3-Bromopyruvate: targets and outcomes.

The pyruvate mimetic 3-bromopyruvate (3-BP) is generally presented as an inhibitor of glycolysis and has shown remarkable efficacy in not only preventing tumor growth, but even eradicating existant tumors in animal studies. We here review reported molecular targets of 3-BP and suggest that the very range of possible targets, which pertain to the altered energy metabolism of tumor cells, contributes both to the efficacy and the tumor specificity of the drug. Its in vivo efficacy is suggested to be due to a combination of glycolytic and mitochondrial targets, as well as to secondary effects affecting the tumor microenvironment. The cytotoxicity of 3-BP is less due to pyruvate mimicry than to alkylation of, e.g., key thiols. Alkylation of DNA/RNA has not been reported. More research is warranted to better understand the pharmacokinetics of 3-BP, and its potential toxic effects to normal cells, in particular those that are highly ATP-/mitochondrion-dependent.

Arsenic trioxide is highly cytotoxic to small cell lung carcinoma cells.

Small cell lung carcinoma (SCLC) is an extremely aggressive form of cancer and current treatment protocols are insufficient. SCLC have neuroendocrine characteristics and show phenotypical similarities to the childhood tumor neuroblastoma. As multidrug-resistant neuroblastoma cells are highly sensitive to arsenic trioxide (As2O3) in vitro and in vivo, we here studied the cytotoxic effects of As2O3 on SCLC cells. As2O3 induced pronounced cell death in SCLC cells at clinically relevant concentrations, and also at hypoxia. SCLC cells were more sensitive than non-SCLC cells to As2O3. Cell death was mainly due to necrosis, although apoptotic responses were also seen. A significant in vivo effect of As2O3 on SCLC growth was shown in a nude mice-xenograft model, although a fraction of the treated tumor-bearing animals did not respond. The nonresponding SCLC tumors differed in morphology and cell organization compared with treatment-responsive tumors, which in turn, showed decreased vascularization and higher expression of neuroendocrine markers compared with control tumors. Our results suggest a potential clinical application of As2O3 in SCLC therapy. In addition to cell death induction, antiangiogenic induction of differentiation may also be part of the in vivo effect of As2O3 on SCLC growth, as suggested by an increase in neuroendocrine markers in cultured cells.

Cisplatin overcomes Bcl-2-mediated resistance to apoptosis via preferential engagement of Bak: critical role of Noxa-mediated lipid peroxidation.

Increased expression of antiapoptotic Bcl-2 proteins confers therapeutic resistance in various cancer types. Targeting Bcl-2 proteins by small molecules or activating alternative pathways to bypass Bcl-2-mediated protection to promote apoptosis are two approaches to overcoming therapeutic resistance. Here, we show that cisplatin triggers a Bak-dependent pathway to induce apoptosis in Bcl-2-overexpressing MCF-7 cells. p53-mediated induction of Noxa expression, generation of lipid peroxidation end products and induction of Noxa-Mcl-1 interaction are necessary for this pathway to function. Although Puma is also induced by cisplatin treatment, it is not required for apoptosis. Similarly, reactive oxygen species production by cisplatin did not have any effect on cisplatin-induced apoptosis in MCF-7 Bcl-2 cells. Furthermore, p53 promotes cisplatin-induced apoptosis by directly binding and counteracting Bcl-x(L) antiapoptotic function. In conclusion, our findings suggest a novel mode of action for cisplatin to overcome Bcl-2-mediated protection against apoptosis, which requires preferential activation of Bak and p53-mediated upregulation of Noxa protein levels and lipid peroxidation.

Restriction of cisplatin induction of acute apoptosis to a subpopulation of cells in a three-dimensional carcinoma culture model.

Cisplatin is a clinically important chemotherapeutical agent used to treat epithelial malignancies. High concentrations (20-100 microM) of cisplatin have been used in numerous studies to induce apoptosis of carcinoma cells grown in monolayer culture over 24-48 hr. These conditions may not be relevant to 3-D tumor tissue in vivo and the importance of apoptosis for tumor response is controversial. We here studied the effects of cisplatin on a 3-D colon carcinoma in vitro model (multicellular spheroids). Cisplatin at a dose of 40 microM induced active caspase-3 preferentially in the peripheral 30 microm cell layer of spheroids, mainly during late stages (72-96 hr). The p53 response to cisplatin was also largely confined to peripheral cell layers. Despite the use of a high cisplatin concentration, a significant fraction of the cells in the spheroids survived treatment. A high proportion of surviving cells stained positive for beta-galactosidase, a marker of premature senescence. Cells growth-arrested by cisplatin treatment showed a higher spontaneous cell death rate than untreated proliferating cells. We propose that acute apoptosis is of minor significance for the overall response of carcinoma cells to cisplatin treatment.

Potentiation of chemotherapeutic drugs by energy metabolism inhibitors 2-deoxyglucose and etomoxir.

Inhibition of energy production as a strategy for potentiation of anticancer chemotherapy was investigated using 1 glycolysis inhibitor and 1 fatty acid beta-oxidation inhibitor-2-deoxyglucose and etomoxir, respectively, both known to be clinically well tolerated. Eighteen anticancer drugs were screened for potentiation by these inhibitors. 2-deoxyglucose potentiated acute apoptosis (24 hr) induced mainly by some, but not all, genotoxic drugs, whereas etomoxir had effect only on cisplatin. By contrast, etomoxir did potentiate the overall, 48 hr effects of some genotoxic drugs, and was in addition more efficient than deoxyglucose in potentiating the overall effects of several non-genotoxic drugs. Both types of potentiation were largely lost in the absence of p53. Because cisplatin was potentiated by both energy inhibitors in both types of assay, it was investigated at additional concentrations and over longer time. Both energy inhibitors strongly potentiated non-apoptotic concentrations of cisplatin in p53-wildtype as well as in p53-deficient, cisplatin-resistant HCT-116 colon carcinoma cells. Reduced ATP levels correlated with, but were not sole determinants, the antiproliferative effects. We conclude that the long-term effects of cisplatin potentiation are important and either p53-independent or improved by a lack of p53. We also conclude that although the potentiated drugs as yet have no obvious mechanistic factor in common, the strategy holds promise with genotoxic as well non-genotoxic anticancer drugs.

Cytokeratin-18 is a useful serum biomarker for early determination of response of breast carcinomas to chemotherapy.

PURPOSE: With a widening arsenal of cancer therapies available, it is important to develop therapy-specific predictive markers and methods to rapidly assess treatment efficacy. We here evaluated the use of cytokeratin-18 (CK18) as a serum biomarker for monitoring chemotherapy-induced cell death in breast cancer. EXPERIMENTAL DESIGN: Different molecular forms of CK18 (caspase cleaved and total) were assessed by specific ELISA assays. Drug-induced release of CK18 was examined from breast carcinoma cells and tissue. CK18 protein composition was examined in serum. CK18 levels were determined in serum from 61 breast cancer patients during docetaxel or cyclophosphamide/epirubicin/5-fluorouracil (CEF) therapy. RESULTS: Caspase-cleaved CK18 molecules were released from monolayer cultures and tumor organ cultures to the extracellular compartment. CK18 was present in complexes with other cytokeratins in serum. Such CK18 protein complexes are remarkably stable, leading to favorable performance of CK18 biomarker assays for clinical investigations. Docetaxel induced increased levels of caspase-cleaved CK18 in serum from breast cancer patients, indicating apoptosis. CEF therapy led to increases predominantly in uncleaved CK18, indicating induction of necrotic cell death in many tumors. The increase in total CK18 at 24 h of the first treatment cycle correlated to the clinical response to CEF therapy (P < 0.0001). CONCLUSIONS: Induction of necrotic cell death may explain the clinical efficacy of anthracycline-based therapy for breast carcinomas with defective apoptosis pathways. We suggest that CK18 biomarkers are useful for early prediction of the response to CEF therapy in breast cancer and may be useful biomarkers for clinical trials.

Doxorubicin requires the sequential activation of caspase-2, protein kinase Cdelta, and c-Jun NH2-terminal kinase to induce apoptosis.

Here, we identified caspase-2, protein kinase C (PKC)delta, and c-Jun NH2-terminal kinase (JNK) as key components of the doxorubicin-induced apoptotic cascade. Using cells stably transfected with an antisense construct for caspase-2 (AS2) as well as a chemical caspase-2 inhibitor, we demonstrate that caspase-2 is required in doxorubicin-induced apoptosis. We also identified PKCdelta as a novel caspase-2 substrate. PKCdelta was cleaved/activated in a caspase-2-dependent manner after doxorubicin treatment both in cells and in vitro. PKCdelta is furthermore required for efficient doxorubicin-induced apoptosis because its chemical inhibition as well as adenoviral expression of a kinase dead (KD) mutant of PKCdelta severely attenuated doxorubicin-induced apoptosis. Furthermore, PKCdelta and JNK inhibition show that PKCdelta lies upstream of JNK in doxorubicin-induced death. Jnk-deficient mouse embryo fibroblasts (MEFs) were highly resistant to doxorubicin compared with wild type (WT), as were WT Jurkat cells treated with SP600125, further supporting the importance of JNK in doxorubicin-induced apoptosis. Chemical inhibitors for PKCdelta and JNK do not synergize and do not function in doxorubicin-treated AS2 cells. Caspase-2, PKCdelta, and JNK were furthermore implicated in doxorubicin-induced apoptosis of primary acute lymphoblastic leukemia blasts. The data thus support a sequential model involving caspase-2, PKCdelta, and JNK signaling in response to doxorubicin, leading to the activation of Bak and execution of apoptosis.

Target specificity and off-target effects as determinants of cancer drug efficacy.

Targeted therapeutics are aimed to hit one or a few key cellular targets. Agents that target single signaling molecules (such as EGFR and IGF-R1) often show limited clinical activities, at least in the major groups of solid tumors. Nevertheless, some signaling inhibitors are effective in the treatment of previously difficult-to-treat diseases such as renal carcinoma. Similarly, these drugs inhibit multiple kinases and/or may display off-target activities. Inhibition of cellular targets such as the proteasome, heat-shock protein 90, and histone deacetylase induces complex cellular effects, and agents that inhibit these targets show promising clinical activities. Clinically effective targeted agents are therefore reminiscent of conventional agents such as cisplatin and doxorubicin, which are known to have several cellular targets. It is becoming increasingly clear that a comprehensive understanding of the spectrum of effects exerted by an anticancer agent is fundamental for understanding its efficacy and toxicity profile.

Mechanisms of action of DNA-damaging anticancer drugs in treatment of carcinomas: is acute apoptosis an "off-target" effect?

DNA damage induces apoptosis of cells of hematological origin. Apoptosis is also widely believed to be the major antiproliferative mechanism of DNA damaging anticancer drugs in other cell types, and a large number of laboratories have studied drug-induced acute apoptosis (within 24 hours) of carcinoma cells. It is, however, often overlooked that induction of apoptosis of carcinoma cells generally requires drug concentrations that are at least one order of magnitude higher than those required for loss of clonogenicity. This is true for different DNA damaging drugs such as cisplatin, doxorubicin and camptothecin. We here discuss apoptosis induction by DNA damaging agents using cisplatin as an example. Recent studies have shown that cisplatin induces caspase activation in enucleated cells (cytoplasts lacking a cell nucleus). Cisplatin-induced apoptosis in both cells and cytoplasts is associated with rapid induction of cellular reactive oxygen species and increases in [Ca(2+)](i). Cisplatin has also been reported to induce clustering of Fas/CD95 in the plasma membrane. Available data suggest that the primary responses to cisplatin-induced DNA damage are induction of long-term growth arrest ("premature cell senescence") and mitotic catastrophe, whereas acute apoptosis may be due to "off-target effects" not necessarily involving DNA damage.

Calpain-mediated Bid cleavage and calpain-independent Bak modulation: two separate pathways in cisplatin-induced apoptosis.

Calpain is a ubiquitous protease with potential involvement in apoptosis. We report that in human melanoma cells, cisplatin-induced calpain activation occurs early in apoptosis. Calpain activation and subsequent apoptosis were inhibited by calpeptin and PD150606, two calpain inhibitors with different modes of action. Furthermore, cisplatin induced cleavage of the BH3-only protein Bid, yielding a 14-kDa fragment similar to proapoptotic, caspase-cleaved Bid. However, Bid cleavage was inhibited by inhibitors of calpain, but not by inhibitors of caspases or of cathepsin L. Recombinant Bid was cleaved in vitro by both recombinant calpain and by lysates of cisplatin-treated cells. Cleavage was calpeptin sensitive, and the cleavage site was mapped between Gly70 and Arg71. Calpain-cleaved Bid induced cytochrome c release from isolated mitochondria. While calpeptin did not affect cisplatin-induced modulation of Bak to its proapoptotic conformation, a dominant-negative mutant of MEKK1 (dnMEKK) inhibited Bak modulation. dnMEKK did not, however, block Bid cleavage. The combination of dnMEKK and calpeptin had an additive inhibitory effect on apoptosis. In summary, calpain-mediated Bid cleavage is important in drug-induced apoptosis, and cisplatin induces at least two separate apoptotic signaling pathways resulting in Bid cleavage and Bak modulation, respectively.

Differentiation between cell death modes using measurements of different soluble forms of extracellular cytokeratin 18.

Cytokeratins are released from carcinoma cells by unclear mechanisms and are commonly used serum tumor markers (TPA, TPS, and CYFRA 21-1). We here report that soluble cytokeratin-18 (CK18) is released from human carcinoma cells during cell death. During necrosis, the cytosolic pool of soluble CK18 was released, whereas apoptosis was associated with significant release of caspase-cleaved CK18 fragments. These results suggested that assessments of different forms of CK18 in patient sera could be used to examine cell death modes. Therefore, CK18 was measured in local venous blood collected during operation of patients with endometrial tumors. In most patient sera, caspase-cleaved fragments constituted a minor fraction of total CK18, suggesting that tumor apoptosis is not the main mechanism for generation of circulating CK18. Monitoring of different CK18 forms in peripheral blood during chemotherapy of prostate cancer patients showed individual differences in the patterns of release. Importantly, several examples were observed where the increase of apoptosis-specific caspase-cleaved CK18 fragments constituted only a minor fraction of the total increase. These results suggest that cell death of epithelially derived tumors can be assessed in patient serum and suggest that tumor apoptosis may not necessarily be the dominating death mode in many tumors in vivo.

Interferon-alpha-induced apoptosis in U266 cells is associated with activation of the proapoptotic Bcl-2 family members Bak and Bax.

We have recently reported that the cytokine interferon-alpha (IFNalpha), commonly used in the treatment of cancer, induced a caspase-dependent apoptosis in tumor cell lines. The signaling mechanisms involved have not been defined. Here, we show that both proapoptotic Bcl-2 family members Bak and Bax were activated by IFNalpha, strictly in correlation with the induction of apoptosis. Using double stainings, we demonstrated that Bak was activated prior to cytochrome c (cyt c) release and caspase-3 activation, whereas activated Bax was only found in cells with released cyt c, mitochondrial depolarization, as well as activated caspase-3. Furthermore, IFNalpha-induced activation of Bak, and to a large extent also of Bax, was dependent on caspase activity. With the use of a panel of specific caspase inhibitors we found, however, that none of caspases-1 to -10 were responsible for this activation. Neither was the Ca(2+)-dependent protease calpain nor the stress-activated p38 SAPK pathway significantly involved. Overexpression of Bcl-2 blocked apoptosis induced by IFNalpha totally abolished Bak activation, as well as decreased the amount of activated Bax. We conclude that IFNalpha induces Bak and Bax activation via distinct mechanisms involving an unknown protease, and that their activation is regulated by Bcl-2.

Induction of endoplasmic reticulum stress by ellipticine plant alkaloids.

Anticancer drugs often show complex mechanisms of action, including effects on multiple cellular targets. Detailed understanding of these intricate effects is important for the understanding of cytotoxicity. In this study, we examined apoptosis induction by ellipticines, a class of cytotoxic plant alkaloids known to inhibit topoisomerase II. The potent ellipticine derivative 6-propanamine ellipticine (6-PA-ELL) induced rapid apoptosis in MDA-MB-231 breast cancer cells, preceded by a conformational change in Bak and cytochrome c release. Experiments using knock-out mouse embryo fibroblasts established that Bak was of particular importance for cytotoxicity. 6-PA-ELL increased the expression of the endoplasmic reticulum chaperones GRP78/BiP and GRP94, suggesting induction of endoplasmic reticulum stress. Induction of GRP78 expression was dependent on the endoplasmic reticulum stress response element (ERSE) of the GRP78 promoter. Examination of different ellipticine derivatives revealed a correlation between pro-apoptotic activity and the ability to induce GRP78 expression. Furthermore, 6-PA-ELL was found to induce splicing of the mRNA encoding the XBP1 transcription factor, characteristic of endoplasmic reticulum stress, and to induce activation of the endoplasmic reticulum-specific caspase-12 in mouse colon cancer cells. We finally demonstrate that 6-PA-ELL induces apoptotic signaling also in enucleated cells, consistent with the existence of a cytoplasmic target for this compound. Our data suggest that induction of endoplasmic reticulum stress may contribute to the cytotoxicity of ellipticines.

Determining tumor apoptosis and necrosis in patient serum using cytokeratin 18 as a biomarker.

Intracellular macromolecules are released from dying tumor cells and may subsequently be detected in patient blood. In this review, we will discuss the use of cytokeratin-18 as a serum biomarker for monitoring therapy-induced cell death. Cytokeratins are abundant intracellular proteins expressed by most types of carcinoma, but not by treatment-sensitive cells from bone marrow and other tissues. Release of cytokeratins into blood is therefore expected to show some specificity for tumor cell death. Cytokeratin-18 (CK18) is cleaved by caspases specifically during apoptosis, and the molecular form of this protein (caspase-cleaved vs. non-cleaved) released from dying tumor cells is therefore diagnostic as to the type of cell death (apoptosis vs. necrosis). Analyses of different CK18 forms in patient sera have suggested that tumor apoptosis may not necessarily be the dominating death mode in many tumors in vivo. Measurements of increased levels of CK18 in serum during therapy of prostate and breast cancer patients have been encouraging with regard to the possible future use of CK18 as a biomarker for monitoring therapy efficiency.

Potentiation of anti-cancer treatment by modulators of energy metabolism.

Oncogene-driven proliferative signaling in tumor cells requires comprehensive upregulation of cellular energy metabolism and macromolecule syntheses. These alterations are now known to include not only upregulated glycolysis, but also increased fatty acid metabolism, glutaminolysis, deregulated mitochondrial function and more. Many prospective targets for tumor-specific pharmacological modulation of metabolism have therefore been identified. While the prospective drugs do not necessarily show very high antitumor activity by themselves, they may by depriving tumor cells of energy and building blocks for repair and proliferation come to be of major clinical use as potentiators of standard chemotherapeutic drugs and/or radiation. To this end, not only inhibitors of specific enzyme functions are being investigated, but also drugs affecting the complex signaling networks that regulate organismal and cellular energy status. This review provides examples of how modulators of energy metabolism (MEMs) targetting different aspects of tumor cell metabolism have been found to potentiate cancer treatment in vitro and in vivo.

Conditional drug screening shows that mitotic inhibitors induce AKT/PKB-insensitive apoptosis.

The phosphatidylinositol 3-kinase (PI3K)/AKT pathway is frequently upregulated in human cancer. Activation of this pathway has been reported to be associated with resistance to various chemotherapeutical agents. We here used a chemical biology/chemical informatic approach to identify apoptotic mechanisms that are insensitive to activation of the PI3K/AKT pathway. The National Cancer Institute (NCI) Mechanistic Set drug library was screened for agents that induce apoptosis in colon carcinoma cells expressing a constitutively active form of AKT1. The cytotoxicity screening data available as self-organized maps at the Developmental Therapeutics Program (DTP) of the NCI was then used to classify the identified compounds according to mechanism of action. The results showed that drugs that interfere with the mitotic process induce apoptosis which is comparatively insensitive to constitutive AKT1 activity. The conditional screening approach described here is expected to be useful for identifying relationships between the state of activation of signaling pathways and sensitivity to anticancer agents.

3-Bromopyruvate as inhibitor of tumour cell energy metabolism and chemopotentiator of platinum drugs.

Tumour cells depend on aerobic glycolysis for adenosine triphosphate (ATP) production, making energy metabolism an interesting therapeutic target. 3-Bromopyruvate (BP) has been shown by others to inhibit hexokinase and eradicate mouse hepatocarcinomas. We report that similar to the glycolysis inhibitor 2-deoxyglucose (DG), BP rapidly decreased cellular ATP within hours, but unlike DG, BP concomitantly induced mitochondrial depolarization without affecting levels of reducing equivalents. Over 24h, and at equitoxic doses, DG reduced glucose consumption more than did BP. The observed BP-induced loss of ATP is therefore largely due to mitochondrial effects. Cell death induced over 24h by BP, but not DG, was blocked by N-acetylcysteine, indicating involvement of reactive oxygen species. BP-induced cytotoxicity was independent of p53. When combined with cisplatin or oxaliplatin, BP led to massive cell death. The anti-proliferative effects of low-dose platinum were strikingly potentiated also in resistant p53-deficient cells. Together with the reported lack of toxicity, this indicates the potential of BP as a clinical chemopotentiating agent.

Charting calcium-regulated apoptosis pathways using chemical biology: role of calmodulin kinase II.

BACKGROUND: Intracellular free calcium ([Ca2+]i) is a key element in apoptotic signaling and a number of calcium-dependent apoptosis pathways have been described. We here used a chemical biology strategy to elucidate the relative importance of such different pathways. RESULTS: A set of 40 agents ("bioprobes") that induce apoptosis was first identified by screening of a chemical library. Using p53, AP-1, NFAT and NF-kappaB reporter cell lines, these bioprobes were verified to induce different patterns of signaling. Experiments using the calcium chelator BAPTA-AM showed that Ca2+ was involved in induction of apoptosis by the majority of the bioprobes and that Ca2+ was in general required several hours into the apoptosis process. Further studies showed that the calmodulin pathway was an important mediator of the apoptotic response. Inhibition of calmodulin kinase II (CaMKII) resulted in more effective inhibition of apoptosis compared to inhibition of calpain, calcineurin/PP2B or DAP kinase. We used one of the bioprobes, the plant alkaloid helenalin, to study the role of CaMKII in apoptosis. Helenalin induced CaMKII, ASK1 and Jun-N-terminal kinase (JNK) activity, and inhibition of these kinases inhibited apoptosis. CONCLUSION: Our study shows that calcium signaling is generally not an early event during the apoptosis process and suggests that a CaMKII/ASK1 signaling mechanism is important for sustained JNK activation and apoptosis by some types of stimuli.

Acute apoptosis by cisplatin requires induction of reactive oxygen species but is not associated with damage to nuclear DNA.

Cisplatin is a broad-spectrum anticancer drug that is also widely used in experimental studies on DNA damage-induced apoptosis. Induction of apoptosis within 24-48 hr requires cisplatin concentrations that are at least one order of magnitude higher than the IC(50). Here, we show that such high, apoptosis-inducing cisplatin concentrations induce cellular superoxide formation and that apoptosis is inhibited by superoxide scavengers. The same concentration limit and the requirement for superoxide are also true for induction of caspase activation in enucleated cells (cytoplasts), showing that cisplatin-induced apoptosis occurs independently of nuclear DNA damage. In contrast, lower cisplatin concentrations, which do not induce acute apoptosis, are sufficient for induction of DNA damage signaling. We propose that the antiproliferative effects of cisplatin at IC(50) doses involve premature senescence and secondary, nonstress-induced apoptosis. The higher doses currently used in in vitro studies lead to acute, stress-induced apoptosis that involves induction of superoxide but is largely DNA damage-independent.