Apoptolidin, a selective cytotoxic agent, is an inhibitor of F0F1-ATPase.

BACKGROUND: Apoptolidin is a macrolide originally identified on the basis of its ability to selectively kill E1A and E1A/E1B19K transformed rat glial cells while not killing untransformed glial cells. The goal of this study was to identify the molecular target of this newly discovered natural product. RESULTS: Our approach to uncovering the mechanism of action of apoptolidin utilized a combination of molecular and cell-based pharmacological assays as well as structural comparisons between apoptolidin and other macrocyclic polyketides with known mechanism of action. Cell killing induced by apoptolidin was independent of p53 status, inhibited by BCL-2, and dependent on the action of caspase-9. PARP was completely cleaved in the presence of 1 microM apoptolidin within 6 h in a mouse lymphoma cell line. Together these results suggested that apoptolidin might target a mitochondrial protein. Structural comparisons between apoptolidin and other macrolides revealed significant similarity between the apoptolidin aglycone and oligomycin, a known inhibitor of mitochondrial F0F1-ATP synthase. The relevance of this similarity was established by demonstrating that apoptolidin is a potent inhibitor of the F0F1-ATPase activity in intact yeast mitochondria as well as Triton X-100-solubilized ATPase preparations. The K(i) for apoptolidin was 4-5 microM. The selectivity of apoptolidin in the NCI-60 cell line panel was found to correlate well with that of several known anti-fungal natural products that inhibit the eukaryotic mitochondrial F0F1-ATP synthase. SIGNIFICANCE: Although the anti-fungal activities of macrolide inhibitors of the mitochondrial F0F1-ATP synthase such as oligomycin, ossamycin and cytovaricin are well-documented, their unusual selectivity toward certain cell types is not widely appreciated. The recent discovery of apoptolidin, followed by the demonstration that it is an inhibitor of the mitochondrial F0F1-ATP synthase, highlights the potential relevance of these natural products as small molecules to modulate apoptotic pathways. The mechanistic basis for selective cytotoxicity of mitochondrial ATP synthase inhibitors is discussed.

Understanding and exploiting the mechanistic basis for selectivity of polyketide inhibitors of F(0)F(1)-ATPase.

Recently, a family of polyketide inhibitors of F(0)F(1)-ATPase, including apoptolidin, ossamycin, and oligomycin, were shown to be among the top 0.1% most cell line selective cytotoxic agents of 37, 000 molecules tested against the 60 human cancer cell lines of the National Cancer Institute. Many cancer cells maintain a high level of anaerobic carbon metabolism even in the presence of oxygen, a phenomenon that is historically known as the Warburg effect. A mechanism-based strategy to sensitize such cells to this class of potent small molecule cytotoxic agents is presented. These natural products inhibit oxidative phosphorylation by targeting the mitochondrial F(0)F(1) ATP synthase. Evaluation of gene expression profiles in a panel of leukemias revealed a strong correlation between the expression level of the gene encoding subunit 6 of the mitochondrial F(0)F(1) ATP synthase (known to be the binding site of members of this class of macrolides) and their sensitivity to these natural products. Within the same set of leukemia cell lines, comparably strong drug-gene correlations were also observed for the genes encoding two key enzymes involved in central carbon metabolism, pyruvate kinase, and aspartate aminotransferase. We propose a simple model in which the mitochondrial apoptotic pathway is activated in response to a shift in balance between aerobic and anaerobic ATP biosynthesis. Inhibitors of both lactate formation and carbon flux through the Embden-Meyerhof pathway significantly sensitized apoptolidin-resistant tumors to this drug. Nine different cell lines derived from human leukemias and melanomas, and colon, renal, central nervous system, and ovarian tumors are also sensitized to killing by apoptolidin.

Gene microarray identification of redox and mitochondrial elements that control resistance or sensitivity to apoptosis.

Multigenic programs controlling susceptibility to apoptosis in response to ionizing radiation have not yet been defined. Here, using DNA microarrays, we show gene expression patterns in an apoptosis-sensitive and apoptosis-resistant murine B cell lymphoma model system both before and after irradiation. From the 11,000 genes interrogated by the arrays, two major patterns emerged. First, before radiation exposure the radioresistant LYar cells expressed significantly greater levels of message for several genes involved in regulating intracellular redox potential. Compared with LYas cells, LYar cells express 20- to 50-fold more mRNA for the tetraspanin CD53 and for fructose-1,6-bisphosphatase. Expression of both of these genes can lead to the increase of total cellular glutathione, which is the principle intracellular antioxidant and has been shown to inhibit many forms of apoptosis. A second pattern emerged after radiation, when the apoptosis-sensitive LYas cells induced rapid expression of a unique cluster of genes characterized by their involvement in mitochondrial electron transport. Some of these genes have been previously recognized as proapoptotic; however others, such as uncoupling protein 2, were not previously known to be apoptotic regulatory proteins. From these observations we propose that a multigenic program for sensitivity to apoptosis involves induction of transcripts for genes participating in mitochondrial uncoupling and loss of membrane potential. This program triggers mitochondrial release of apoptogenic factors and induces the "caspase cascade." Conversely, cells resistant to apoptosis down-regulate these biochemical pathways, while activating pathways for establishment and maintenance of high intracellular redox potential by means of elevated glutathione.

Redox aspects of Bcl-2 function.

The oncogene Bcl-2 has attracted recent research attention as recognition of the importance of Bcl-2 control over apoptosis commitment in disease development and clinical response to therapy has been targeted for pharmacological intervention. Much of the basic science research regarding Bcl-2 has focused on the role that Bcl-2 plays in directly regulating mitochondrial function. This has come about because of Bcl-2's localization to mitochondrial membranes and its reported interaction with the mitochondrial megachannel. During the time that the mitochondrial function of Bcl-2 was being investigated, a smaller, yet potentially as important, role for Bcl-2 was being pursued by investigators who were following up the initial study of Bcl-2 knockout mice. These mice expressed a phenotype consistent with that of mice exposed to chronic oxidative stress. This research into the redox aspects of Bcl-2 function has led to a hypothesis that Bcl-2-expressing cells have enhanced antioxidant capacities that suppress oxidative stress signals generated during the initiation phase of many apoptotic pathways. This review will further develop the idea of Bcl-2's role in regulating cellular redox pathways associated with apoptosis, as well as integrate recently reported evidence that ties the antioxidant effects of Bcl-2 to mitochondrial function, thereby unifying both mitochondrial and redox aspects of Bcl-2 function.

BCL-2 and glutathione: alterations in cellular redox state that regulate apoptosis sensitivity.

The importance of intracellular glutathione (GSH) in the pathology of disease, particularly cancer, has long been appreciated. However the ubiquitous nature of GSH has made it difficult to ascribe to a specific molecular mechanism in disease fulfillment. In addition, in all but a few cases, the underlying genetic regulation of the cellular redox state disrupted in disease has not been well described. Early identification of the importance of intracellular GSH to detoxification reactions has now led to investigating the potential importance that glutathione chemistry has on signal transduction and molecular regulation of cellular physiology. Here new relationships between the cellular redox state and the apoptotic regulatory protein BCL-2 will be described with emphasis on potential mechanisms by which GSH can alter cellular physiology in addition to its role in detoxification.

Analysis of redox regulation of cytochrome c-induced apoptosis in a cell-free system.

In this study, we investigated the importance of redox and Bcl-2 status on cytochrome c-mediated apoptosis. Two mouse lymphoma cell lines, LYas and LYar that express Bcl-2 protein at different levels, were used to reconstitute a cell-free system. Cytoplasmic extracts made from apoptosis-sensitive LYas cells 2.5 h after exposure to 5 Gy gamma-radiation were able to induce apoptosis in isolated nuclei, whereas extracts made from LYas cells at time points earlier than 2. 5 h, or from Bcl-2-overexpressing, apoptosis-resistant LYar cells at all time points after irradiation were inactive. Apoptotic activity was restored to inactive extracts by the addition of oxidized but not reduced cytochrome c. Cytochrome c reductase was able to inhibit apoptosis in extracts made from LYas cells 2.5 h after irradiation and LYar extracts activated by addition of oxidized cytochrome c. Antioxidants, but not oxidant defensive enzymes, blocked apoptosis implying that antioxidants might alter the redox state of factors important in mediating apoptosis. These findings confirm the importance of cellular redox state during apoptosis and are consistent with a role for Bcl-2 in regulating this redox state.

Bcl-2 expression causes redistribution of glutathione to the nucleus.

In this study we used HeLa cells transfected with a conditional Bcl-2 expression construct to study the effects of Bcl-2 on reduced glutathione (GSH) metabolism. Our previous work demonstrated that depletion of GSH by culturing cells in tissue culture medium lacking the amino acids cysteine and methionine, essential for GSH biosynthesis, caused cells overexpressing Bcl-2 to become sensitized to apoptotic induction. Here we report that Bcl-2 also dramatically alters GSH compartmentalization. Cellular distribution of GSH, assayed by confocal microscopy, revealed that when Bcl-2 expression was suppressed GSH was uniformly distributed primarily in the cytosol, whereas overexpression of Bcl-2 led to a relocalization of GSH into the nucleus. Isolated nuclei readily accumulated radiolabeled GSH and maintained higher nuclear GSH concentration in direct relation to Bcl-2 nuclear protein levels. Moreover, exogenous GSH blocked apoptotic changes and caspase activity in isolated nuclei exposed to the pro-apoptotic protease granzyme B. Our results indicate that one of the functions of Bcl-2 is to promote sequestration of GSH into the nucleus, thereby altering nuclear redox and blocking caspase activity as well as other nuclear alterations characteristic of apoptosis. We speculate that this mechanism contributes to the suppression of apoptosis in cells with elevated Bcl-2 levels.

Reversing drug resistance in bcl-2-expressing tumor cells by depleting glutathione.

The immense research effort in cancer cell physiology has led to an appreciation of the molecular and biochemical pathways that regulate cellular responses to endogenous and exogenous insults. Similarly, in tumor cells, there are multiple overlapping pathways that, once activated, impart resistance to therapeutic intervention. The multi drug resistance pathway is one such pathway. In this review, we will present current ideas concerning a mechanism of tumor cell resistance that involves the inability to undergo apoptosis. The bcl-2 family of proteins are known to regulate apoptosis in response to a wide variety of toxic agents. Additionally, recent evidence points to bcl-2 involvement in the regulation of antioxidant pathways mediated by glutathione. This new information will be discussed in some detail and strategies for overcoming these resistance mechanisms that may have clinical utility will be presented.

Resistance to radiation-induced apoptosis in Bcl-2-expressing cells is reversed by depleting cellular thiols.

The mechanism by which Bcl-2 oncogene expression inhibits radiation-induced apoptosis has been investigated in two mouse lymphoma cell lines: line LY-as is radiation sensitive, displays substantial radiaton-induced apoptosis, and expresses low levels of Bcl-2; line LY-ar is radiation-resistant, displays a low apoptosis propensity, and expresses 30-fold higher amount of Bcl-2 protein than does the sensitive line. We observed that upon incubation in cystine/methionine-free (C/M-) medium, radiation-induced apoptosis in the LY-ar cells was restored to levels comparable to that seen in the LY-as cells. lntracellular glutathione (GSH) concentrations in LY-ar cells incubated in C/M- medium plummeted to 50% of control values within 2 h. LY-ar cells treated with diethyl maleate (DEM) or diamide, agents that deplete cellular thiols, had increased susceptibility to radiation-induced apoptosis in a manner similar to C/M- medium. These results are consistent with the general idea that Bcl-2 expression blocks apoptosis through an antioxidant pathway that involves cellular thiols. That Bcl-2-expressing tumor cells can be sensitized by exogeneous agents that modify cellular thiols offers strategies for overcoming such resistance.

Modulating Ca2+ in radiation-induced apoptosis suppresses DNA fragmentation but does not enhance clonogenic survival.

The role of intracellular Ca2+ in radiation-induced apoptosis was studied in a cell line derived from a mouse B-cell lymphoma (LY-TH). These cells had previously been shown to be sensitive to radiation and to die by apoptosis. The cell permeant Ca2+ chelator (acetyoxymethyl-)1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tet raacetic acid (BAPTA/AM) reduced the DNA fragmentation characteristic of apoptosis but had no effect on clonogenic survival. Intracellular Ca2+ concentrations measured using the fluorescent indicator fura-2 only slowly increased over control values after cells were irradiated unlike the rapid increase observed in other systems. Our results indicate that modulating the endpoint of DNA fragmentation using some agents may not necessarily alter the cells' commitment to death as determined by clonogenic survival assays. This suggests that such agents play a role downstream of early initiation steps in apoptosis and modulate only particular features of apoptosis after the cell is committed to die.

Radiation-induced apoptosis in sensitive and resistant cells isolated from a mouse lymphoma.

Cells were isolated from a mouse lymphoma (LY-TH) and grown in vitro. They were susceptible to radiation-induced apoptosis after low doses with the appearance of endonucleolytically fragmented DNA 1 h after irradiation. Four hours after receiving 5 Gy, 80% of the DNA was endonucleolytically cleaved. Apoptosis induction by DNA double-strand break (dsb) formation was more effective compared with induction by single-strand break (ssb) formation. After long-term culturing, LY-TH cultures became refractory to apoptosis. Apoptosis-permissive cells (LY-as, cloned from LY-TH cells) were three times more radiosensitive than clonally expanded apoptosis-refractory cells (LY-ar). Low dose-rate irradiation and maintenance at 25 degrees C for 5 h postirradiation was sparing in LY-ar but not LY-as cells, suggesting a repair deficiency in LY-as cells. Analysis of dsb rejoining kinetics revealed no difference in the initial phase of dsb rejoining. After 1 h, however, relative dsbs in the LY-as variant increased as endonucleolytic cleavage was initiated. Signalling for radiation-induced apoptosis in LY-as cells was independent of the DNA dsb repair pathway and appeared determined by initial events, whereas in LY-ar cells, because of an inhibition in the apoptotic pathway, survival was enhanced and modifiable by repair processes.

Biochemical modulation of radiation-induced apoptosis in murine lymphoma cells.

Considerable effort in our laboratory has been directed toward characterizing the role of apoptosis as a mode of cell death in model tumors irradiated in vivo. These studies have shown that apoptosis is an important response in some tumors, correlating with tumor growth delay and tumor cure. However, the response is heterogeneous among both the various tumors examined and the cells in a given tumor, suggesting that the propensity for cells to undergo apoptosis upon irradiation is regulated by unknown factors in tumors. To develop a model system for investigating these regulatory pathways in vitro at the molecular and biochemical levels, we have established cells from a tumor that displays a dramatic apoptotic response in vivo, the TH lymphoma, in cell culture. In this article, we review some of the results of our studies using this model system. To date, we have shown that the dose-response relationship and kinetics of the development of apoptosis for these cells in culture are similar to what we observed for the tumor response in vivo. Moreover, the roles of calcium and signal transduction pathways as important regulatory factors in radiation-induced apoptosis have been defined in this system. Ultimately such investigations may yield the insight necessary for designing protocols to modulate apoptosis biochemically in irradiated normal and tumor tissues to therapeutic advantage.

L-asparaginase kills lymphoma cells by apoptosis.

Microscopic examination of histological sections of lymph nodes from a canine case of malignant lymphoma at 4 h after treatment with L-asparaginase revealed massive destruction of neoplastic cells by what was consistent with apoptosis morphologically. Apoptosis as the mode of cell death after asparaginase treatment was confirmed in a mouse lymphoma cell line (LY-TH) by the characteristic fragmentation of DNA into oligonucleosome-sized pieces and by the morphological changes consistent with apoptosis following treatment in vitro. Applied to these cells, asparaginase was found to be most cytotoxic over the range of 1-10 IU/ml. Even after 4 h of asparaginase treatment at 100 IU/ml, protein synthesis was reduced by only one-half, yet DNA fragmentation reached 40%. Other agents that affect protein synthesis (cycloheximide and actinomycin D) caused apoptosis as well; however, agents (radiation, prednisolone, and VP-16) whose mechanisms are different from inhibition of protein synthesis also caused apoptosis. As such, it seems unlikely that protein depletion per se and/or the elimination of specific short-lived proteins is the triggering event that leads to cell death. It is more likely that the suspension of cellular proliferation commits cells to apoptosis after asparaginase treatment.