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Compositions and methods of sphingosine kinase inhibitors in radiation therapy of various cancers

Inactive Publication Date: 2008-10-23
UNIV OF SOUTHERN CALIFORNIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0013]In a further embodiment, the invention relates to SPK inhibitors that silence or knock out the gene responsible for SPK over-expression potentiate the effect of radiation in a variety of cancer cells.
[0016]In yet another embodiment, the invention relates to compositions comprising SPK inhibitors that silence or knock out the gene responsible for SPK over-expression potentiate the effect of radiation in a variety of cancer cells.
[0018]In accordance with another embodiment, the invention relates to a method of using SPK inhibitors to potentiate the effect of radiation. The method comprises irradiating cancer cell lines that have been transfected with and without SPK inhibitors. If the cell viability percentage of irradiated SPK transfected cancer cells decreases more than the cell viability of irradiated cancer cells that have not been transfected with SPK inhibitor, then the SPK inhibitor has reduced the amount of radiation needed to decrease cancer cell viability and the effect of radiation has been potentiated.
[0019]In a closely related embodiment, the invention relates to a method of using SPK inhibitors to potentiate the effect of radiation in vivo. The method comprises injecting a mammal with cancer cells that have been transfected with and without SPK inhibitors and irradiating said mammal. If the tumor volume of the animal treated with the SPK inhibitor decreases more than the tumor volume of the mammal treated without SPK inhibitor, then the SPK inhibitor has reduced the amount of radiation needed to decrease tumor size in vivo and the effect of radiation has been potentiated.

Problems solved by technology

High doses of radiation is required to kill all the cancer cells, but the radiation injures both the tumor cells as well as normal tissues that are in its path and treatment causes many side effects.
Side effects include soreness, diarrhea, nausea, edema, infertility, fatigue, fibrosis, hair loss, dryness of mouth, or damage to salivary glands.
In some cases, radiation itself can lead to the formation of cancer.
Repetitive use high doses of radiation is limited because the chances of damage to vital organs such as the spinal cord increases as exposure to radiation increases.
However, the formation of S1P antagonizes the formation of ceramide.
However, these pharmacological agents exhibit moderate levels of antitumor activity or exhibit toxicity that makes the use of pharmacological inhibitors undesirable.

Method used

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  • Compositions and methods of sphingosine kinase inhibitors in radiation therapy of various cancers
  • Compositions and methods of sphingosine kinase inhibitors in radiation therapy of various cancers
  • Compositions and methods of sphingosine kinase inhibitors in radiation therapy of various cancers

Examples

Experimental program
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Effect test

example 1

Immunohistochemistry Staining for SPK Expression

[0058]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 (21). 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 stain...

example 2

Western Blotting for SPK Expression in Tumor Tissue

[0059]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.

[0060]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 us...

example 3

Western Blotting for SPK-1 Expression in siRNA Treated Cells

[0061]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 ...

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Abstract

The present invention relates to Sphingosine kinase inhibitors that are useful for treating various cancers. The invention further relates to compositions and methods of SPK inhibitors, including siRNAs, which specifically block gene expression of SPK and potentiates the effect of radiation in the treatment of various cancers.

Description

[0001]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[0002]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 in radiation therapy for the treatment of various cancers.BACKGROUND OF THE INVENTION[0003]There are millions of patients worldwide afflicted with cancer who are treated with radiation therapy. Radiation therapy is used as a primary therapy or in combination with surgery and / or chemotherapy and / or hormone therapy. Most common cancer types may be treated with radiation therapy in some way. The precise treatment or radiation dose depends upon the tumor type, location, stage, as well as the general health of the patient. In most cases, radiation therap...

Claims

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Application Information

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IPC IPC(8): A61K31/7105C07H21/02A61P35/00C12N15/113
CPCC12N15/1137C12N2310/14C12Y207/01091A61P31/00A61P35/00
Inventor SINHA, UTTAM K.MASOOD, RIZWAN
Owner UNIV OF SOUTHERN CALIFORNIA
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