WO2012111900A1 - Method for treating breast cancer by decreasing the expression of adenine nucleotide translocator 2 mrna - Google Patents
Method for treating breast cancer by decreasing the expression of adenine nucleotide translocator 2 mrna Download PDFInfo
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- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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Definitions
- the present invention relates to a method for treating breast cancer by decreasing the expression of adenine nucleotide translocator 2 mRNA. More specifically, the present invention relates to a method for treating breast cancer by ANT2 siRNA (small interfering RNA) or ANT2 shRNA (short hairpin RNA).
- ANT2 siRNA small interfering RNA
- ANT2 shRNA short hairpin RNA
- Tumor is a result of abnormal, incontrollable and disordered cell proliferation including excessive abnormal cell proliferation.
- a tumor exhibits destructive proliferation, infiltration and metastasis, it is classified as a malignant tumor.
- a tumor is considered as a genetic disease caused by mutation of a gene.
- three treatment methods which are surgical operation, radiotherapy and chemotherapy have been conducted either separately or together.
- surgical operation is a method to eliminate most of pathogenic tissues, which is thus very effective to remove tumors growing in the breast, colon and skin but not so effective to treat tumors in spine and dispersive tumors.
- Radiotherapy has been performed to treat acute inflammatory, benign or malignant tumors, endocrine disorders and allergies, and it has been effective to treat such malignant tumors resulted from abnormal rapid cell division.
- the ratio therapy carries serious side effects such as functional disorder or defect of normal cells, outbreak of cutaneous disorders on the treated area and particularly retardation and anostosis in children.
- Chemotherapy is a method to disturb duplication or metabolism of cancer cells, which has been performed to treat breast cancer, lung cancer and testicular tumor.
- the biggest problem of this treatment method is the side effect carried by systemic chemotherapy. Side effects of such chemotherapy are lethal and thus increase anxiety and fear for the treatment.
- One of the representative side effects of chemotherapy is dose limiting toxicity (DLT).
- DLT dose limiting toxicity
- Mucositis is one of examples of DLT for various anticancer agents (antimetabolic agents such as 5-fluorouracil and methotrexate, and antitumor antibiotics such as doxorubicin).
- anticancer agents antimetabolic agents such as 5-fluorouracil and methotrexate, and antitumor antibiotics such as doxorubicin.
- Most cases of side effects require hospitalization or at least need pain killers. So, side effects by chemotherapy and radiotherapy are such as are serious matters for the treatment of cancer patients.
- gene therapy is based on the DNA recombination technique, which is the method to insert a therapeutic gene into cancer patient cells to correct gene defect or to endow a novel functions to disordered cells to treat or prevent various genetic diseases caused by mutations diseases, infective of genes, diseases, cancer, autoimmune cardiovascular diseases, etc. More particularly, gene therapy is a method to treat the said diseases by inducing intracellular expressions of normal proteins or therapeutic target proteins by conveying a therapeutic gene into a target organ. Gene therapy has an excellent selectivity, compared with other treatment methods using drugs and can be applied for a long term with improved treatment effect on difficult diseases. To enhance the therapeutic effect of gene therapy, gene transfer technique is essential for the realization of high efficient gene expression in target cells.
- a gene carrier is a mediator for the insertion of a therapeutic gene into a target cell.
- a preferable gene carrier is the one that is not harmful for human, can be mass-produced and has ability to transmit a therapeutic gene effectively and induce constant expression of the therapeutic gene.
- gene transfer technique is a key factor for gene therapy and representative gene carriers most wanted for gene therapy so far are exemplified by viral carriers such as adenovirus, adeno-associated virus (AAV), and retrovirus; and non-viral carriers such as liposome and polyethyleneimine.
- the method using a tumor suppressor gene is to treat cancer by conveying the original form of a tumor suppressor gene such as p53, which is deficient or mutated in many cancer patients.
- the method using a tumor-specific killer virus is to treat cancer by conveying a virus gene carrier that can be proliferated selectively in tumor cells into cancer patients by taking advantage of the activity of a tumor suppressor gene transformed in cancer tissues.
- the basic strategy of the above two methods is to kill tumor cells directly.
- the method using a suicide gene is to induce suicide of tumor cells by inserting sensitive genes such as HSK-TK.
- the method using an immunoregulation gene is to treat disease indirectly by stimulating T-cell mediated tumor cell recognition by delivering a gene increasing antitumor immune response such as interleukin 12 (IL12) , interleukin 4 (IL4) , interleukin 7 (IL7), ⁇ -interferon and tumor necrosis factor (TNF) or by inducing apoptosis by interrupting tumor inducing proteins.
- IL12 interleukin 12
- IL4 interleukin 4
- IL7 interleukin 7
- TNF tumor necrosis factor
- ANT 2 adenine nucleotide translocator 2
- ANT (adenine nucleotide translocator) is an enzyme found in inner membrane (IM) of mitochondria, which imports ADP from cytoplasm through VDAC (voltage dependent anion channel) of outer membrane (OM) of mitochondria and exports ATP generated in electron transfer chain system into cytoplasm (HLA Vieira, et ale , Cell Death and Differentiation, 7, 1146-1154, 2000).
- ANT playing a key role in energy metabolism of cells is classified into ANTI, ANT2 and ANT3.
- Particularly ANT2 exhibits low expression rate in normal cells but is highly expressed in cancer cells or similarly highly proliferated cells, which seems to be closely related to glycolysis under anaerobic condition, so that ANT2 is rising up as a new target for cancer treatment (Chevrollier, A, et al., Med. Sci., 21 (2), 156-161, 2005).
- the object of the present invention is to provide a composition for treating breast cancer and a method for treating stem cells of breast cancer.
- the present invention provides a composition for treating breast cancer or stem cells of breast cancer comprising ANT2 siRNA or ANT2 shRNA as an active ingredient.
- the present invention provides a composition for treating breast cancer, comprising adenine nucleotide translocator 2 (ANT2) small interfering RNA (siRNA) or adenine nucleotide translocator 2 (ANT2) short hairpin RNA (shRNA)as an active ingredient, characterized by ability to suppress metastasis of breast cancer cells.
- ANT2 adenine nucleotide translocator 2
- siRNA small interfering RNA
- ANT2 adenine nucleotide translocator 2
- shRNA short hairpin RNA
- the ANT2 siRNA or ANT2 shRNA induces degradation of ANT2 mRNA by interacting an anti-sense sequence represented by SEQ ID NO:3 with a sense sequence represented by SEQ ID NO:1.
- the composition suppresses the expression of HER2/neu (human epidermal growth factor receptor 2).
- the composition enhances an effect of TNF-related apoptosis-inducing ligand (TRAIL) for treating the breast cancer .
- TRAIL TNF-related apoptosis-inducing ligand
- the composition enhances an expression of death receptor 4 (DR4) and death receptor 5 (DR5) and suppresses an expression of death decoy receptor 1 (DcR1) and death decoy receptor 2 (DcR2).
- DR4 death receptor 4
- DcR1 death decoy receptor 1
- DcR2 death decoy receptor 2
- the composition increases the expression and activity of p53 protein.
- the present invention also provides a composition for treating stem cells of breast cancer comprising ANT2 siRNA or ANT2 shRNA as an active ingredient.
- the composition inhibits expression and activity of ATP-binding cassette sub-family G member 2 (ABCG2).
- the composition when the present composition is coadministered with an anti-cancer agent, the composition enhances the effect of the anti-cancer agent by improving the response of the stem cells to the anti-cancer agent and reducing development of tolerance toward the anti-cancer agent.
- the anti-cancer agent comprises doxorubicin.
- the present invention also provides a method for treating breast cancer comprising administering to a patient in need ANT2 siRNA or ANT2 shRNA as an active ingredient, which is characterized in inhibiting metastasis of breast cancer cells.
- the present invention also provides a method for treating stem cells of breast cancer, comprising administering to a patient in need ANT2 siRNA or ANT2 shRNA.
- the ANT2 siRNA or ATN2 shRNA according to the present invention decreases the expression of ANT2 mRNA and thus effectively induces cell death.
- the present invention gives a solution to TRAIL resistance.
- the ANT2 siRNA or ATN2 shRNA according to the present invention inhibits the metastasis of breast cancer cells and is effective to treat stem cells of breast cancer.
- the present invention provides therapeutics for breast cancer, which are highly effective for inhibiting the metastasis of breast cancer cells and overcoming the tolerance toward an anti-cancer agent. Further, the present invention could be used to develop therapeutics for stem cells of breast cancer.
- FIG. 1 shows that when the breast cancer cell line SK-BR3 overexpressing HER2/neu is transfected with ANT2 shRNA, its HER2/neu expression level is reduced.
- FIG. 2A shows that when the breast cancer cell line SK-BR3 overexpressing HER2/neu is transfected with ANT2 shRNA, the introduced ANT2 shRNA promotes the degradation of HSP90 resulting in the reduction of the expression level of HSP90.
- FIG. 2B shows that when the breast cancer cell line SK-BR3 overexpressing HER2/neu is transfected with ANT2 shRNA, its HER2 expression level is reduced.
- FIG. 3A shows that when the breast cancer cell line SK-BR3 overexpressing HER2/neu is transfected with ANT2 shRNA, its Akt activity is reduced.
- FIG. 3B shows that the breast cancer cell line SK-BR3 overexpressing HER2/neu is decreased in Akt activity when transfected with ANT2 shRNA, and recovers its Akt activity by ANT2 overexpression.
- FIG. 3C shows that when the breast cancer cell line SK-BR3 overexpressing HER2/neu is transfected with ANT2 shRNA, HSP90 weakly interacts with Akt and the activity of Akt is reduced.
- FIG. 4A shows that when the breast cancer cell line SK-BR3 overexpressing HER2/neu is transfected with ANT2 shRNA, there is a decrease in the mRNA expression level of VEGF.
- FIG. 4B shows that when the breast cancer cell line SK-BR3 overexpressing HER2/neu is transfected with ANT2 shRNA, there is a decrease in the intracellular level of VEGF.
- FIG. 4C shows that the breast cancer cell line SK-BR3 overexpressing HER2/neu is decreased in the mRNA expression level of VEGF when transfected with ANT2 shRNA, and recovers VEGF mRNA levels by the overexpression of ANT2.
- FIG. 5A shows that when the breast cancer cell line SK-BR3 overexpressing HER2/neu is transfected with ANT2 shRNA, there is a decrease in the mRNA expression level of MT1-MMP.
- FIG. 5B shows that when the breast cancer cell line SK-BR3 overexpressing HER2/neu is transfected with ANT2 shRNA, there is a decrease in the protein expression level of MT1-MMP.
- FIG. 6A shows that the breast cancer cell line SK-BR3 overexpressing HER2/neu is decreased in the mRNA expression level of MT1-MMP when transfected with ANT2 shRNA, and recovers the mRNA expression level by the overexpression of PI3K.
- FIG. 6B shows that the breast cancer cell line SK-BR3 overexpressing HER2/neu is decreased in the protein expression level of MT1-MMP when transfected with ANT2 shRNA, and recovers the protein expression level by the overexpression of PI3K.
- FIG. 7A shows that when the breast cancer cell line SK-BR3 overexpressing HER2/neu is transfected with ANT2 shRNA, there is a decrease in the mRNA expression level of both MMP2 and MMP9.
- FIG. 7B shows that when the breast cancer cell line SK-BR3 overexpressing HER2/neu is transfected with ANT2 shRNA, there is a decrease in the activity of both MMP2 and MMP9.
- FIG. 8A shows that when the breast cancer cell line SK-BR3 overexpressing HER2/neu is transfected with ANT2 shRNA, its invasion ability is reduced.
- FIG. 8B shows that when the breast cancer cell line SK-BR3 overexpressing HER2/neu is transfected with ANT2 shRNA, its migration ability is reduced.
- FIG. 9A shows a result of the resistance of MCF7 and the sensitivity (apoptosis) of MDA-MB-231 to TRAIL.
- FIG. 9B shows a result of Western blotting, wherein while the breast cancer cell line MCF7 is resistant to TRAIL, the introduction of ANT2 shRNA induces the sensitivity of MCF7 to TRAIL (apoptosis).
- FIG. 9C shows that the breast cancer cell line MCF7 is resistant to TRAIL, but the introduction of ANT2 shRNA induces the cells to undergo apoptosis as observed in cell photographs.
- FIG. 10A shows that the breast cancer cell lines T47D and BT474 are resistant to TRAIL, but the introduction of ANT2 shRNA effectively induces the cells to undergo apoptosis.
- FIG. 10B shows that the breast cancer cell line MCF7 is resistant to TRAIL, but the introduction of ANT2 shRNA makes the cells highly sensitive to TRAIL (apoptosis) and the overexpression of ANT2 by transfection with an ANT2 expression vector eliminates the apoptotic effect of ANT2 shRNA.
- FIG. 10C shows that the breast cancer cell lines T47D and BT474 are resistant to TRAIL, but they are induced to undergo TRAIL-mediated apoptosis when transfected with ANT2 shRNA, with the concomitant accompaniment of events including the cleavage of caspase-8/9/7, bid truncation, and increased release of cytochrome c due to mitochondrial destruction.
- FIG. 11A shows that the breast cancer cell line MCF7, resistant to TRAIL, is low in the expression level of the TRAIL receptors DR4 and DR5 and high in the expression level of the decoy receptors DcR1 and DcR2, which interfere with the TRAIL signaling pathway and that the breast cancer cell line MDA-MB-231, sensitive to TRAIL, is high in the expression level of the TRAIL receptors DR4 and DR5 and low in the expression level of the decoy receptors DcR1 and DcR2.
- FIG. 11B shows that the breast cancer cell line MCF7, resistant to TRAIL, is low in the expression level of the TRAIL receptors DR4 and DR5 and high in the expression level of the decoy receptors DcR1 and DcR2, the introduction of ANT2 shRNA increases the expression of DR5 and decreases the expression of DcR2.
- FIG. 11C shows that the breast cancer cell line MDA-MB-231, sensitive to TRAIL, is low in the expression level of the TRAIL receptors DR4 and DR5 and high in the expression level of the decoy receptors DcR1 and DcR2, the introduction of ANT2 shRNA increases the expression of DR5 and decreases the expression of DcR2.
- FIG. 12A shows that the breast cancer cell line MCF7, resistant to TRAIL, is increased in the expression and phosphorylation/activation of p53 protein when transfected with ANT2 shRNA.
- FIG. 12B shows that the breast cancer cell line MCF7, resistant to TRAIL, is increased in the transcriptional activity of p53 protein when transfected with ANT2 shRNA.
- FIG. 12C shows that the breast cancer cell line MCF7, resistant to TRAIL, is increased in the expression level of the TRAIL receptors DR4 and DR5 when transfected with ANT2 shRNA, indicating that the increased expression is attributed to the activation of p53.
- FIG. 13 shows that the breast cancer cell line MCF7, resistant to TRAIL, is increased in sensitivity to TRAIL when transfected with ANT2 shRNA, indicating that the increased sensitivity is attributed to the activation of p53.
- FIG. 14A shows that after the breast cancer cell line MCF7 is transplanted to immunodeficient Balb/c nude mice, the growth of the resulting tumor was slightly suppressed by treatment with TRAIL alone, moderately by treatment with ANT2 shRNA alone, and greatly by treatment with a combination of TRAIL and ANT2 shRNA.
- FIG. 14B shows that after the breast cancer cell line MCF7 is transplanted to immunodeficient Balb/c nude mice, the growth of the resulting tumor was greatly suppressed by treatment with a combination of TRAIL and ANT2 shRNA and the tumor increased in the expression level of the TRAIL receptors DR4 and DR5.
- FIG. 15A shows a result of RT-PCR, wherein the progenitor cells (CD44+/CD24-) sorted from the breast cancer cell lines MCF7 and MDA-MB-231 and unsorted cells are found to express high levels of ANT2.
- FIG. 15B shows a result of real-time RT-qPCR, wherein the progenitor cells (CD44+/CD24-) sorted from the breast cancer cell lines MCF7 and MDA-MB-231 and unsorted cells are found to express high levels of ANT2.
- FIG. 16A shows the morphological change when the normal breast epithelial cell line MCF10A is mesenchymally transdifferentiated by transfection with E-cadherin shRNA.
- FIG. 16B shows that when the normal breast epithelial cell line MCF10A is mesenchymally transdifferentiated by transfection with E-cadherin shRNA, the cells are decreased in the expression level of E-cadherin and increased in the expression level of ANT2.
- FIG. 17A shows that when the progenitor cells (CD44+/CD24-) isolated from the breast cancer cell line MDA-MB-231 are transfected with ANT2 shRNA, they are effectively induced to undergo apoptosis.
- FIG. 17B shows that when the progenitor cells (CD44+/CD24-) isolated from the breast cancer cell line MCF7 are transfected with ANT2 shRNA, the progenitor cells are effectively induced to undergo apoptosis.
- FIG. 18A shows that when the normal breast epithelial cell line MCF10A is transfected with ANT2 shRNA, the breast epithelial cell line is not induced to undergo apoptosis.
- FIG. 18B shows that when the normal breast epithelial cell line MCF10A is mesenchymally transdifferentiated by transfection with E-cadherin shRNA, the introduction of ANT2 shRNA can effectively induce apoptosis.
- FIG. 19A shows that MDA-MB-231 with a high proportion of the breast cancer progenitor cells (CD44+/CD24-) and MCF7 with a low proportion of the breast cancer progenitor cells (CD44+/CD24-) show high and low abilities to form cell masses, respectively, and that the ability to form cell masses is reduced in both the cells by the transfection with ANT2 shRNA.
- FIG. 19B shows that MDA-MB-231 with a high proportion of the breast cancer progenitor cells (CD44+/CD24-) and MCF7 with a low proportion of the breast cancer progenitor cells (CD44+/CD24-) show high and low abilities to form cell masses, respectively, and that the ability to form cell masses is reduced in both the cells by the transfection with ANT2 shRNA.
- FIG. 20 shows that MDA-MB-231 treated with ANT2 shRNA, wherein while MDA-MB-231 with a high proportion of the breast cancer progenitor cells (CD44+/CD24-) is highly resistant to doxorubicin, the introduction of ANT2 shRNA makes the cells, whether sorted for CD44+/CD24- cells or unsorted, sensitive to doxorubicin.
- FIG. 21 shows the result of low doxorubicin resistance breast cancer cell lines, MCF7 treated with ANT2 shRNA, wherein MCF7 with a low proportion of the breast cancer progenitor cells (CD44+/CD24-) is sensitive to doxorubicin (adherent cells) while only the progenitor cells (CD44+/CD24-) shows resistance to doxorubicin (floating cells) and that the introduction of ANT2 shRNA makes the cells, whether sorted for CD44+/CD24- cells or unsorted, sensitive to doxorubicin.
- FIG. 22 shows that the progenitor cells (CD44+/CD24-) isolated from both the breast cancer cell lines MCF7 and MDA-MB-231 are high in the mRNA expression level of ABCG2 involved in drug resistance, in contrast to unsorted cells (left panel) and that when the normal breast epithelial cell line MCF10A is mesenchymally transdifferentiated by transfection with E-cadherin shRNA, the resulting cells are increased in the mRNA level of ABCG2 (right panel).
- FIG. 23 shows that the progenitor cells (CD44+/CD24-) isolated from both the breast cancer cell lines MCF7 and MDA-MB-231 are high in the protein expression level of ABCG2 involved in drug resistance, in contrast to unsorted cells, and decreased in the protein expression level of ABCG2 when transfected with ANT2 shRNA and that when the normal breast epithelial cell line MCF10A is mesenchymally transdifferentiated by transfection with E-cadherin shRNA, the resulting cells are increased in the protein level of ABCG2.
- FIG. 24 shows that the progenitor cells (CD44+/CD24-) isolated from both the breast cancer cell lines MCF7 and MDA-MB-231 are higher in the activity of ABCG2 involved in drug resistance than are unsorted cells and that when the normal breast epithelial cell line MCF10A is mesenchymally transdifferentiated by transfection with E-cadherin shRNA, the resulting cells are increased in the activity of ABCG2.
- FIG. 25 shows that the progenitor cells (CD44+/CD24-) isolated from both the breast cancer cell lines MCF7 and MDA-MB-231 are higher in the activity of ABCG2 involved in drug resistance than are unsorted cells, the introduction of ANT2 shRNA decreases the activity of ABCG2 and that the cells mesenchymally transdifferentiated from the normal breast epithelial cell line MCF10A by transfection with E-cadherin shRNA show an increased activity of ABCG2, but are decreased in the activity of ABCG2 by introduction of ANT2 shRNA thereinto.
- FIG. 26 shows that the nanocomplexes targeting CD44, which is highly expression on the surface of progenitor cells of breast cancer, selectively delivers ANT2 shRNA to the cell line expressing CD44.
- the present invention provides a composition for treating breast cancer comprising ANT2 siRNA or ANT2 shRNA as an active ingredient, which is characterized in inhibiting the metastasis of breast cancer cells.
- ANT2 siRNA or ANT2 shRNA inhibits the migration and invasion of breast cancer cells into other tissues.
- Breast cancer cells are known to activate certain signal transduction pathways through the cell receptor EGFR (epidermal growth factor receptor) for their proliferation, survival, and migration and invasion into other tissues.
- EGFR epidermal growth factor receptor
- HER2/neu a member of the EGFR family
- HER2/neu is known to be overexpressed on the surface of the cancer cells, up to 30% of breast cancer patients.
- the overexpression of HER2/neu gives rise to a poor prognosis for breast cancer patients and shows multi-drug resistance in chemotherapy.
- Representative among the HER2/neu-mediated signal transduction pathway is the PI3K/Akt signaling pathway, the activation of which causes breast cancer cells to proliferate, survive, and migrate and invade into other tissues. Therefore, the treatment of breast cancer has focused on down-regulating the expression of HER2/neu or interfering with the activity of the HER2/neu-mediated signaling pathway.
- the present invention provides a method wherein, when the expression of the ANT2 gene, which is known to be overexpressed in breast cancer cells and plays a crucial role in the ATP production of breast cancer cells, is inhibited by ANT2 siRNA or ANT2 shRNA.
- the cancer cells are not only induced to undergo cell death due to an insufficient supply of the energy necessary for the survival thereof, but also prevented from migration or invasion into other tissues, resulting in the suppression of cancer metastasis.
- HSP90 heat shock protein 90
- HER2/neu his shock protein 90
- the present invention expected that when the intracellular ATP level of breast cancer cells is depleted by ANT2 siRNA or ANT2 shRNA, HSP90 cannot bind sufficiently to ATP and thus cannot perform its functions, leading to the degradation of HER2/neu. From this expectation, the present inventors framed a hypothesis that the degradation of HER2/neu causes a decrease in the activity of HER2/neu-mediated PI3K/Akt signaling pathway, resulting in weakening the ability of breast cancer cells to migrate and invade into other tissues.
- the human breast cancer cell line SK-BR3, known to overexpress HER2/neu was induced to decrease in ANT2 expression level by the transfection of ANT2 shRNA thereinto. It was observed that the lack of intracellular ATP decreases HSP90 activity, resulting in suppressed HER2/neu expression. In addition, it was experimentally proved that the HER2/neu-mediated PI3K/Akt signaling pathway is inhibited, resulting in suppressing the ability of breast cancer cells to migrate and invade into other tissues.
- the present invention provides a pharmaceutical composition for the treatment of breast cancer, comprising ANT2 siRNA or ANT2 shRNA as an active ingredient. It is designed to control the expression of TRAIL receptors to increase the therapeutic effect of TRAIL on breast cancer.
- the TRAIL/apo2 ligand a member of tumor necrosis factor, can selectively kill cancer cells, but not normal cells.
- TRAIL a member of tumor necrosis factor
- chemotherapy makes cancer cells resistant to TRAIL.
- This resistance is currently a popular research topic. Accordingly, the present inventors tried to slove the resistance problem using ANT2 shRNA.
- the present invention presents that the introduction of ANT2 siRNA or ANT2 shRNA into breast cancer cells promotes TRAIL-induced apoptosis of tumor cells and amplifies the suppressive effect of TRAIL on tumor growth both in cell culture conditions (in vitro) and in animals (in vivo).
- the regulation of TRAIL receptor expression was found to account for the mechanism of ANT2 siRNA or ANT2 shRNA in promoting the TRAIL-induced apoptosis of breast cancer cells.
- TRAIL receptors there are DR4 (TRAILR1; TRAIL receptor 1) and DR5 (TRAILR2: TRAIL receptor 2).
- TRAIL When TRAIL binds to the DR4 and DR5, the apoptosis signal is transduced into the cancer cell, so that the cell dies.
- the TRAIL receptors DcR1 (decoy receptors 1: TRAILR3) and DcR2 (decoy receptors 2: TRAILR4) function to protect tumor cells from DR4 and DR5-mediated apoptosis signals. That is, if TRAIL binds to DcR1 and DcR2, the apoptosis signal is not transduced into the tumor cells. There are various reasons for the resistance of tumor cells to TRAIL.
- DcR1 and DcR2 are up-regulated on the surface of tumor cells while DR4 and DR5 is down-regulated.
- the present inventors have verified that ANT2 siRNA or ANT2 shRNA cause an alteration in the intracellular signal transduction pathways of cancer cells to up-regulate DR4 and DR5 with the concomitant down-regulation of DcR1 and DcR2, thus increasing the sensitivity of tumor cells to TRAIL and overcoming the drug resistance of tumor cells.
- the present invention provide a method wherein, through a cell culture experiment (in vitro) and an animal test (in vivo) that the knock-down of ANT2 by ANT2 siRNA or ANT2 shRNA interference promotes the therapeutic effect of TRAIL on breast cancer and gives a solution to the problem of resistance to TRAIL, which is based on the regulation of TRAIL receptor expression.
- the present invention provides a composition for the treatment of stem cells of breast cancer, comprising ANT2 siRNA or ANT2 shRNA as an active ingredient.
- cancer stem cells e.g., breast cancer stem cells. It is therefore very important to effectively eliminate breast cancer stem cells.
- ANT2 shRNA was proven to be able to kill breast cancer stem cells.
- breast cancer cell line-derived side populations CD44+/CD24-
- breast cancer cell line-derived side populations CD44+/CD24-
- MCF10A breast epithelial cells mesenchymally-transdifferentiated through E-cadherin knockdown were used. This was on the basis of the fact that when transdifferentiated from epitheilial cells (epithelial-to-mesenchymal transition, EMT), the mesenchymal cells show characteristics of breast cancer stem cells.
- EMT epitheilial cells
- an adenovirus system was utilized to effectively carry ANT2 shRNA into breast cancer stem cells.
- adenovirus expressing ANT2 shRNA was constructed and infected into breast cancer stem cells.
- ANT2 shRNA efficiently induced apoptotic cell death in both of the two cell groups of breast cancer stem cells characteristic.
- ANT2 shRNA solved the problem of resistance and thus sensitized both cells to the drug.
- the composition enhances the effect of the anti-cancer agent by improving the response of the stem cells to the anti-cancer agent and reducing development of tolerance toward the anti-cancer agent.
- the anti-cancer agent comprises doxorubicin.
- the present invention also provides a method for the treatment of breast cancer comprising administering to a patient in need ANT2 siRNA or ANT2 shRNA as an active ingredient, which is characterized in inhibiting metastasis of breast cancer cells.
- the present invention also provides a method for treatment of stem cells of breast cancer, comprising administering to a patient in need ANT2 siRNA or ANT2 shRNA.
- the administration dose vary depending on the patient s weight, age, gender, health state and diet, frequency of administration, route of administration, and rate of the excretion of the composition, and the severity of disease.
- ANT2 mRNA sequence (GenBank Accession No. NM_001152) was obtained from National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/).
- the candidate sets of ANT2 siRNA were obtained by introducing the above ANT2 mRNA sequence to siRNA prediction program (http://www.ambion.com/technical, resources/siRNA target finder).
- siRNA binding to sequence 5'-GCAGAUCACUGCAGAUAAG-3' corresponding to second exon of the ANT2 mRNA, was prepared to detect the silencing effect on ANT2 mRNA in vitro test. [The synthesis of ANT2 siRNA was conducted at Bioneer (Korea)].
- ANT2 shRNA was prepared. Specifically, two stranded oligomers with following sequences were synthesized. [The synthesis of ANT2 siRNA was conducted at Bioneer (Korea)].
- SEQ ID NO 1 5'-GCAGAUCACUGCAGAUAAGUU-3' (sense sequence of ANT2 shRNA)
- SEQ ID NO 2 5'-UUCAAGAGA-3' (loop sequence of ANT2 shRNA)
- SEQ ID NO 3 5'-AACUUAUCUGCAGUGAUCUGC-3' (anti-sense sequence of ANT2 shRNA)
- ANT2 shRNA was found to decrease 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).
- Lipofectamine TM 2000 (Invitrogen), a non-viral delivery system, was used.
- the positively charged, hydrophilic components of Lipofectamine TM 2000 interact with the negative charged shRNA to form a complex. This complex fuses with the cell membranes to deliver the shRNA into the cell.
- HER2/neu expressed on cell surfaces was bound with a antibody [primary antibody: anti-human HER2/neu antibody(Santa Cruz biotechnology, Heidelberg, Germany)] which was subsequently reacted with a secondary antibody (FITC-conjugated-anti--rabbit-IgG(Santa Cruz biotechnology, Heidelberg, Germany)). Because the secondary antibody was conjugated with a fluorescent material, its detection by FACS allowed the HER2/neu-expressed cells to be sorted from non-expressed cells.
- SK-BR3 expressed a high expression level of HER2/neu (sc shRNA) whereas the expression of HER2/neu was down-regulated by the introduction of ANT2 shRNA into cells (FIG. 1).
- proteins were extracted from the human breast cancer cell line SK-BR3 transfected with ANT2 shRNA.
- the proteins were immunoprecipitated with anti-human HER2/neu antibody (Santa Cruz biotechnology, Heidelberg, Germany), followed by Western blotting with an anti-human HSP90 antibody (Santa Cruz biotechnology, Heidelberg, Germany) to determine protein expression levels.
- anti-human HER2/neu antibody Santa Cruz biotechnology, Heidelberg, Germany
- Western blotting with an anti-human HSP90 antibody Santa Cruz biotechnology, Heidelberg, Germany
- ANT2 shRNA When the expression of ANT2 was down regulated by ANT2 shRNA, as shown in FIG. 2, weak interaction between HER2/neu and HSP90 was detected while an increased level of ubiquitination, a process of marking a protein with ubiquitin before degradation, was detected. That is, the knockdown of ANT2 by shRNA induced a decreased interaction between HER2/neu and HSP90 to increase the ubiquitination of HER2/neu, resulting in a decrease in the expression level of HER2/neu.
- an HSP inhibitor/17-AAG (17-allylamino-17-demethoxygeldanamycin (A.G. Scientific Inc., San Diego, CA)
- Beta-actin a cytoskeletal protein
- Beta-actin was used as a loading control because it generally remains the same proportion of total cell protein except under circumstances that experimental conditions are dramatically affecting cytoskeletal rearrangement or cell adhesion.
- an anti-beta-actin antibody (Cell signaling Tech., Beverly, MA) was used.
- Akt In the breast cancer cell line SK-BR3, highly expressed HER2/neu activates Akt.
- the activated Akt is known to be involved in the mechanism of survival of breast cancer cells as well as in the migration and invasion of breast cancer cells into other tissues.
- the activation of Akt can be determined by phosphorylation at tyrosine residues.
- HSP90 was immunoprecipitated with the antibody [HSP inhibitor/17-AAG (17-allylamino-17-demethoxygeldanamycin): A.G. Scientific Inc. (San Diego, CA)] followed by Western blotting with anti-Akt antibody (Cell signaling Tech. , Beverly, MA) to determine the expression level.
- ANT2 shRNA was found to interfere with the interaction between HSP90 and Akt and decrease the level of phospho-Akt (FIG. 3C). The data indicate that the suppression of HSP90 activity by ANT2 shRNA leads to a weak interaction between HSP90 and Akt as well as a reduced activity of Akt.
- ANT2 shRNA introduced into the human breast cancer cell line SK-BR3 causes a reduction in Akt activity (suppressing the PI3K/Akt signaling pathway), resulting in the down-regulation of VEGF, which is involved in angiogenesis around tumor cells.
- RNAs transfected with ANT2 shRNA were cultured for 24 hrs, followed by isolation of total RNAs. From the mRNA of them, cDNA was synthesized through reverse transcription with oligo-dT. Using primers specific for VEGF (vascular endothelial growth factor), the expression of VEGF was analyzed.
- VEGF vascular endothelial growth factor
- the knockdown of ANT2 by shRNA in the breast cancer cell line SK-BR3 was observed to reduce the mRNA level of VEGF, which is involved in angiogenesis, as measured by RT-PCR.
- VEGF protein levels were decreased using FACS (flow cytometry and cell sorting). VEGF is produced within cells and released from cells. To prevent this extracellular secretion, the cells were treated with BFA (brefeldin A). When VEGF protein was accumulated, its amounts were compared. In this regard, after the intracellular accumulation of VEGF by treatment with BFA, the cell membranes were perforated to form holes through which the primary anti-VEGF antibody (BD Pharmingen, San Diego, CA) was introduced into the cells.
- BFA feldin A
- a secondary antibody [FITC-conjugated-anti-mouse IgG antibody (BD Pharmingen, San Diego, CA)] was allowed to bind to the primary antibody complexed with the VEGF, followed by FACS analysis (FIG. 4B).
- ANT2 shRNA was experimentally reconfirmed to down-regulate the mRNA expression of VEGF.
- the introduction of an ANT2-overexpressing vector (pcDNA3.0-ANT2 expression vector) increased the expression of ANT2, with the concomitant recovery of VEGF mRNA levels.
- An inhibitory drug (LY294002) against PI3K that induces the phosphorylation of Akt (FIG. 4C) was used as a positive control, on the basis of the fact that the mRNA expression of VEGF is induced by the activation of the PI3K/Akt signal transduction pathway.
- MMPs MT1-MMP, known to be controlled in expression by VEGF, and MMP2 and MMP9, both known as being controlled in activity by MT1-MMP, were examined for expression and activity.
- the breast cancer cell line SK-BR3 transfected with ANT2 shRNA was found to decrease in the mRNA level of MT1-MMP, which is responsible for the degradation of ECM (extracellular matrix), as measured by RT-PCR (FIG. 5A), and also in the protein level, as measured by Western blotting (FIG. 5B). Furthermore, ANT2 shRNA was re-confirmed to decrease the mRNA expression of MT1-MMP.
- a PI3K-overexpressing vector [wild-type PI3K/p110 vector (provided by Dr.
- MT1-MMP was introduced to artificially activate the PI3K/Akt signal transduction pathway, MT1-MMP was observed to be recovered in mRNA level, as measured by RT-PCR (FIG. 6A), and in protein level, as measured by Western blotting (FIG. 6B).
- RT-PCR showed that the introduction of ANT2 shRNA into the breast cancer cell line SK-BR3 decreased the level of MMP2 mRNA and MMP9 mRNA, both controlled by MTI-MMP (FIG. 7A).
- GAPDH mRNA was used as a loading control to show the uniform quantities of the mRNAs used because GAPDH remains constant irrespective of intra- and extracellular conditions.
- the introduction of ANT2 shRNA into the breast cancer cell line SK-BR3 was found to deactivate MMP2 and MMP9, both controlled by MT1-MMP, as measured by gelatin zymography. In the zymogram, the higher gelatinase activity of MMP2 and MMP9, which accounts for the greater population of the active forms of MMP2 and MMP9, allowed the appearance of more intensive white bands (FIG. 7B).
- ANT2 shRNA decreased the expression and activity of MMPs (matrix metalloproteinases), which are responsible for the migration and invasion of tumor cells, as demonstrated in Example 4.
- MMPs matrix metalloproteinases
- a matrigel invasion assay is to analyze the invasion ability of tumor cells by staining the cells which degrade and proceed through matrigel.
- the human breast cancer cell line SK-BR3 which was transfected with ANT2 shRNA was incubated for 18hours. After that, 100 ⁇ l of 0.1% BSA, ⁇ -MEM containing 2 ⁇ 10 4 cells therein was put in an upper side of chamber (Becton Dickinson Labware, USA), followed by incubation. 24 hours later, the medium was removed from the upper sied of the chamber, and the cells were washed three times with PBS and taken with a cotton swab. The cells which passed through the matrigel and adhered to the outside bottom of the well were fixed for 10 min with 10% formalin and stained for 20 min with 0.1% crystal violet. Stained cells were counted under a microscope.
- a transwell migration assay was utilized to examine the migration ability of tumor cells.
- the lower side of a transwell insert with a 8 ⁇ m pore size (Corning) was coated with gelatin (Sigma) before a cell suspension was placed into the transwell insert.
- a serum-free medium containing bFGF (Sigma) at a concentration of 10 ng/mL was put in the external vessel.
- the inside of the insert was wiped with a cotton swab to take endothelial cells which did not migrate.
- the cells which migrated into the lower layer were stained with a Diff-Quick solution and counted under a 200x microscopic sight.
- MCF7 resistant to TRAIL and MDA-MB-231 highly sensitive to TRAIL were employed.
- MCF7 and MDA-MB-231 were treated with TRAIL at various concentrations after which cell death was analyzed with a CCK8 reagent.
- the CCK8 assay is a cell viability assay. Specifically, 100 ⁇ L of a cell suspension was placed onto each well (1 ⁇ 10 4 cells/well). The cells were treated with 10-fold serial dilutions of TRAIL starting from 1 ⁇ g/mL to zero, followed by incubation for 12 hrs. After the addition of 10 ⁇ L of the CCK8 reagent, the cells were incubated for a proper time (4 hours) in a CO 2 incubator and measured for absorbance at 450 nm to analyze cell death.
- TRAIL When TRAIL was added at a concentration of as high as 100 ng/mL, MCF7 cells, which are resistant to TRAIL, were not induced to undergo apoptosis. However, the introduction of ANT2 shRNA into MCF7 cells induced apoptosis ( ⁇ 25%). In combination with TRAIL, ANT2 shRNA was found to induce more potential apoptosis ( ⁇ 65%) as measured by the CCK8 reagent (FIG. 9B). Also, the introduction of ANT2 shRNA was observed to decrease the intracellular protein level of ANT2 as measured by Western blotting. As for a negative control, it was PBS for TRAIL, and sc shRNA (scrambled shRNA) for ANT2 shRNA.
- FIG. 9C After being treated with TRAIL alone, ANT2 shRNA alone, and TRAIL and ANT2 shRNA in combination, MCF7 cells, resistant to TRAIL, were examined for apoptosis, and the results are shown in FIG. 9C. As shown in FIG. 9C, the strongest apoptosis was induced by a combination of ANT2 shRNA and TRAIL.
- TRAIL-mediated apoptosis was found to be reduced as measured by a CCK8 assay (FIG. 10B).
- the empty vector pcDNA was introduced as a negative control for pcDNA-ANT2.
- cytochrome C which is released from mitochondria to cytoplasm, was quantitatively monitored upon the ANT2 shRNA-triggered, TRAIL-mediated apoptosis.
- the mitochondrial protein COX IV was used as a loading control to guarantee the use of mitochondrial proteins in the same amount. No changes in the pattern of apoptosis-induced proteins were observed when TRAIL was used alone. Greater changes in the protein pattern were observed when the cells were treated with ANT2 shRNA in combination with TRAIL than ANT shRNA alone (FIG. 10C). These changes of intracellular protein patterns confirmed that ANT2 shRNA enhanced TRAIL-mediated apoptosis.
- the breast cancer cell lines MCF7 and MDA-MB-231 which are different in sensitivity to TRAIL from each other, were analyzed for expression levels of the receptors DR4, DR5, DcR1, and DcR2, all binding to TRAIL, by Western blots.
- DR4 and DR5 When bound to TRAIL, DR4 and DR5 deliver normal apoptotic signals into the cells whereas DcR1 and DcR2 do not. Accordingly, higher sensitivity to TRAIL can be obtained by higher expression levels of DR4 and DR5 and lower expression levels of DcR1 and DcR2.
- MCF7 cells which are low in sensitivity to TRAIL, are observed to have low levels of DR4 and DR5 and high levels of DcR1 and DcR2 while high levels of DR4 and DR5 and low levels of DcR1 and DcR2 were detected in MDA-MB-231 cells, which are high in sensitivity to TRAIL.
- FIG. 11B Upon the introduction of ANT2 shRNA, expression patterns of TRAIL receptors were examined, and the results are given in FIG. 11B. As shown in FIG. 26B, the introduction of ANT2 shRNA up-regulated DR4 and DR5, and down-regulated DcR1 and DcR2. In T47D and BT474 cell lines, which are known to have TRAIL resistance, the same effects as in MCF7 were observed. That is, the introduction of ANT2 shRNA increased the expression of DR4 and DR5, but decreased the expression of DcR1 and DcR2 (FIG. 11C).
- a reporter gene assay showed that the up-regulation of p53 by ANT2 shRNA is attributed to increased affinity for the promoter of p53.
- This assay is generally designed in such a manner that when a certain protein binds to DNA to induce the expression of a gene of interest, the reporter is concurrently expressed and quantitatively analyzed by absorbance.
- cells were co-transfected with ANT2 shRNA and a pGL-p53 binding site-luciferase expression vector, and incubated for a certain period of time.
- a substrate was reacted with the expressed luciferase to measure the activity of the luciferase using a luminometer (FB12 luminometer; Berthold Detection Systems, Pforzheim, Germany).
- ANT2 shRNA increases the gene expression inducting ability of p53.
- p53 inhibitor pifithrin-alpha
- ANT2 shRNA induced the expression and activity of p53, leading to an increase in sensitivity to TRAIL.
- the cell death was quantified using a CCK8 assay, with the use of PBS as a negative control for the p53 inhibitor (FIG. 13).
- Example 9 In Vivo Assay of ANT2 shRNA for Ability to Suppress Tumor Growth by Increasing Sensitivity to TRAIL
- the breast cancer cell line MCF7 resistant to TRAIL was transplanted into immunedeficient Balb/c nude mice.
- a tumor grew to a volume of 100 mm 3 , they were treated with ANT2 shRNA and TRAIL.
- TRAIL was intraperitoneally injected (10 mg/kg) while ANT2 shRNA was directly injected into the tumor (100 ⁇ g: supplemented 200 ⁇ l Lipofectamine TM 2000).
- Lipofectamine TM 2000 was used to deliver ANT2 shRNA into tumor cells.
- the size of tumor was monitored.
- a same volume of PBS and sc shRNA was used as respective negative controls for TRAIL and sc shRNA.
- TRAIL alone had no significant influences on tumor growth, as in the cell culture tests.
- ANT2 alone was observed to suppress tumor growth.
- the greatest suppressive effect on tumor growth was obtained when the animal was treated ANT2 shRNA and TRAIL at the same time.
- Adenovirus system was used for the effective tranfection of progenitor cells with ANT2 shRNA (ANT2 shRNA sequence and loop sequence targeting ANT2 are the same as sequences used in the construction of ANT2 shRNA expression vector).
- the pSilencer-ANT2shRNA DNA was subcloned into the EcoR I /Hind III site of the Pca14 shuttle vector, which is designed to facilitate cloning into an adenovirus vector.
- a real-clone was detected by Pvu I enzyme mapping (DNA was enzymatically digested and electrophoresed to detect a DNA fragment at a suitable size position).
- a Pca14-mANT2shRNA DNA linearized by Pca I was subjected to homologous recombination with an adenovirus-dl324 vector (E1 deleted: replication-defective vector) DNA linearized by BstBI.
- the resulting recombinant DNA was transformed into DH5 ⁇ cells and amplified.
- a real-clone was transfected into a 293a packaging cell (a cell designed to readily introduce DNA thereinto, and to allow the mass production of cloned adenovirus therein due to the presence of the adenoviral replication gene E1) to induce the proliferation of adenovirus within the cell.
- a 293a packaging cell a cell designed to readily introduce DNA thereinto, and to allow the mass production of cloned adenovirus therein due to the presence of the adenoviral replication gene E1
- the enriched adenovirus-mANT2shRNA was separated to purity using a PEG-CsCl (density gradient layer separation) method before use in experiments.
- a breast cancer cell line is composed of progenitor cells and non-progenitor cells.
- ANT2 protein is highly expressed in both cell populations.
- apoptosis according to the introduction of ANT2 shRNA was examined in both cell populations.
- the progenitor cells of a tumor are characterized by CD44+/CD24- on their surface.
- MDA-MB-231 is composed predominantly of progenitor cells (CD44+/CD24-) (more than 80%), while MCF7 contains a minor proportion (less than 10%) of progenitor cells.
- the two cell lines and the progenitor cells (CD44+/CD24-) separated therefrom were analyzed for ANT2 gene expression using RT-PCR.
- Real-time PCR was also used to confirm the results of RT-PCR.
- the two cell lines were treated with an CD44 monoclonal antibody[anti-PE-conjugated CD44 monoclonal antibody (BD-PharMingen, San Diego, California, USA) and anti-PE microbeads [MiltenyiBiotec. (BergischGladbach, Germany)], followed by sorting necessary cells with the aid of MACS (Magnetic Activating cell sorter).
- MACS Magnetic Activating cell sorter
- both cell lines whether sorted into the progenitor cells (CD44+/CD24-) or not, were high in ANT2 expression level.
- the cells which had normally grown in a densely adherent pattern changed to show a loosely adherent growth pattern, which indicates the conversion of the normal epithelial cells into mesenchymal cells.
- the cells were found to increase in E-cadherin expression and decrease in ANT2 expression, as measured by RT-PCR (FIG. 16B).
- ANT2 shRNA was introduced into only the progenitor cells (CD44+/CD24-) separated from the breast cancer cell lines MDA-MB-231 and MCF7, after which apoptosis was analyzed using FACS with Annexin V-FITC and PI staining. This assay is based on the fact that when cells are dead, intracellular proteins and DNA are released from the cells (CD44+/CD24-) and reacted with Annexin V-FITC and PI, respectively. The results are given in FIG. 17. Even the progenitor cells separated from the two tumor cell lines were observed to be effectively induced to undergo apoptosis by ANT2 shRNA. For this, sc shRNA was used as a negative control.
- ANT2 shRNA was examined to determine whether it can induce effective apoptosis in the breast cancer stem-like cells obtained by introducing E-cadherin shRNA into the normal breast epithelial cell line (MCF10A).
- MCF10A normal breast epithelial cell line
- FIG. 18 For analysis, the cells were stained with annexin-V-FITC and PI. This is based on the fact that cell membranes at an early stage of programmed cell death are destroyed to release phospholipids such as phosphatydylserine from the cells and Annexin V is associated with the phospholipids, which is used as a proof for the early stage of programmed cell death.
- PI propium iodide
- MCF10A normal breast epithelial cell line
- ANT2 shRNA was introduced, little apoptosis was induced in the normal breast epithelial cell line (MCF10A) having low level of ANT2 expression (FIG. 18A).
- the meshechymally transdifferentiated cells obtained by introducing E-cadherin shRNA into MCF10A were improved in ANT2 expression level and, when ANT2 shRNA was introduced thereinto, the cells (E-cad shRNA transfected-MCF10A) were effectively induced to undergo apoptosis (FIG. 18B).
- ANT2 shRNA can effectively kill breast cancer stem cells without influence on normal epithelial cells.
- progenitor cells CD44+/CD24-
- MDA-MB-231 and MCF7 in the same manner as in Example 10
- ANT2 shRNA the attachment of cell to culture was minimized to promote the formation of cell mass
- FIG. 19A a large number of cell masses were formed when ANT2 shRNA was not introduced whereas the introduction of ANT2 shRNA allowed the formation of almost no or few cell masses.
- FIG. 19B the cell masses, although formed, were very small in volume.
- ANT2 shRNA inhibits the tumor growth activity of the progenitor cells which play a crucial role in the recurrence of tumor.
- the progenitor cells of breast cancer exhibit high resistance to drugs (anti-cancer agents).
- ANT2 shRNA The effect of ANT2 shRNA on the sensitivity of progenitor cells to drugs was examined.
- the MDA-MB-231 cell line which has a predominant proportion of progenitor cells (CD44+/CD24-) was treated with 10-fold serial dilutions of doxorubicin, a widely used anticancer agent starting from 10 ⁇ M to zero, followed by incubation (24 hrs).
- the apoptosis was quantitatively analyzed using a CCK8 assay and the results are given in FIG. 20.
- the assay was conducted in the same manner as in Example 6.
- the breast cancer cell line MCF7 which has a minor proportion of progenitor cells (CD44+/CD24-), was treated with various concentrations of doxorubicin, widely used for the treatment of breast cancer, after which apoptosis was quantitatively analyzed using a CCK8 assay.
- the results are given in FIG. 21.
- the progenitor cells CD44+/CD24-
- the introduction of ANT2 shRNA was found to induce apoptosis in a dose-dependent manner in both cell populations. The results indicate that the introduction of ANT2 shRNA makes the cells more sensitive to doxorubicin, giving a solution to the problem of drug resistance.
- Example 13 Effect of ANT2 shRNA on the Expression of the Receptor ABCG2 responsible for Drug Resistance of Breast Cancer Progenitor Cells
- MDR multi-drug resistance receptors
- ABCG2 ABCG2 which is highly expressed particularly in breast cancer cells.
- the breast cancer cell lines MDA-MB-231 and MCF7 which have high and low proportions of the progenitor cells (CD44+/CD24-), respectively, were used in this experiment.
- Progenitor cells (CD44+/CD24-) sorted from the breast cancer cells lines, unsorted cells, and the mesenchymally transdifferentiated-breast epithelial cell line (MCF10A) by the knockdown of E-cadherin were analyzed for ABCG2 mRNA expression using RT-PCR.
- ABCG protein levels were determined by Western blotting with an anti-ABCG2 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).
- the mRNA expression level of ABCG2 is increased in progenitor cells (CD44+/CD24-) sorted from the two tumor cell lines, non-sorted cells, and the mesenchymally transdifferentiated-breast epithelial cell line (MCF10A) by the knockdown of E-cadherin.
- the protein expression level of ABCG2 is increased in progenitor cells (CD44+/CD24-) sorted from the two tumor cell lines, non-sorted cells, and the mesenchymally transdifferentiated-breast epithelial cell line (MCF10A) by the knockdown of E-cadherin.
- the increased level of ABCG2 was observed to be reduced when ANT2 shRNA was introduced.
- the results indicate that breast cancer progenitor cells and breast cancer stem cells, both expressing a high level of ABCG2, can be reduced in ABCG2 expression by the introduction of ANT2 shRNA thereinto.
- progenitor cells of breast cancer were examined to determine if there was a practical increase in the activity of ABCG2.
- progenitor cells CD44+/CD24-
- sorted from the breast cancer cells lines MDA-MB-231 and MCF7, unsorted cells, and the mesenchymally transdifferentiated-breast epithelial cell line (MCF10A) by the knockdown of E-cadherin were analyzed for ABCG2 activity using Hoechst 33342.
- the extent of accumulation of Hoechst 33342 gives a quantitative index for the activity of ABCG2. The results are given in FIG. 24.
- ABCG2 activity is increased in progenitor cells (CD44+/CD24-) sorted from the two tumor cell lines, non-sorted cells, and the mesenchymally transdifferentiated-breast epithelial cell line (MCF10A) by the knockdown of E-cadherin.
- Example 14 Selective Delivery of ANT2 shRNA to Breast Cancer Progenitor Cells and Apoptotic Effect thereof
- ANT2 shRNA can effectively kill progenitor cells of breast cancer.
- ANT2 shRNA When patients with breast cancer are treated by using ANT2 shRNA, it is very important to effectively deliver ANT2 shRNA to target cells.
- nano-complexes[PEI/hyaluronic acid (HA) nano-complexes] were prepared to use as a delivery material targeting CD44 which is highly expressed on the progenitor cells of breast cancer.
- ANT2 shRNA fluorescence-conjugated nanoparticle [PEI/hyaluronic acid (HA) nano-complexes]was complexed with ANT2 shRNA.
- PEI/hyaluronic acid (HA) nano-complexes fluorescence-conjugated nanoparticle [PEI/hyaluronic acid (HA) nano-complexes]was complexed with ANT2 shRNA.
- the resulting nano-complexes were applied to each cell line and then incubated, followed by analyzing intracellular fluorescent intensity to determine the level of the ANT2 shRNA introduced into the cells.
- ANT2 shRNA can be selectively delivered to the progenitor cells and induce the cells to undergo apoptosis.
- the present invention provides therapeutics for breast cancer, which are highly effective for inhibiting the metastasis of breast cancer cells and overcoming the tolerance toward an anti-cancer agent. Further, the present invention could be used to develop therapeutics for stem cells of breast cancer.
- SEQ ID NO 1 5'-GCAGAUCACUGCAGAUAAGUU-3' (sense sequence of ANT2 shRNA)
- SEQ ID NO 2 5'-UUCAAGAGA-3' (loop sequence of ANT2 shRNA)
- SEQ ID NO 3 5'-AACUUAUCUGCAGUGAUCUGC-3' (anti-sense sequence of ANT2 shRNA)
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US20090202623A1 (en) * | 2006-04-11 | 2009-08-13 | Chul Woo Kim | Gene therapy for cancer using small interfering rna specific to ant2 and a method to overcome tolerance to antitumor agent |
WO2009126335A2 (en) * | 2008-04-08 | 2009-10-15 | University Of Florida Research Foundation, Inc. | Ant2 inhibitor compounds and methods of use thereof |
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JI-YOUNG JANG ET AL.: "Degradation of HER2/neu by ANT2 shRNA suppresses migration and invasiveness of breast cancer cells", BMC CANCER, vol. 10, 23 July 2010 (2010-07-23), pages 391, XP021075213 * |
JI-YOUNG JANG ET AL.: "Short-hairpin RNA-induced suppression of adenine nucleotide translocase-2 in breast cancer cells restores their susceptibility to TRAIL-induced apoptosis by activating JNK and modulating TRAIL receptor expression", MOL CANCER, vol. 28, no. 9, 28 September 2010 (2010-09-28), pages 262, XP021078000 * |
JI-YOUNG JANG ET AL.: "Suppression of adenine nucleotide translocase-2 by vector-based siRNA in human breast cancer cells induces apoptosis and inhibits tumor growth in vitro and in vivo", BREAST CANCER RESEARCH, vol. 10, no. 1, 12 February 2008 (2008-02-12), pages R11, XP021041293 * |
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CN103263676A (zh) * | 2013-02-07 | 2013-08-28 | 魏敏杰 | 一种应用miR-487a逆转乳腺癌耐药的方法 |
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CN103379914B (zh) | 2016-02-17 |
EP2675468A4 (en) | 2014-10-22 |
JP2014506888A (ja) | 2014-03-20 |
KR20120095263A (ko) | 2012-08-28 |
CN103379914A (zh) | 2013-10-30 |
EP2675468A1 (en) | 2013-12-25 |
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