent to tumors, tumor, and lymph nodes were collected from several patients. SPK expression was observed in each of the tumor samples. Similarly, all tumor-positive lymph nodes showed SPK expression that was equal to or slightly greater than the primary tumor (FIG. 3). No or minimal expression was observed in the normal tissue that was a adjacent to the tumor. Quantitative analysis by stage and lymph node status showed that SPK levels were substantially higher in stage III/IV compared to stage I/II.
Western blotting was performed by adding tissues to 0.5 ml of cold lysis buffer (50 mM Tris, pH 8, 150 mM NaCl, 1% Triton X-100, 0.5 mM EDTA, containing Halt Protease Inhibitor cocktail [Pierce, Rockford Ill.]) and homogenizing them on ice using a PowerGen 125 homogenizer (Fisher Scientific). Homogenized samples were transferred to 1.7 ml microcentrifuge tubes and centrifugated at 10,000×g for 10 min at 4° C. to clear the lysates. Protein extracts were gently removed and put into fresh tubes. Total protein was determined by Dc colorimetric assay (BioRad, Richmond, Calif.). Protein samples (25 μg protein) were fractionated on 4-20% Tris-glycine polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad) by electroblotting. Membranes were blocked with 5% non-fat milk prior to incubation with primary SPK antibody at 4° C., for 16 hr. Secondary antibody (1:100,000 dilution) conjugated with horseradish peroxidase was applied for 1 hr at 25° C. The membranes were developed using the SuperSignal West Femto Maximum sensitivity chemiluminescent substrate (Pierce, Rockford, Ill.) according to the manufacturer's instructions. The same membranes were stripped and re-probed with B-actin and the chemiluminescent signal was quantitated using BioRad QuantityOne software analysis and specific proteins normalized to β-actin in each sample.

EXAMPLE 3

Western Blotting for SPK-1 Expression in siRNA Treated Cells

SCC cell lines (SCC-04, SCC-15, SCC-25 and SCC-71) from ATCC were seeded on six well plates. Lipofectamine™ 2000 was used according to the manufacturer's instructions to introduce siRNA (10-100 nM of HNB-001) into the SCC cells. Control SCC cells were transfected with lipofectamine only. Four hours post-transfection, the cells were returned to growth media (DMEM/10% FCS). Three days after transfection, the cells were harvested and washed once with cold PBS. The cell pellet was lysed in lysis buffer and the extracted protein was tested for SPK expression by Western Blotting. SPK expression in SCC cells treated with siRNA was reduced by 95% as compared to the control (FIG. 4, left image). This clearly demonstrates that, the siRNA inhibitor has silenced or knocked out the gene responsible for SPK expression. In addition, the sphingolipid rheostat teaches that the concentration of S1P is decreased while the concentration of ceramide is increased (FIG. 4, right image).

EXAMPLE 4

Viability of SCC-71 siRNA Transfected Cells

SCC-71 cells were seeded in T₂₅flasks. Lipofectamine™ 2000 was used according to the manufacturer's instructions to introduce siRNA (50 nM HNB-001) or lipofectamine into the cells. Four hours post-transfection, the cells were returned to growth media (DMEM/10% FCS) and two days later cell viability was determined using trypan blue staining. There was about a 35% decrease in viability for cells transfected with siRNA as compared to the lipofectamine transfected cells (FIG. 5). These results clearly demonstrates that siRNA (SPK specific inhibitor) may be used to knock SPK gene expression and induce apoptosis in cancer cells.

EXAMPLE 5

Comparison of the Viability of SCC-71 and SCC-15 siRNA Transfected Cells

SCC-71 and SCC-15 cells were compared to determine the viability of siRNA (100 nM HNB-002) transfected cells. This experiment was performed as in Example 4. The results show over a 50% reduction in cell viability of both the siRNA transfected SCC-15 and SCC-71 cells as compared to the lipofectamine transfected cells (FIG. 6).

EXAMPLE 6

Viability of siRNA Transfected MCF-7 Cells

The MCF-7 cell line, which is derived from estrogen sensitive human breast cancer, was used to demonstrate the effect of siRNA (50 and 100 nM HNB-001) on cell viability. Lipofectamine and GFP siRNA (250 nM) transfected MCF-7 cells were used as controls. The cell viability for both the lipofectamine and GFP siRNA transfected cells were the same (FIG. 7). In contrast, there was about a 20% and 50% reduction in cell viability for the 50 nM and 100 nM siRNA transfected cells respectively.

EXAMPLE 7

Viability of siRNA Transfected PC-3 Cells

The PC-3 cell line, which is derived from human prostate cancer, was used to demonstrate the effect of siRNA (50 nM and 100 nM HNB-001) on cell viability. Non-treated PC-3 and lipofectamine and GFP siRNA (250 nM) transfected cells) were used as controls (FIG. 8). The cell viability decreased by about 20% and 50% for 50 nM and 100 nM siRNA transfected cells respectively.

EXAMPLE 8

Viability of siRNA Transfected HT-29 Cells

The HT-29 cell line, which is derived from colon carcinoma, was used to demonstrate the effect of siRNA (100 nM HNB-001) on cell viability. Lipofectamine and GFP siRNA (250 nM) transfected cells) were used as controls (FIG. 9). Cell viability was decreased by about 20% in siRNA transfected cells.

EXAMPLE 9

Effect of siRNA on Tumor Size in Mice

In vivo experiments were performed in mice to determine whether the decrease in cell viability displayed by the various cell lines exemplified above (Examples 4-8) would occur in tumor bearing mice (Balb/C, athymic).
SCC-15 cells were transfected with either 250 nM of GFP siRNA or 50 nM HNB-001. The cells were transfected using Lipofectamine™ 2000 according to the manufactures instructions. Forty-eight hours after transfection, cells were counted using trypan blue staining. Two million viable cells were injected subcutaneously into mice. Tumors were allowed to grow in mice for 8 days and were measured on day 9 through day 18 using Digital calipers. The tumor volume was calculated using formula: a×b2 (squared)×0.52 (22).
By day 18, the tumors in group 1 had grown 250 times in volume from when they were first measured on day 9 (FIG. 11). Tumors in group II grew to a volume that was 250 times its size, while group II tumors grew 60 times in volume. Accordingly, about 75% of tumor growth was inhibited by treatment with 50 nM of siRNA (HNB-001) (FIGS. 10 and 11). Thus, the same cancer cell inhibition that occurs in various cell lines, also occur in mice.
Obviously, many modifications and variation of the invention as hereinbefore set forth can be made without departing from the spirit and scope thereof and therefore only such limitations should be imposed as are indicated by the appended claims.
All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

REFERENCES

1. Visentin B, Vekich J A, Sibbald B J, Cavalli A L, Moreno K M, Matteo R G, Garland W A, Lu Y, Yu S, Hall H S, Kundra V, Mills G B, Sabbadini R A. Validation of an anti-sphingosine-1-phosphate antibody as a potential therapeutic in reducing growth, invasion, and angiogenesis in multiple tumor lineages. Cancer Cell. 2006; 9:225-38
2. Evan G I, Vousden K H. Proliferation, cell cycle and apoptosis in cancer. Nature. 2001; 411:342-348.
3. Shu X, Wu W, Mosteller R D, Broek D. Sphingosine kinase mediates vascular endothelial growth factor-induced activation of ras and mitogen-activated protein kinases. Mole. Cell. Bio. 2002; 22:77587768
4. Sankaranarayanan, R., Masuyer, E., Swaminathan, R., Ferlay, J., & Whelan S. Head and neck cancer: a global perspective on epidemiology and prognosis. Anticancer Res. 1998; 18:4779-4786.
5. Brachman DG. Molecular biology of head and neck cancer. Semin Oncol. 1994; 21:320-329.
6. Doll F, Pfeilschifter J, Huwiler A. The epidermal growth factor stimulates sphingosine kinase-1 expression and activity in the human mammary carcinoma cell line MCF7. Biochim Biophys Acta. 2005; 1738:72-81.
7. Sarkar S, Maceyka M, Hait N C, Paugh S W, Sankala H, Milstien S, Spiegel S. Sphingosine kinase 1 is required for migration, proliferation and survival of MCF-7 human breast cancer cells. FEBS Lett. 2005; 579:5313-7.
8. Nava V E, Hobson J P, Murthy S, Milstien S, Spiegel S. Sphingosine kinase type 1 promotes estrogen-dependent tumorigenesis of breast cancer MCF-7 cells. Exp Cell Res. 2002; 281:115-27
9. Kohno M, Momoi M, Oo M L, Paik J H, Lee Y M, Venkataraman K, Ai Y, Ristimaki A P, Fyrst H, Sano H, Rosenberg D, Saba J D, Proia R L, Hla T. Intracellular role for sphingosine kinase 1 in intestinal adenoma cell proliferation. Mol Cell Biol. 2006; 26:7211-23.
10. Pchejetski D, Golzio M, Bonhoure E, Calvet C, Doumerc N, Garcia V, Mazerolles C, Rischmann P, Teissie J, Malavaud B, Cuvillier O. Sphingosine kinase-1 as a chemotherapy sensor in prostate adenocarcinoma cell and mouse models. Cancer Res. 2005; 65:11667-75.
11. Kawamori T, Osta W, Johnson K R, Pettus B J, Bielawski J, Tanaka T, Wargovich M J, Reddy B S, Hannun Y A, Obeid L M, Zhou D. Sphingosine kinase 1 is up-regulated in colon carcinogenesis. FASEB J. 2006; 20:386-8.
12. Johnson K R, Johnson K Y, Crellin H G, Ogretmen B, Boylan A M, Harley R A, Obeid L M. Immunohistochemical distribution of sphingosine kinase 1 in normal and tumor lung tissue. J Histochem Cytochem. 2005; 53:1159-66.
13. Le Scolan E, Pchejetski D, Banno Y, Denis N, Mayeux P, Vainchenker W, Levade T, Moreau-Gachelin F. Overexpression of sphingosine kinase 1 is an oncogenic event in erythroleukemic progression. Blood. 2005; 106:1808-16.
14. Mosteller R D, Broek D. VEGF receptor expression and signaling in human bladder tumors. Oncogene. 2003; 22:3361-70
15. French K J, Schrecengost R S, Lee B D, Zhuang Y, Smith S N, Eberly J L, Yun J K, Smith C D. Discovery and evaluation of inhibitors of human sphingosine kinase. Cancer Res. 2003; 63:5962-9.
16. Gamble J R, Xia P, Hahn C N, Drew J J, Drogemuller C J, Brown D, Vadas M A. Phenoxodiol, an experimental anticancer drug, shows potent antiangiogenic properties in addition to its antitumour effects. Int J. Cancer. 2006; 118:2412-20.
17. Kim J W, Kim Y W, Inagaki Y, Hwang Y A, Mitsutake S, Ryu Y W, Lee W K, Ha H J, Park C S, Igarashi Y. Synthesis and evaluation of sphingoid analogs as inhibitors of sphingosine kinases. Bioorg Med Chem. 2005; 13:3475-85
18. Edsall L C, Van Brocklyn J R, Cuvillier O, Kleuser B, Spiegel S. N,N-Dimethylsphingosine is a potent competitive inhibitor of sphingosine kinase but not of protein kinase C: modulation of cellular levels of sphingosine 1-phosphate and ceramide. Biochemistry. 1998; 37:12892-8
19. Kono K, Tanaka M, Ogita T, Kohama T. Characterization of B-5354c, a new sphingosine kinase inhibitor, produced by a marine bacterium. J Antibiot (Tokyo). 2000; 53:759-64
20. Kono K, Tanaka M, Ogita T, Hosoya T, Kohama T. F-12509A, a new sphingosine kinase inhibitor, produced by a discomycete. J Antibiot (Tokyo). 2000; 53:459-66.
21. Taha T A, Kitatanik K, El-Alwani M, Bielawski J, Hannum Y A, Obeid L H. Loss of sphingosine kinae-1 activities the inrinstic pathway of programmed cell ealth:modulation of sphingolipid levels of and the induction of apoptosis. FASEB J. 2-6:20:482-84.
22. Pchejetski D, Golzio M, Bonhoure E, Calvet C, Doumerc N, Garcia V, Mazerolles C, Rischmann P, Teissie J, Malavaud B, Cuvillier O. Sphinogsine Kinase-1 as a Chemotherapy Sensor in Prostate Adenocarcinoma Cell and Mouse Models. Cancer Research 2005, 65, 11667-675.
23. Atkins D, Reiffen K A, Tegtmeier C L, Winther H, Bonato M S, Storkel S. Immunohistochemical detection of EGFR in paraffin-embedded tumor tissues: variation in staining intensity due to choice of fixative and storage time of tissue sections. J Histochem Cytochem. 2004; 52:893-901.
24. Masood R, Kumar S R, Sinha U K, Crowe D L, Krasnoperov V, Reddy R K, Zozulya S, Singh J, Xia G, Broek D, Schonthal A H, Gill P S. EphB4 provides survival advantage to squamous cell carcinoma of the head and neck. Int J. Cancer. 2006 Sep. 15; 119(6):1236-48.

Claims

1. A composition comprising siRNA that specifically blocks the expression of sphingosine kinase and induces apoptosis in more than one type of cancer cell, wherein said cancer cell is a breast, prostate, colorectal, lung, bladder, head and neck, intestine, ovarian, skin or the like.

2. The composition according to claim 1, wherein said cancer cell is in a mammal.

3. The composition according to claim 1, wherein said siRNA comprises 19-25 oligonucleotides.

4. The composition according to claim 3, wherein said oligonucleotide sequence is double stranded and comprises the sense sequence of SEQ ID NO 1 and antisense sequence of SEQ ID NO 2.

5. The composition according to claim 4, wherein said sense sequence is SEQ ID NO 3 and antisense sequence is SEQ ID NO 4.

6. A therapeutic agent comprising siRNA, that specifically inhibits the expression of sphingosine kinase and induces apoptosis in more than one type of cancer cell, wherein said cancer cell is a breast, prostate, colorectal, lung, bladder, head and neck, intestine, ovarian, skin or the like.

7. The therapeutic agent according to claim 6 wherein siRNA comprises 19-25 oligonucleotides.

8. The therapeutic agent according to claim 6, wherein said siRNA is double stranded and comprises the sense sequence of SEQ ID NO: 1 and antisense sequence of SEQ ID NO: 2.

9. The therapeutic agent according to claim 6, wherein said siRNA is double stranded and comprises the sense sequence of SEQ ID NO: 3 and antisense sequence of SEQ ID NO: 4.

10. A composition comprising siRNA with a sense sequence comprising SEQ ID NO: 1 and an antisense sequence comprising SEQ ID NO: 2, and wherein said composition induces apoptosis in a variety of cancer cells.

11. A composition comprising siRNA with a sense sequence comprising SEQ ID NO: 3 and an antisense sequence comprising SEQ ID NO: 4, and wherein said composition induces apoptosis in a variety of cancer cells.

12. The composition according to claim 10, wherein said cancer cell is a breast, prostate, colorectal, lung, bladder, colorectal, head and neck, intestine, ovarian, skin or the like.

13. The composition according to claim 11, wherein said cancer cell is a breast, prostate, colorectal, lung, bladder, colorectal, head and neck, intestine, ovarian, skin or the like.

14. A therapeutic agent comprising a composition comprising siRNA with a sense sequence comprising SEQ ID NO: 1 and an antisense sequence comprising SEQ ID NO: 2, and wherein said composition induces apoptosis in a variety of cancer cells.

15. A therapeutic agent comprising a composition comprising siRNA with a sense sequence comprising SEQ ID NO: 3 and an antisense sequence comprising SEQ ID NO: 4, and wherein said composition induces apoptosis in a variety of cancer cells.

16. The composition according to claim 14, wherein said cancer cell is a breast, prostate, colorectal, lung, bladder, colorectal, head and neck, intestine, ovarian, skin or the like.

17. The composition according to claim 15, wherein said cancer cell is a breast, prostate, colorectal, lung, bladder, colorectal, head and neck, intestine, ovarian, skin or the like.