Assays for non-apoptotic cell death and uses thereof

Apoptosis, or programmed cell death, is a highly regulated way for an organism to selectively eliminate cells. This process plays an important role in embryogenesis, maintaining an organism’s size, and eliminating damaged or aberrant cells. The importance of apoptosis in human health is underscored by the many diseases resulting from aberrant apoptosis. Dysregulation of apoptosis has been linked to various cancers, neurological and cardiovascular disorders, and autoimmune diseases. Email:marketing@medicilon.com.cn web:www.medicilon.com

The invention relates to methods for identifying agents, which induce oxidative cell death in a tumor cell. The invention further relates to methods for identifying tumor cells, which are sensitive to agents that induce oxidative cell death. The invention also relates to methods for identifying subjects who are suffering from tumors, which are sensitive to agents that induce oxidative cell death.

The invention is directed to, inter alia, a non-apoptotic mechanism and assays for cell death induced by genotype-selective anti-tumor drugs. The invention is further directed to uses of assays of non-apoptotic cell death assay to identify agents which are genotype-selective anti-tumor drugs.

In certain aspects, the invention provides methods for identifying an agent, which induces oxidative cell death in tumor cells, the method comprising: determining VDAC level in a tumor cell, contacting the tumor cell with an agent, and determining whether the tumor cell dies via oxidative cell death, and wherein a tumor cell which dies via oxidative death indicates that the agent induces oxidative cell death. In certain embodiments, oxidative cell death is defined by the presence of oxidative species, and/or decreased protein level of VDAC1, VDAC 2, VDAC 3 or any combination thereof. In other embodiments, oxidative cell death is defined by the presence of oxidative species, and/or decreased protein level of VDAC1, VDAC 2, VDAC 3 or any combination thereof, and the absence of any one of a number of molecular markers which are associated with cell death mechanisms such as apoptosis, necrosis, autophagy, and so forth. Determining of VDAC level can be done by any suitable known method in the art.

In other aspects, the invention provides methods for identifying an agent, which induces oxidative cell death in tumor cells, the method comprising: increasing VDAC level in a cell, contacting the cell with an agent, and determining whether the cell dies via oxidative cell death, wherein a cell which dies via oxidative death indicates that the agent induces oxidative cell death. In certain embodiments, the method increases the expression of VDAC1, VDAC 2, VDAC 3 or any combination thereof. In certain embodiments, VDAC expression can be increased by any suitable method known in the art, including nucleofection with vector carrying nucleic acid encoding VDAC, stable transfection with nucleic acid encoding VDAC, treatment with any suitable agent which increases VDAC expression. In some embodiments, such agents can upregulate VDAC expression by targeting or downregulating inhibitors of VDAC expression. In other embodiments, such agents can target upstream effectors of VDAC function and expression.

In other aspects, the invention provides a method for identifying an agent which induces oxidative cell death in tumor cells, the method comprising: providing a tumor cell, contacting the tumor cell with an agent, and determining whether the tumor cell dies via oxidative cell death assay, wherein oxidative cell death is determined by (I) detecting: (i) an increased level of oxidative species in the cell; or (ii) a decreased level of VDAC expression in the cell, and (II) identifying one or more of: (i) a lack of caspase 3 cleavage or activation; (ii) a lack of cytochrome C release; (iii) a lack of PARP cleavage or activation; (iv) a lack of Annexin V staining; (v) lack of alterations in chromatin morphology; (vi) a lack of nuclear DNA laddering; (vii) a lack of TUNEL staining of nuclear DNA; (viii) a lack of depletion of ATP levels.

In certain embodiments, the methods of the present invention can be optionally performed in the presence or absence of a second agent selected from the group consisting of: inhibitors of mitochondria-generated oxidative species, iron chelators, and anti-oxidants. In certain embodiments of the methods of the present invention, determining cell viability compares viability in the presence or absence of the second agent, wherein loss of cell viability in the absence of the second agent is indicative of an agent which induces oxidative cell death. An agent which induces cell death only in the absence of inhibitors of mitochondria-generated oxidative species, iron chelators, and anti-oxidants, is indicative of an agent which induces oxidative cell death.

In certain embodiments, the methods of the present invention further comprise determining whether mitochondrial morphology is altered, wherein altered mitochondrial morphology is indicative of an agent which induced an oxidative cell death. In certain embodiments, altered morphology can be detected when mitochondria are enlarged, or fused.

In other aspects, the present invention provides methods for identifying an agent which induces oxidative cell death in tumor cells, the method comprising: providing isolated mitochondria expressing VDAC protein, wherein VDAC protein is VDAC1, VDAC2, or VDAC3, or any isoform thereof, or any combination thereof; contacting the cellular fraction with an agent; and determining whether the agent alters permeability of the outer mitochondrial membrane, wherein an increase in the permeability of the outer mitochondrial membrane is indicative of an agent which induces a non-apoptotic oxidative cell death. In certain embodiments, the isolated mitochondria are in a cellular fraction comprising mitochondria. In other embodiments, the isolated mitochondria are purified mitochondria in a lipid bi-layer. In certain embodiments, determining whether the agent alters permeability of the outer mitochondrial membrane is done by measuring the levels NADH transport. In other embodiments, whether the agent alters permeability of the outer mitochondrial membrane is done by measuring the levels ATP transport.

In other aspects, the invention provides methods for identifying an agent which induces oxidative cell death in tumor cells, the method comprising: providing a tumor cell expressing a fluorescently labeled VDAC protein, wherein the VDAC protein is VDAC1, VDAC2, or VDAC3, or any isoform thereof, or any combination thereof; contacting the tumor cell with an agent; determining cell viability, and measuring the fluorescent signal due to the fluorescently labeled VDAC protein, wherein a decrease in cell viability and a decrease in fluorescence due to the fluorescently labeled VDAC protein is indicative of an agent which induces an oxidative cell death.

In other aspects, the invention provides methods for identifying an agent which induces oxidative cell death in tumor cells, the method comprising: providing a tumor cell expressing a fluorescently labeled VDAC protein comprising two different fluorescent labels, wherein the labeled VDAC protein exhibits fluorescent emission at a first and second wavelength when the channel is open, or a first, second and third (FRET) wavelength when the channel is closed; contacting the tumor cell with an agent; determining cell viability, and measuring the fluorescent signal due to the fluorescently labeled VDAC protein, wherein a decrease in cell viability and a decrease in fluorescence due to FRET in the labeled VDAC protein is indicative of an agent which induces an oxidative cell death. In certain embodiments, the fluorescently labeled VDAC protein is VDAC1, VDAC 2, or VDAC3, or any isoform thereof, or any combination thereof.

In certain aspects, the present invention provides methods for determining susceptibility of a tumor cell to an agent which induces an oxidative cell death, the method comprising: providing a tumor cell and a syngeneic non-tumor cell, and measuring a level of VDAC in the tumor cell and the non-tumor cell, wherein an increase in the level of VDAC in the tumor cell compared to the VDAC level in the non-tumor cell is indicative of a tumor cell, which is susceptible to an agent that induces oxidative cell death. In certain embodiments, VDAC protein level is measured by any suitable method known in the art. In another embodiment, VDAC mRNA level is measured by any suitable method known in the art. In certain embodiments, VDAC protein is VDAC1, VDAC2 or VDAC3, or any isoform, or any combination thereof.