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Method for treating breast cancer by reducing expression of adenine nucleotide transporter 2 mRNA

An adenine nucleotide and transporter technology, applied in the field of breast cancer treatment, can solve problems such as side effects and achieve the effect of overcoming tolerance

Active Publication Date: 2016-02-17
BIOINFRA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the side effects of chemotherapy and radiotherapy are important issues in the treatment of cancer patients

Method used

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  • Method for treating breast cancer by reducing expression of adenine nucleotide transporter 2 mRNA
  • Method for treating breast cancer by reducing expression of adenine nucleotide transporter 2 mRNA
  • Method for treating breast cancer by reducing expression of adenine nucleotide transporter 2 mRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] Example 1: Effect of ANT2shRNA on HER2 / neu expression

[0106] The present inventors found that the introduction of ANT2 shRNA reduced the expression level of HER2 / neu on the surface of the breast cancer cell line SK-BR3 as measured by FACS (flow cytometry and cell sorting).

[0107] Lipo-fectamine TM 2000 (Invitrogen, a non-viral delivery system) was used to introduce ANT2 shRNA into the breast cancer cell line SK-BR3. In this regard, Lipofectamine TM The positively charged hydrophilic component of 2000 interacts with the negatively charged shRNA to form a complex. This complex fuses with the cell membrane, thereby delivering the shRNA into the cell.

[0108] For FACS analysis, HER2 / neu expressed on the cell surface was conjugated to an antibody [primary antibody: anti-human HER2 / neu antibody (Santa Cruz Biotechnology, Heidelberg, Germany)] followed by secondary antibody (FITC-conjugated anti-rabbit-IgG (SantaCruz Biotechnology, Heidelberg, Germany)) reacted. Beca...

Embodiment 2

[0113] Example 2: Effect of ANT2shRNA on Akt activity

[0114] In breast cancer cell line SK-BR3, highly expressed HER2 / neu activates Akt. Activated Akt is known in the art to be involved in breast cancer cell survival mechanisms and in breast cancer cell migration and invasion into other tissues. Activation of Akt can be determined by phosphorylation of tyrosine residues.

[0115] Activated Akt (phosphorylated) was quantified after ANT2 shRNA transfection into the human breast cancer cell line SK-BR3 [anti-Akt antibody and anti-phospho-Akt antibody (CellsignalingTech., Beverly, MA)]. Higher Akt activity maintained in breast cancer cell lines was reduced by introduction of ANT2 shRNA. 17-AAG, known to inhibit HSP90 and thus the HER2 / neu-mediated Akt signal transduction pathway, was used as a positive control ( image 3 A).

[0116] In addition, inhibition of Akt by ANT2 shRNA was determined by another experiment. The activity of phospho-Akt was decreased after knockdown o...

Embodiment 3

[0119] Example 3: Effect of ANT2shRNA on VEGF production

[0120] In the present invention, it was found that the introduction of ANT2 shRNA into the human breast cancer cell line SK-BR3 resulted in a decrease in Akt activity (inhibition of the PI3K / Akt signaling pathway), resulting in downregulation of VEGF, which is involved in angiogenesis around tumor cells.

[0121] Specifically, cells transfected with ANT2 shRNA were cultured for 24 hours, followed by isolation of total RNA. From the mRNA therein, cDNA was synthesized by reverse transcription with oligo-dT. The expression of VEGF was analyzed using primers specific for VEGF (vascular endothelial growth factor).

[0122] Such as Figure 4 As shown in A, knockdown of ANT2 by shRNA in the breast cancer cell line SK-BR3 was observed to decrease mRNA levels of VEGF, which is involved in angiogenesis, as measured by RT-PCR.

[0123] In addition, decreased intracellular VEGF protein levels were observed using FACS (flow cyto...

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Abstract

The present invention relates to a method for treating breast cancer by decreasing the expression of ANT2 (adenine nucleotide translocator 2) mRNA. Particularly, the present invention provides a composition for the treatment of breast cancer, comprising ANT2 siRNA or ANT2 shRNA as an active ingredient, characterized by ability to suppress metastasis of breast cancer cells. The present composition enhances an effect of TRAIL (TNF-related apoptosis-inducing ligand) for treating the breast cancer. Also, the present invention provides a composition for treating stem cells of breast cancer, comprising ANT2 (adenine nucleotide translocator 2) siRNA or ANT2 shRNA as an active ingredient. Therefore, the present invention provides a therapeutic for breast cancer, which is highly effective for suppressing the metastasis of breast cancer cells and overcoming drug resistance. Furthermore, the therapeutic is applicable to the prevention of stem cells of breast cancer.

Description

technical field [0001] The invention relates to a method for treating breast cancer by reducing the expression of adenine nucleotide transporter 2 mRNA. More specifically, the present invention relates to a method for treating breast cancer by ANT2siRNA (small interfering RNA) or ANT2shRNA (short hairpin RNA). Background technique [0002] Tumors result from abnormal, uncontrolled, and unregulated cell proliferation, including excessive abnormal cell proliferation. Tumors are classified as malignant when they exhibit destructive proliferation, infiltration and metastasis. Specifically, from the perspective of molecular biology, tumors are considered to be genetic diseases caused by gene mutations. For the treatment of malignant tumors, three methods of treatment, surgery, radiotherapy, and chemotherapy, have been performed alone or in combination. Specifically, surgery is a method of eradicating most of the disease-causing tissue, so it is very effective in removing tumor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/16C12N15/113A61P35/00
CPCA61P35/00A61P35/04A61P43/00C12N15/1138C12N2310/14C12N2310/531
Inventor 金哲右张支映
Owner BIOINFRA
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