WO2008150388A1 - Inhibiton of mitochnodrial uncoupling protein - Google Patents
Inhibiton of mitochnodrial uncoupling protein Download PDFInfo
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- WO2008150388A1 WO2008150388A1 PCT/US2008/006582 US2008006582W WO2008150388A1 WO 2008150388 A1 WO2008150388 A1 WO 2008150388A1 US 2008006582 W US2008006582 W US 2008006582W WO 2008150388 A1 WO2008150388 A1 WO 2008150388A1
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- A—HUMAN NECESSITIES
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- Cancer is a leading cause of death in the United States. Despite the availability of numerous chemotherapeutic drugs, some tumors are or become resistant to their anti-neoplastic effects.
- a combination cancer therapeutic formulation contains an inhibitor of UCP2 expression or activity and an antineoplastic agent in amounts that inhibit tumor cell proliferation.
- the antineoplastic agent is selected from the group consisting of irinotecan, topotecan, 9-nitro-20(S)-camptothecin, and combinations thereof.
- Cancer type is selected from the group consisting of leukemia, breast cancer, skin cancer, bone cancer, prostate cancer, liver cancer, lung cancer, brain cancer, cancer of the larynx, gallbladder, pancreas, rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck, colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma of both ulcerating and papillary type, metastatic skin carcinoma, osteosarcoma, Ewing's sarcoma, veticulum cell sarcoma, myeloma, giant cell tumor, small-cell lung tumor, gallstones, islet cell tumor, primary brain tumor, acute and chronic lymphocytic and granulocytic tumors, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuronms, intestinal ganglioneuromas, hyperplastic corneal nerve tumor, marfanoid habitus tumor, Wilm's
- Also within the invention is a method of identifying an inhibitor of UCP2 expression.
- the method includes the steps of contacting a cell expressing UCP2 with a candidate compound and detecting UCP2 transcript or protein, wherein a reduction in the amount of transcript or protein in the presence of said compound compared to that in the absence of the compound indicates that said compound inhibits UCP2 expression or activity.
- compositions described herein used for treatment of cell proliferative disorders are substantially pure.
- substantially pure is meant a nucleic acid, polypeptide, or other molecule that has been separated from the components that naturally accompany it.
- the polypeptide is substantially pure when it is at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated.
- a substantially pure polypeptide may be obtained by extraction from a natural source, by expression of a recombinant nucleic acid in a cell that does not normally express that protein, or by chemical synthesis.
- Some UCP2 inhibitory compounds are nucleic acids.
- isolated nucleic acid is meant DNA that is free of the genes which, in the naturally occurring genome of the organism from which the given nucleic acid is derived, flank the DNA.
- isolated nucleic acid encompasses cloned nucleic acids or synthetic nucleic acids (RNA, RNAi, DNA).
- Candidate compounds may include, for example, peptides, polypeptides, synthetic organic molecules, naturally occurring organic molecules, nucleic acid molecules, peptide nucleic acid molecules, and components and derivatives thereof.
- Figs IA and IB are photographs of electrophoretic gels showing UCP2 expression
- Figs. 2A-F are a series of photographs of electrophoretic gels, a graph, and a dot plot showing that UCP2 expression protects cancer cells from apoptosis.
- Figs. 3A-D are a series of photographs of electrophoretic gels and graphs showing that the effects of UCP2 on camptothecin-induced oxidative stress and apoptosis are reproduced by artificial uncouplers.
- Figs. 4A-E are a series of photographs of electrophoretic gels showing that the anti- apoptotic effect of UCP2 in cancer cells is p53 dependent.
- Fig. 5 is a dot plot showing that UCP2 silencing enhances drug-induced apoptosis in cancer cells.
- Fig. 6 is a diagram of UCP2 activity.
- Fig. 7 is a diagram showing UCP2 -dependent intracellular processes.
- the invention represents a major advance in the treatment of tumors by increasing the sensitivity of tumor cells to chemotherapeutic/antineoplastic drugs.
- Mitochondrial uncoupling protein-2 (UCP2) is a widely expressed inner mitochondrial membrane protein (Fleury, C. et al. Nat Genet 1997; 15: 269-272).
- UCP2 expression correlates with neoplastic changes in human colon cancer (Horimoto, M. et al. Clin Cancer Res 2004; 10: 6203-6207).
- Chemoresistant cancer cells often overexpress UCP2 (Harper, M.E. et al. FASEB J 2002; 16: 1550-155).
- UCP2 is a sensor and negative regulator of oxidative stress (Brand, M.D. et al.
- ROS Reactive oxygen species
- UCP2 By controlling ROS production, UCP2 abrogates p53-dependent apoptosis induced by DNA damage in cancer cells. Experiments were carried out to assess the degree of apoptosis in UCP2 overexpressing HCTl 16 colon cancer cells and to study the mechanisms of increased chemoresistance in these cells.
- UCP2 attenuated ROS-mediated p53 stabilization and activation (e.g., by limiting N-terminal phosphorylation). These data indicate that inhibition of UCP2 is useful in the treatment of chemoresistant tumors.
- UCP2 an endogenously occurring cellular modulator of oxidative stress, provides important benefits when combined with chemotherapeutics or other currently available cancer treatment modalities. Advantages of this combination therapy protocol include better efficacy of the antineoplastic drug and reduced side effects. UCP2 activity in cells
- UCP2 is a transmembrane protein that controls leakage of protons across the inner mitochondrial membrane. Inhibiting UCP2 slows the stream of protons, improving membrane potential and allowing mitochondria to produce greater quantities of ATP. As a result, potassium channels close, depolarizing the cell, which in turn causes calcium channels to open.
- oxidative stress due to hypoxia, nutritional deprivation, and host defense responses.
- a major intracellular source of oxidative stress is the mitochondrial respiratory chain that generates superoxide, a major form of reactive oxygen species (ROS). Since excessive oxidative stress may induce cell growth arrest, senescence, and programmed cell death (apoptosis), survival and proliferation of cancer cells depends on effective control of this process.
- ROS reactive oxygen species
- UCP2 plays a key role in the adaptation of cancer cells to oxidative stress. Increased expression and function of UCP2 protects cancer cells from oxidative stress and becomes an important determinant of chemoresistance. Inhibition of UCP2 expression and function makes cancer cells more vulnerable to oxidative stress and the action of other neoplastic agents.
- UCP2 is a mitochondrial carrier protein located in the inner mitochondrial membrane (1) and acts as a negative regulator of mitochondrial ROS production. When overexpressed, UCP2 reduces ROS production and oxidative damage of the cell (2-5), while inhibition or genetic ablation of UCP2 results in increased generation of ROS, release of pro-inflammatory cytokines, and persistent activation of the pleiotropic transcription factor NF-kB (6-9). This protective effect of UCP2 has been demonstrated in a number of cell types by using both in vitro and in vivo experimental paradigms.
- UCP2 modulates the sensitivity of cancer cells to oxidative stress induced by chemotherapeutic agents
- UCP2 was overexpressed in several human-derived cancer cell lines to determine how UCP2 affects the response of cancer cells to various chemotherapeutic agents.
- An expression plasmid containing the full-length cDNA of the human ucp2 gene (hUCP2-pcDNA3.1/Zeo(-)) was constructed using standard cloning techniques.
- hUCP2-pcDNA3.1/Zeo(-) was constructed using standard cloning techniques.
- the following cells were transfected: HCTl 16 cells, a human colon cancer cell line, and RBE, a human cholangiocarcinoma cell line, with the hUCP2-pcDNA3.1/Zeo(-) plasmid.
- apoptosis programmed cell death
- camptothecin a frequently used chemotherapeutic agent that inhibits the activity of topoisomerases and also induces oxidative stress.
- Apoptosis of HCTl 16 cells was detected (Fig. 2). Camptothecin-induced apoptosis was diminished in HCTl 16 cells overexpressing UCP2 in comparison to mock-transfected controls as assessed by DNA-ladder gel electrophoresis and annexin V flow cytometry (combined with propidium iodide to detect non-apoptotic in addition to apoptotic cell death).
- camptothecin-induced activation of caspase-3 was diminished in HCTl 16 cells overexpressing UCP2.
- the abundance of several pro-apoptotic proteins (e.g., PUMA) and anti- apoptotic proteins (e.g., BcI-XL) in HCTl 16 cells was analyzed in response to camptothecin treatment and UCP2 overexpression was found to be associated with decreased levels of pro- apoptotic proteins, but the level of anti-apoptotic proteins was increased.
- p53 was stabilized in HCTl 16 cells upon camptothecin treatment and immunoblot analysis showed p53 abundance in these preparations (Fig. 4).
- UCP2 overexpression in HCTl 16 cells resulted in diminished accumulation of p53 both at baseline and in response to camptothecin. This effect could also be replicated by FCCP treatment of HCTl 16 cells exposed to camptothecin.
- FCCP treatment of HCTl 16 cells exposed to camptothecin To determine the mechanism of limited p53 accumulation, the cellular levels of MDM2, a protein with E3 ubiquitin ligase properties that is responsible for targeting p53 to the proteasome.
- FCCP had no effect on MDM2 levels in HCTl 16 cells prior or after camptothecin treatment, indicating that enhanced degradation of p53 is likely not the result of impaired ubiquitination.
- HCTl 16 cells were pretreated with MGl 32, a known inhibitor of the proteasome, and results showed that MG132 completely prevented the effect of FCCP on limiting p53 at baseline and in response to camptothecin.
- mitochondrial uncoupling primarily modulates p53 abundance in HCTl 16 cells by altering the function of proteasome.
- P53-deficient (p53-/-) HCTl 16 cells were used to analyze the effect of FCCP on the response of these cells to camptothecin in comparison to p53-wild type HCTl 16 cells. The effect of camptothecin was significantly diminished in p53-/- HCTl 16 cells and FCCP had no apparent further effect on the degree of apoptosis in these cells. Inhibition of UCP2 in cancer cells
- Plasmid-encoded UCP2 was properly targeted to the mitochondrial inner membrane of HCTl ⁇ 6 ⁇ CP2 cells, which displayed lower A ⁇ m and consumed more O 2 .
- ROS levels in HCTl ⁇ 6 UCP2 cells were consistently lower at baseline and in response to neoplastic agents.
- HCTl ⁇ UCP2 cells treated with the agents displayed decreased DNA laddering, annexinV staining, and caspase-3 cleavage in addition to a shift from PUMA to Bcl-X L expression, indicating diminished apoptosis.
- Plasmid-encoded UCP2 was properly targeted to the mitochondrial inner membrane and caused lower A ⁇ m and increased O 2 consumption indicating increased uncoupling in resting HCTl 16 ⁇ 2 cells.
- HCTl 16 ⁇ 2 cells showed decrease in DNA laddering, subGl fraction, annexin V expression, and caspase-3 cleavage in addition to decreased PUMA expression and increased BCI-X L expression.
- ROS levels before and after CPT treatment were consistently lower in HCTl I6 ⁇ cp2 cells indicating a negative effect of UCP2 on oxidative stress.
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Abstract
The invention provides methods of inhibiting tumors by contacting a tumor cell with an antineoplastic agent and an inhibitor of UCP2 expression or activity.
Description
INHIBITION OF MITOCHONDRIAL UNCOUPLING PROTEIN
Statement as to Federally Sponsored Research
This invention was made with U.S. Government support under Grant Number R-17695 from National Center for Research Resources/National Institutes of Health and Grant Number DK-61890 from National Institute of Diabetes and Digestive and Kidney Diseases/National Institutes of Health. The government has certain rights in the invention.
Field of the Invention
The present invention relates to the field of cancer therapy.
Background of the Invention
Cancer is a leading cause of death in the United States. Despite the availability of numerous chemotherapeutic drugs, some tumors are or become resistant to their anti-neoplastic effects.
Summary of the Invention
The invention provides a solution to a persistent problem in the field of cancer therapy - chemoresistance of tumors. Enhancing the effects of chemotherapeutics in cancer cells is accomplished by inhibition of mitochondrial uncoupling protein-2 (UCP2).
A method for augmenting the antineoplastic effect of chemotherapy is carried out by contacting a tumor cell with an antineoplastic composition and an inhibitor of UCP2 expression or activity. The antineoplastic composition is an inhibitor of cell proliferation and/or a cytotoxic agent. The UCP2 inhibitor inhibits expression of UCP2 in a tumor cell (e.g., a UCP2-specific RNAi composition) or inhibits an activity of UCP2 (e.g., genipin, which inhibits UCP2 mediated proton leak. Preferably, the inhibitory compound is cell permeable. The UCP2 inhibitory compound and the antineoplastic compound are administered together or sequentially.
A combination cancer therapeutic formulation contains an inhibitor of UCP2 expression or activity and an antineoplastic agent in amounts that inhibit tumor cell proliferation. For
example, the antineoplastic agent is selected from the group consisting of irinotecan, topotecan, 9-nitro-20(S)-camptothecin, and combinations thereof.
The invention includes a method for treating a cell proliferative disorder such as tumor growth in a mammal by administering to the mammal a therapeutically effective amount of a combination of a UCP2 inhibitor and an antineoplastic agent. A combination therapy regimen involves administering to a patient suffering from cancer a therapeutically effective amount of a neoplastic agent and an inhibitor of UCP2, wherein the therapy is enhanced by the synergistic effect of said neoplastic agent and said UCP2 inhibitor. Cancer type is selected from the group consisting of leukemia, breast cancer, skin cancer, bone cancer, prostate cancer, liver cancer, lung cancer, brain cancer, cancer of the larynx, gallbladder, pancreas, rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck, colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma of both ulcerating and papillary type, metastatic skin carcinoma, osteosarcoma, Ewing's sarcoma, veticulum cell sarcoma, myeloma, giant cell tumor, small-cell lung tumor, gallstones, islet cell tumor, primary brain tumor, acute and chronic lymphocytic and granulocytic tumors, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuronms, intestinal ganglioneuromas, hyperplastic corneal nerve tumor, marfanoid habitus tumor, Wilm's tumor, seminoma, ovarian tumor, leiomyomater tumor, cervical dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion, mycosis fungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic and other sarcoma, malignant hypercalcemia, renal cell tumor, polycythemia vera, adenocarcinoma, glioblastoma multiforma, leukemias, lymphomas, malignant melanomas, and epidermoid carcinoma.
Also within the invention is a method of identifying an inhibitor of UCP2 expression. The method includes the steps of contacting a cell expressing UCP2 with a candidate compound and detecting UCP2 transcript or protein, wherein a reduction in the amount of transcript or protein in the presence of said compound compared to that in the absence of the compound indicates that said compound inhibits UCP2 expression or activity.
The compositions described herein used for treatment of cell proliferative disorders such as cancer are substantially pure. By "substantially pure" is meant a nucleic acid, polypeptide, or other molecule that has been separated from the components that naturally accompany it.
Typically, the polypeptide is substantially pure when it is at least 60%, 70%, 80%, 90%, 95%, or
even 99%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. For example, a substantially pure polypeptide may be obtained by extraction from a natural source, by expression of a recombinant nucleic acid in a cell that does not normally express that protein, or by chemical synthesis. Some UCP2 inhibitory compounds are nucleic acids. The term "isolated nucleic acid" is meant DNA that is free of the genes which, in the naturally occurring genome of the organism from which the given nucleic acid is derived, flank the DNA. Thus, the term "isolated nucleic acid" encompasses cloned nucleic acids or synthetic nucleic acids (RNA, RNAi, DNA).
An effective amount is an amount of a compound, alone or in a combination, required to reduce or prevent the growth or invasiveness of a tumors. The effective amount of active compound(s) varies depending upon the route of administration, age, body weight, and general health of the subject.
By a "candidate compound" is meant a chemical, be it naturally-occurring or artificially- derived. Candidate compounds may include, for example, peptides, polypeptides, synthetic organic molecules, naturally occurring organic molecules, nucleic acid molecules, peptide nucleic acid molecules, and components and derivatives thereof.
The term "pharmaceutical composition" is meant any composition, which contains at least one therapeutically or biologically active agent and is suitable for administration to the patient. Any of these formulations can be prepared by well-known and accepted methods of the art. See, for example, Remington: The Science and Practice of Pharmacy, 20th edition, (ed. A. R. Gennaro), Mack Publishing Co., Easton, Pa., 2000. Parenteral administration, such as intravenous, subcutaneous, intramuscular, and intraperitoneal delivery routes, may be used to deliver the compounds. Alternatively, the compounds are administered locally, e.g., directly to a tumor site. For example, skin cancers are treated by administering the compounds topically. For treatment of CNS tumors, direct infusion into cerebrospinal fluid is useful. The blood-brain barrier may be compromised in cancer patients, allowing systemically administered drugs to pass through the barrier into the CNS. Liposome formulations of therapeutic compounds may also facilitate passage across the blood-brain barrier.
Dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular nucleic acid to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. References cited are hereby incorporated by reference.
Brief Description of the Drawings
Figs IA and IB are photographs of electrophoretic gels showing UCP2 expression Figs. 2A-F are a series of photographs of electrophoretic gels, a graph, and a dot plot showing that UCP2 expression protects cancer cells from apoptosis.
Figs. 3A-D are a series of photographs of electrophoretic gels and graphs showing that the effects of UCP2 on camptothecin-induced oxidative stress and apoptosis are reproduced by artificial uncouplers. Figs. 4A-E are a series of photographs of electrophoretic gels showing that the anti- apoptotic effect of UCP2 in cancer cells is p53 dependent.
Fig. 5 is a dot plot showing that UCP2 silencing enhances drug-induced apoptosis in cancer cells.
Fig. 6 is a diagram of UCP2 activity. Fig. 7 is a diagram showing UCP2 -dependent intracellular processes.
Detailed Description
The invention represents a major advance in the treatment of tumors by increasing the sensitivity of tumor cells to chemotherapeutic/antineoplastic drugs. Mitochondrial uncoupling protein-2 (UCP2) is a widely expressed inner mitochondrial membrane protein (Fleury, C. et al. Nat Genet 1997; 15: 269-272). UCP2 expression correlates with neoplastic changes in human colon cancer (Horimoto, M. et al. Clin Cancer Res 2004; 10: 6203-6207). Chemoresistant cancer cells often overexpress UCP2 (Harper, M.E. et al. FASEB J
2002; 16: 1550-155). UCP2 is a sensor and negative regulator of oxidative stress (Brand, M.D. et al. Free Radic Biol Med 2004; 37: 755-76) Reactive oxygen species (ROS) enhance p53 stabilization and subsequent induction of apoptosis after DNA damage (Hwang, P.M. et al. Nat Med 2001; 7: 1111-1117). Reduction of mitochondrial ROS attenuates p53-dependent apoptosis in human T-lymphocytes and MOLT-3 leukemia cells (Karawajew, L. et al. Blood 2005; 105: Al 61 -All 5). The mechanism of UCP2 activity is illustrated in Figs. 6 and 7.
By controlling ROS production, UCP2 abrogates p53-dependent apoptosis induced by DNA damage in cancer cells. Experiments were carried out to assess the degree of apoptosis in UCP2 overexpressing HCTl 16 colon cancer cells and to study the mechanisms of increased chemoresistance in these cells.
Mitochondrial uncoupling was established in p53 wild type and p53-deficient HCTl 16 cells by transfection of hUCP2-pcDNA3.1/Zeo(-) plasmid (pUCP2) or by 24 h FCCP (chemical uncoupler) treatment. UCP2 overexpression was verified by mitochondrial fractionation, Western blotting and functional assays (JCl: mitochondrial membrane potential (Dym), O2 consumption, ATP measurement, DCF assay: reactive oxygen specied (ROS). HCT 116 cells were treated with an antineoplastic agent, topoisomerase I inhibitor camptothecin (CPT) to induce apoptosis, which was assessed by DNA laddering, annexin V flow cytometry, caspase-3 cleavage, and cell cycle analysis. p53 degradation was inhibited by MGl 32 proteasome inhibitor. JNK phosphorylation was assessed by ELISA. Mitochondrial uncoupling was found to protect tumor cells, e.g., HCTl 16 colon cancer cells, against oxidative stress and apoptosis induced by chemotherapeutic drugs. Overexpression of the endogenously occurring UCP2 increased the proteasomal degradation of p53 at baseline and following CPT treatment. By decreasing oxidative stress, UCP2 attenuated ROS-mediated p53 stabilization and activation (e.g., by limiting N-terminal phosphorylation). These data indicate that inhibition of UCP2 is useful in the treatment of chemoresistant tumors.
Inhibiting UCP2, an endogenously occurring cellular modulator of oxidative stress, provides important benefits when combined with chemotherapeutics or other currently available cancer treatment modalities. Advantages of this combination therapy protocol include better efficacy of the antineoplastic drug and reduced side effects.
UCP2 activity in cells
UCP2 is a transmembrane protein that controls leakage of protons across the inner mitochondrial membrane. Inhibiting UCP2 slows the stream of protons, improving membrane potential and allowing mitochondria to produce greater quantities of ATP. As a result, potassium channels close, depolarizing the cell, which in turn causes calcium channels to open.
Cancer cells frequently exhibit increased oxidative stress due to hypoxia, nutritional deprivation, and host defense responses. A major intracellular source of oxidative stress is the mitochondrial respiratory chain that generates superoxide, a major form of reactive oxygen species (ROS). Since excessive oxidative stress may induce cell growth arrest, senescence, and programmed cell death (apoptosis), survival and proliferation of cancer cells depends on effective control of this process. UCP2 plays a key role in the adaptation of cancer cells to oxidative stress. Increased expression and function of UCP2 protects cancer cells from oxidative stress and becomes an important determinant of chemoresistance. Inhibition of UCP2 expression and function makes cancer cells more vulnerable to oxidative stress and the action of other neoplastic agents.
UCP2 is a mitochondrial carrier protein located in the inner mitochondrial membrane (1) and acts as a negative regulator of mitochondrial ROS production. When overexpressed, UCP2 reduces ROS production and oxidative damage of the cell (2-5), while inhibition or genetic ablation of UCP2 results in increased generation of ROS, release of pro-inflammatory cytokines, and persistent activation of the pleiotropic transcription factor NF-kB (6-9). This protective effect of UCP2 has been demonstrated in a number of cell types by using both in vitro and in vivo experimental paradigms.
UCP2 controls ROS production by mediating proton leak and by modulating the mitochondrial inner membrane potential (ΔΨm) (10). Mitochondrial respiration uses the energy of electron transport to build a proton gradient across the inner membrane and to fuel ATP synthesis (oxidative phosphorylation). Superoxide, a key ROS radical, is produced during this process by incomplete reduction of molecular oxygen (11-13). This pathway normally involves less than 1% of the transported electrons, but may escalate if the electron transport becomes sluggish due to elevated ΔΨm (11, 12). Modulation of ΔΨm is therefore a powerful tool to control mitochondrial ROS production. This task is achieved by uncoupling proteins that mediate proton leak (i.e., reentry of protons into the mitochondrial matrix without driving ATP
synthesis), reduce ΔΨ m, and diminish the formation of superoxide. Of all uncoupling proteins, UCP2 has the widest tissue distribution. Chemoresistance of tumor cells
Drug-resistant sub-lines of various tumor cells have increased expression of UCP2 in association with lower ΔΨ m and diminished susceptibility to oxidative stress (14).
Furthermore, overexpression of UCP2 in HepG2 cells, a transformed hepatocellular cell line, results in increased resistance to apoptosis induced by hypoxia and oxidative stress (15). These findings indicate that increased presence of UCP2 in cancer cells correlates with increased ability of cancer cells to withstand the effects of chemotherapy. UCP2 modulates the sensitivity of cancer cells to oxidative stress induced by chemotherapeutic agents
UCP2 was overexpressed in several human-derived cancer cell lines to determine how UCP2 affects the response of cancer cells to various chemotherapeutic agents. An expression plasmid containing the full-length cDNA of the human ucp2 gene (hUCP2-pcDNA3.1/Zeo(-)) was constructed using standard cloning techniques. By use of a highly efficient nucleofection system based electroporation optimized to the cell type (Amaxa, Cologne, Germany), the following cells were transfected: HCTl 16 cells, a human colon cancer cell line, and RBE, a human cholangiocarcinoma cell line, with the hUCP2-pcDNA3.1/Zeo(-) plasmid. To optimize transfection and minimize non-specific cytotoxic effects, various amounts of plasmid were used for transfection. To confirm the appropriate targeting of exogenous UCP2 into the mitochondrial inner membrane, mitochondrial sub-fractionation was performed by digitonin and alkali treatment. As expected, the exogenous UCP2 protein localized to the inner membrane fraction as assessed by immunoblot analysis (Fig. 1). To demonstrate the functional impact of exogenous UCP2 in cancer cells, ΔΨ m was assessed semi-quantitatively by using JC-I, a fluorescent dye that accumulates in the mitochondria and forms J-aggregates proportional to ΔΨm. UCP2 overexpression caused lower ΔΨ m, but no apparent changes occurred in mock-transfected cancer cells.
The effect of UCP2 overexpression on programmed cell death (apoptosis) was determined in HCTl 16 cells in response to camptothecin, a frequently used chemotherapeutic agent that inhibits the activity of topoisomerases and also induces oxidative stress. Apoptosis of HCTl 16 cells was detected (Fig. 2). Camptothecin-induced apoptosis was diminished in
HCTl 16 cells overexpressing UCP2 in comparison to mock-transfected controls as assessed by DNA-ladder gel electrophoresis and annexin V flow cytometry (combined with propidium iodide to detect non-apoptotic in addition to apoptotic cell death). In addition, camptothecin-induced activation of caspase-3, a key effector of apoptosis, was diminished in HCTl 16 cells overexpressing UCP2. The abundance of several pro-apoptotic proteins (e.g., PUMA) and anti- apoptotic proteins (e.g., BcI-XL) in HCTl 16 cells was analyzed in response to camptothecin treatment and UCP2 overexpression was found to be associated with decreased levels of pro- apoptotic proteins, but the level of anti-apoptotic proteins was increased. These findings indicate that UCP2 overexpression confers increased resistance to the apoptosis-inducing effects of camptothecin in HCT 116 cells. UCP2 overexpression in HCTl 16 cells also has a protective effect against apoptosis induced by other chemotherapeutics, such as etoposide and cisplatin.
Experiments were carried out to determine the biochemical mechanism(s) by which UCP2 overexpression exerts its protective effect against chemotherapeutics-induced apoptosis in cancer cells. Experiments were conducted to determine camptothecin-induced apoptosis after pre-treating HCTl 16 cells with FCCP, an artificial uncoupling agent (Fig. 3). A defined concentration range (0.1 μM to 5μM), FCCP provided protection to HCTl 16 cells from camptothecin-induced apoptosis and therefore to partially replicate the effects of UCP2 overexpression. These results are evidence that the effects of UCP2 relate to its ability of mediating proton leak and modulating ΔΨm, since these are the known biochemical effects of FCCP. Second, the protective effect of UCP2 overexpression was associated with decreased oxidative stress, since the level of ROS in camptothecin-treated HCTl 16 cells was significantly diminished when compared to mock-transfected cells.
To further analyze the apoptosis-inhibitory effect of UCP2 overexpression, experiments were carried out to determine its impact on cellular p53 abundance in HCTl 16 cells treated with camptothecin. P53 is a major tumor suppressor protein and acts as the guardian of genomic integrity. P53 initiates DNA repair, but it induces apoptosis in cells with irreparable DNA damage. P53 has both transcriptional and non-transcriptional targets. While genetic mutations of p53 occur in -50% of all cancers, HCTl 16 cells have wild type p53. HCTl 16 cells provide therefore an appropriate and recognized model to study the physiologic functions of p53. The abundance of p53 is primarily regulated by its degradation in the proteasome. p53 was stabilized in HCTl 16 cells upon camptothecin treatment and immunoblot analysis showed p53 abundance
in these preparations (Fig. 4). However, UCP2 overexpression in HCTl 16 cells resulted in diminished accumulation of p53 both at baseline and in response to camptothecin. This effect could also be replicated by FCCP treatment of HCTl 16 cells exposed to camptothecin. To determine the mechanism of limited p53 accumulation, the cellular levels of MDM2, a protein with E3 ubiquitin ligase properties that is responsible for targeting p53 to the proteasome. FCCP had no effect on MDM2 levels in HCTl 16 cells prior or after camptothecin treatment, indicating that enhanced degradation of p53 is likely not the result of impaired ubiquitination. HCTl 16 cells were pretreated with MGl 32, a known inhibitor of the proteasome, and results showed that MG132 completely prevented the effect of FCCP on limiting p53 at baseline and in response to camptothecin. These findings indicate that mitochondrial uncoupling primarily modulates p53 abundance in HCTl 16 cells by altering the function of proteasome. P53-deficient (p53-/-) HCTl 16 cells were used to analyze the effect of FCCP on the response of these cells to camptothecin in comparison to p53-wild type HCTl 16 cells. The effect of camptothecin was significantly diminished in p53-/- HCTl 16 cells and FCCP had no apparent further effect on the degree of apoptosis in these cells. Inhibition of UCP2 in cancer cells
Experiments were carried out to determine whether reducing endogenous UCP2 expression in HCTl 16 cells affects cellular sensitivity to camptothecin-induced apoptosis. Altering UCP2 expression to the opposite direction may result in reduced ability of cancer cells to resist chemotherapeutics-induced apoptosis. RNA interference (silencing) was used to test this function. HCTl 16 cells were transfected with a plasmid containing UCP2-specific short- hairpin RNA (shRNA) able to induce silencing. Using annexin V flow cytometry, data was generated to demonstrate that UCP2 silencing enhances the apoptosis-inducing effect of camptothecin in HCTl 16 cells (Fig. 5). These data indicate that inhibiting or silencing of endogenous UCP2 augments the anti-cancer effects of camptothecin. This combination therapy approach may increases the efficacy of chemotherapy. It also permits the clinician to use lower effective doses of the antineoplastic agent, e.g., camptothecin, thereby reducing adverse side effects that are otherwise associated with therapeutic doses of the drug.
The data establish the protective effect of UCP2 in cancer cells and the effect of UCP2 overexpression on the tumor suppressor p53 as well as the link between UCP2, a negative regulator of ROS production and p53, a tumor suppressor protein inducing apoptosis in
association with oxidative stress. UCP2 overexpression is a mechanism by which cancer cells adapt to oxidative stress. UCP2 inhibition or silencing results in enhanced apoptosis induced by camptothecin in HCTl 16 cells. These data indicate that altering endogenous UCP2 expression is a powerful approach to treating cancer. UCP2 is inhibited at the nucleic acid level, e.g., by antisense (DeSousa et al., 2007,
FASEB J. 21:1153-1163), RNA interference (siRNA or RNAi, e.g., as described in U.S. Patent Nos. 7,056,704; 7,078,196; 5,898,031; and 6,107,094) or other strategies known in the art. Regulators of UCP2 gene expression are identified using know nucleic acid sequences and methods (e.g., as described in U.S. Patent No. 5,807,740). Alternatively, the function of UCP2 is inhibited. For example, genipin (Zhang et al.,
2006, Cell Metabolism 3 :417-427) is a cell permeable inhibitor of UCP2 activity. Other inhibitors are identified by contacting cells with a candidate inhibitory compound and measuring level of UCP2 gene expression or a UCP2 activity and comparing the level of expression or activity to that detected in the absence of the compound. Optionally, the cells overexpress UCP2, e.g., they have been transfected with an exogenous UCP2 gene sequence. A decrease in UCP expression or activity indicates that the compound inhibits UCP2 and is useful to augment the antineoplastic activity of a chemotherapeutic drug for the treatment of cancer. Example 1 : Mitochondrial uncoupling protein-2 alters chemoresistance by decreasing ROS production and apoptosis rates in colon cancer cells ROS have a complex role in cancer development and growth. While excessive oxidative stress promotes senescence and apoptosis, surviving cancer cells adapt and utilize mechanisms that limit intracellular ROS production. Experiments were carried out to test whether uncoupling protein-2 (UCP2), a negative regulator of mitochondrial ROS production, supports this adaptive response and whether inhibition of UCP2 is a feasible target to restore chemosensitivity in cancer cells.
Endogenous UCP2 levels were altered in the human colon cancer cell lines HCTl 16 and CaCo-2, either by overexpressing the full-length human UCP2 or by RNAi-mediated silencing. The effect of various chemotherapeutic agents was tested on cells that either overexpress the full- length human UCP2 (HCTl 16^^) or have their endogenous UCP2 silenced. Such antineoplastic agents included camptothecin, etoposide, cisplatin, and doxorubicin. Changes
were monitored in the mitochondrial membrane potential (Δ y/m), O2 consumption, and intracellular levels of ATP and ROS.
Plasmid-encoded UCP2 was properly targeted to the mitochondrial inner membrane of HCTl \6υCP2 cells, which displayed lower Aψm and consumed more O2. ROS levels in HCTl \6UCP2 cells were consistently lower at baseline and in response to neoplastic agents. Moreover, HCTl \βUCP2 cells treated with the agents displayed decreased DNA laddering, annexinV staining, and caspase-3 cleavage in addition to a shift from PUMA to Bcl-XL expression, indicating diminished apoptosis. Pretreatment of HCTl 16 cells with the chemical uncoupler FCCP, a pure protonophore, reproduced the protective effect of UCP2 overexpression, confirming the primary role of proton leak in the anti-apoptotic action of UCP2. Finally, UCP2 silencing by siRNA and shRNA molecules in HCTl 16 and CaCo-2 cells resulted in higher Aψm, elevated ROS levels, and markedly increased rates of apoptosis in response to the agents.
The results indicated that UCP2 is a major negative determinant of chemotherapeutic drug-induced oxidative stress and apoptosis in HCTl 16 and CaCo-2 cells. Inhibition of UCP2 was found to reduce chemoresistance in cancer cells.
Example 2: Mitochondrial uncoupling protein-2 inhibits oxidative stress and p53-mediated apoptosis in HCTl 16 cells
The role of ROS in cancer development and growth involves effects on the activation and execution response orchestrated by the tumor suppressor p53 protein. Studies were carried out to determine whether UCP2 alters p53 -mediated apoptosis and promotes chemoresistance in
HCTl 16 cells. Endogenous UCP2 levels were altered by overexpressing the full-length human UCP2 in HCTl 16 cells (HCTl \6UCP2). Uncoupling-mediated changes, such as mitochondrial membrane potential (Aψm), O2 consumption, and intracellular levels of NADH/NAD, ATP, and ROS were monitored. Apoptosis induced by camptothecin (CPT) was evaluated using known methods.
Plasmid-encoded UCP2 was properly targeted to the mitochondrial inner membrane and caused lower Aψm and increased O2 consumption indicating increased uncoupling in resting HCTl 16^2 cells. In response to CPT, HCTl 16^2 cells showed decrease in DNA laddering, subGl fraction, annexin V expression, and caspase-3 cleavage in addition to decreased PUMA expression and increased BCI-XL expression. Moreover, ROS levels before and after CPT
treatment were consistently lower in HCTl I6υcp2 cells indicating a negative effect of UCP2 on oxidative stress. Surprisingly, camptothecin-induced p53 accumulation was markedly diminished in HCTl \6UCP2 cells, but the proteasome inhibitor MG132 prevented this effect, indicating that UCP2 overexpression enhances p53 degradation. UCP2 had no apparent effect on CPT-induced apoptosis in p53-/- HCTl 16 cells. The artificial uncoupler FCCP, which is a pure protonophore, reproduced the effects of UCP2 overexpression on ROS, apoptosis, and p53 accumulation in HCTl \6UCP2 cells, thus confirming the primary role of proton leak in these events.
The results indicate that UCP2 is a major determinant of CPT-induced oxidative stress and apoptosis in HCTl 16 cells. The marked antioxidant effect of uncoupling promotes p53 degradation indicates that ROS represents a major link between mitochondrial uncoupling proteins and the p53 response and identifies UCP2 as target for the prevention and treatment of cancer. References 1. Boss, O., Hagen, T., and Lowell, B. B. (2000) Uncoupling proteins 2 and 3. Potential regulators of mitochondrial energy metabolism. Diabetes 49, 149-156
2. Ryu, J. W., Hong, K. H., Maeng, J. H., Kim, J. B., Ko, J., Park, J. Y., Lee, K. U., Hong, M. K., Park, S. W., Kim, Y. H., and Han, K. H. (2004) Overexpression of uncoupling protein 2 in THPl monocytes inhibits beta2 integrin-mediated firm adhesion and transendothelial migration. Arterioscler Thromb Vase Biol 24, 864-870
3. Blanc, J., Alves-Guerra, M. C, Esposito, B., Rousset, S., Gourdy, P., Ricquier, D., Tedgui, A., Miroux, B., and Mallat, Z. (2003) Protective role of uncoupling protein 2 in atherosclerosis. Circulation 107, 388-390
4. Teshima, Y., Akao, M., Jones, S. P., and Marban, E. (2003) Uncoupling protein-2 overexpression inhibits mitochondrial death pathway in cardiomyocytes. Circ Res 93, 192-200
5. Mattiasson, G., Shamloo, M., Gido, G., Mathi, K., Tomasevic, G., Yi, S., Warden, C. H., Castilho, R. F., Melcher, T., Gonzalez-Zulueta, M., Nikolich, K., and Wieloch, T. (2003) Uncoupling protein-2 prevents neuronal death and diminishes brain dysfunction after stroke and brain trauma. Nat Med 9, 1062-1068
6. Negre-Salvayre, A., Hirtz, C, Carrera, G., Cazenave, R., Troly, M., Salvayre, R., Penicaud, L., and Casteilla, L. (1999) A role for uncoupling protein-2 as a regulator of mitochondrial hydrogen peroxide generation. FASEB J. 11, 809-815
7. Arsenijevic, D., Onuma, H., Pecqueur, C, Raimbault, S., Manning, B. S., Miroux, B., Couplan, E., Alves-Guerra, M. C, Goubern, M., Surwit, R., Bouillaud, F., Richard, D., Collins, S., and Ricquier, D. (2000) Disruption of the uncoupling protein-2 gene in mice reveals a role in immunity and reactive oxygen species production. Nat Genet 26, 435-439.
8. Horimoto, M., Fulop, P., Derdak, Z., Wands, J. R., and Baffy, G. (2004) Uncoupling protein-2 deficiency promotes oxidant stress and delays liver regeneration in mice. Hepatology 39, 386-392
9. Bai, Y., Onuma, H., Bai, X., Medvedev, A. V., Misukonis, M., Weinberg, J. B., Cao, W., Robidoux, J., Floering, L. M., Daniel, K. W., and Collins, S. (2005) Persistent NF-kappa B activation in Ucp2-/-mice leads to enhanced nitric oxide and inflammatory cytokine production. J Biol Chem 280, 19062-19069 10. Krauss, S., Zhang, C. Y., and Lowell, B. B. (2005) The mitochondrial uncoupling-protein homologues. Nat Rev MoI Cell Biol 6, 248-261
11. Turrens, J. F. (2003) Mitochondrial formation of reactive oxygen species. J Physiol 552, 335-344
12. Brand, M. D., Affourtit, C, Esteves, T. C, Green, K., Lambert, A. J., Miwa, S., Pakay, J. L., and Parker, N. (2004) Mitochondrial superoxide: production, biological effects, and activation of uncoupling proteins. Free Radic Biol Med 37, 755-767
13. Lenaz, G. (2001) The mitochondrial production of reactive oxygen species: mechanisms and implications in human pathology. IUBMB Life 52, 159-164
14. Harper, M. E., Antoniou, A., Villalobos-Menuey, E., Russo, A., Trauger, R., Vendemelio, M., George, A., Bartholomew, R., Carlo, D., Shaikh, A., Kupperman, J., Newell, E. W., Bespalov, I. A., Wallace, S. S., Liu, Y., Rogers, J. R., Gibbs, G. L., Leahy, J. L., Camley, R. E., Melamede, R., and Newell, M. K. (2002) Characterization of a novel metabolic strategy used by drug-resistant tumor cells. Faseb J 16, 1550-1557
15. Collins, P., Jones, C, Choudhury, S., Damelin, L., and Hodgson, H. (2005) Increased expression of uncoupling protein 2 in HepG2 cells attenuates oxidative damage and apoptosis.
Liver Int 25, 880-887
Other Embodiments
While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
What is claimed is:
Claims
1. A method for augmenting the antineoplastic effect of chemotherapy, comprising contacting a tumor cell with an antineoplastic composition and an inhibitor of UCP2 expression or activity.
2. The method of claim 1, wherein said antineoplastic composition is an inhibitor of cell proliferation.
3. The method of claim 1, wherein said antineoplastic composition is a cytotoxic agent.
4. The method of claim 1, wherein said antineoplastic composition is selected from the group consisting of irinotecan, topotecan, 9-nitro-20(S)-camptothecin and combinations thereof.
5. The method of claim 1, wherein said UCP2 inhibitor comprises genipen.
6. A composition comprising an inhibitor of UCP2 expression or activity and an antineoplastic agent in amounts that inhibit tumor cell proliferation.
7. A method for treating a cell proliferative disorder in a mammal comprising administering to the mammal a therapeutically effective amount of a combination of a UCP2 inhibitor and an antineoplastic agent.
8. A method for treating cancer with a combination therapy, comprising administering to a patient suffering from cancer a therapeutically effective amount of a neoplastic agent and an inhibitor of UCP2, wherein the therapy is enhanced by the synergistic effect of said neoplastic agent and said UCP2 inhibitor.
9. The method of claims 1, 6, 7, or 8, wherein the cancer is selected from the group consisting of leukemia, breast cancer, skin cancer, bone cancer, prostate cancer, liver cancer, lung cancer, brain cancer, cancer of the larynx, gallbladder, pancreas, rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck, colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma of both ulcerating and papillary type, metastatic skin carcinoma, osteosarcoma, Ewing's sarcoma, veticulum cell sarcoma, myeloma, giant cell tumor, small-cell lung tumor, gallstones, islet cell tumor, primary brain tumor, acute and chronic lymphocytic and granulocytic tumors, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuronms, intestinal ganglioneuromas, hyperplastic corneal nerve tumor, marfanoid habitus tumor, Wilm's tumor, seminoma, ovarian tumor, leiomyomater tumor, cervical dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion, mycosis fungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic and other sarcoma, malignant hypercalcemia, renal cell tumor, polycythemia vera, adenocarcinoma, glioblastoma multiforma, leukemias, lymphomas, malignant melanomas, and epidermoid carcinoma.
10. A method of identifying an inhibitor of UCP2 expression, comprising contacting a cell expressing UCP2 with a candidate compound and detecting UCP2 transcript or protein, wherein a reduction in the amount of transcript or protein in the presence of said compound compared to that in the absence of the compound indicates that said compound inhibits UCP2 expression.
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WO2017151855A1 (en) * | 2016-03-03 | 2017-09-08 | Mayo Foundation For Medical Education And Research | Materials and methods for increasing immune responses |
WO2019202473A1 (en) * | 2018-04-16 | 2019-10-24 | Université de Lausanne | Ucp2 inducing agents for the treatment of cancer resistant to immune checkpoint blockade |
CN117205225A (en) * | 2023-09-12 | 2023-12-12 | 湖南普瑞玛药物研究中心有限公司 | Application of geniposide in preparation of medicament for treating myeloproliferative neoplasm |
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Cited By (5)
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WO2017151855A1 (en) * | 2016-03-03 | 2017-09-08 | Mayo Foundation For Medical Education And Research | Materials and methods for increasing immune responses |
US11141432B2 (en) | 2016-03-03 | 2021-10-12 | Mayo Foundation For Medical Education And Research | Materials and methods for increasing immune responses |
WO2019202473A1 (en) * | 2018-04-16 | 2019-10-24 | Université de Lausanne | Ucp2 inducing agents for the treatment of cancer resistant to immune checkpoint blockade |
CN117205225A (en) * | 2023-09-12 | 2023-12-12 | 湖南普瑞玛药物研究中心有限公司 | Application of geniposide in preparation of medicament for treating myeloproliferative neoplasm |
CN117205225B (en) * | 2023-09-12 | 2024-05-31 | 湖南普瑞玛药物研究中心有限公司 | Application of geniposide in preparation of medicament for treating myeloproliferative neoplasm |
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