Mechanisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT-737 in acute myeloid leukemia.

BCL-2 proteins are critical for cell survival and are overexpressed in many tumors. ABT-737 is a small-molecule BH3 mimetic that exhibits single-agent activity against lymphoma and small-cell lung cancer in preclinical studies. We here report that ABT-737 effectively kills acute myeloid leukemia blast, progenitor, and stem cells without affecting normal hematopoietic cells. ABT-737 induced the disruption of the BCL-2/BAX complex and BAK-dependent but BIM-independent activation of the intrinsic apoptotic pathway. In cells with phosphorylated BCL-2 or increased MCL-1, ABT-737 was inactive. Inhibition of BCL-2 phosphorylation and reduction of MCL-1 expression restored sensitivity to ABT-737. These data suggest that ABT-737 could be a highly effective antileukemia agent when the mechanisms of resistance identified here are considered.

Small-molecule antagonists of apoptosis suppressor XIAP exhibit broad antitumor activity.

Apoptosis resistance commonly occurs in cancers, preventing activation of Caspase family cell death proteases. XIAP is an endogenous inhibitor of Caspases overexpressed in many cancers. We developed an enzyme derepression assay, based on overcoming XIAP-mediated suppression of Caspase-3, and screened mixture-based combinatorial chemical libraries for compounds that reversed XIAP-mediated inhibition of Caspase-3, identifying a class of polyphenylureas with XIAP-inhibitory activity. These compounds, but not inactive structural analogs, stimulated increases in Caspase activity, directly induced apoptosis of many types of tumor cell lines in culture, and sensitized cancer cells to chemotherapeutic drugs. Active compounds also suppressed growth of established tumors in xenograft models in mice, while displaying little toxicity to normal tissues. These findings validate IAPs as targets for cancer drug discovery.

An inhibitor of Bcl-2 family proteins induces regression of solid tumours.

Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-X(L) and Bcl-2, are overexpressed in many cancers and contribute to tumour initiation, progression and resistance to therapy. Bcl-X(L) expression correlates with chemo-resistance of tumour cell lines, and reductions in Bcl-2 increase sensitivity to anticancer drugs and enhance in vivo survival. The development of inhibitors of these proteins as potential anti-cancer therapeutics has been previously explored, but obtaining potent small-molecule inhibitors has proved difficult owing to the necessity of targeting a protein-protein interaction. Here, using nuclear magnetic resonance (NMR)-based screening, parallel synthesis and structure-based design, we have discovered ABT-737, a small-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w, with an affinity two to three orders of magnitude more potent than previously reported compounds. Mechanistic studies reveal that ABT-737 does not directly initiate the apoptotic process, but enhances the effects of death signals, displaying synergistic cytotoxicity with chemotherapeutics and radiation. ABT-737 exhibits single-agent-mechanism-based killing of cells from lymphoma and small-cell lung carcinoma lines, as well as primary patient-derived cells, and in animal models, ABT-737 improves survival, causes regression of established tumours, and produces cures in a high percentage of the mice.

Apogossypol derivatives as antagonists of antiapoptotic Bcl-2 family proteins.

Guided by a combination of nuclear magnetic resonance binding assays and computational docking studies, we synthesized a library of 5,5' substituted Apogossypol derivatives as potent Bcl-XL antagonists. Each compound was subsequently tested for its ability to inhibit Bcl-XL in an in vitro fluorescence polarization competition assay and exert single-agent proapoptotic activity in human cancer cell lines. The most potent compound BI79D10 binds to Bcl-XL, Bcl-2, and Mcl-1 with IC50 values of 190, 360, and 520 nmol/L, respectively, and potently inhibits cell growth in the H460 human lung cancer cell line with an EC50 value of 680 nmol/L, expressing high levels of Bcl-2. BI79D10 also effectively induces apoptosis of the RS11846 human lymphoma cell line in a dose-dependent manner and shows little cytotoxicity against bax-/-bak-/- mouse embryonic fibroblast cells, in which antiapoptotic Bcl-2 family proteins lack a cytoprotective phenotype, implying that BI79D10 has little off-target effects. BI79D10 displays in vivo efficacy in transgenic mice, in which Bcl-2 is overexpressed in splenic B cells. Together with its improved plasma and microsomal stability relative to Apogossypol, BI79D10 represents a lead compound for the development of novel apoptosis-based therapies for cancer.

Bcl-B expression in human epithelial and nonepithelial malignancies.

PURPOSE: Apoptosis plays an important role in neoplastic processes. Bcl-B is an antiapoptotic Bcl-2 family member, which is known to change its phenotype upon binding to Nur77/TR3. The expression pattern of this protein in human malignancies has not been reported. EXPERIMENTAL DESIGN: We investigated Bcl-B expression in normal human tissues and several types of human epithelial and nonepithelial malignancy by immunohistochemistry, correlating results with tumor stage, histologic grade, and patient survival. RESULTS: Bcl-B protein was strongly expressed in all normal plasma cells but found in only 18% of multiple myelomas (n = 133). Bcl-B immunostaining was also present in normal germinal center centroblasts and centrocytes and in approximately half of diffuse large B-cell lymphoma (n = 48) specimens, whereas follicular lymphomas (n = 57) did not contain Bcl-B. In breast (n = 119), prostate (n = 66), gastric (n = 180), and colorectal (n = 106) adenocarcinomas, as well as in non-small cell lung cancers (n = 82), tumor-specific overexpression of Bcl-B was observed. Bcl-B expression was associated with variables of poor prognosis, such as high tumor grade in breast cancer (P = 0.009), microsatellite stability (P = 0.0002), and left-sided anatomic location (P = 0.02) of colorectal cancers, as well as with greater incidence of death from prostate cancer (P = 0.005) and shorter survival of patients with small cell lung cancer (P = 0.009). Conversely, although overexpressed in many gastric cancers, Bcl-B tended to correlate with better outcome (P = 0.01) and more differentiated tumor histology (P < 0.0001). CONCLUSIONS: Tumor-specific alterations in Bcl-B expression may define subsets of nonepithelial and epithelial neoplasms with distinct clinical behaviors.

Gambogic acid is an antagonist of antiapoptotic Bcl-2 family proteins.

The natural product gambogic acid (GA) has been reported to have cytotoxic activity against tumor cells in culture and was identified as an active compound in a cell-based high-throughput screening assay for activators of caspases, proteases involved in apoptosis. Using the antiapoptotic Bcl-2 family protein, Bfl-1, as a target for screening of a library of natural products, we identified GA as a competitive inhibitor that displaced BH3 peptides from Bfl-1 in a fluorescence polarization assay. Analysis of competition for BH3 peptide binding revealed that GA inhibits all six human Bcl-2 family proteins to various extents, with Mcl-1 and Bcl-B the most potently inhibited [concentrations required for 50% inhibition (IC(50)), < 1 micromol/L]. Competition for BH3 peptide binding was also confirmed using a time-resolved fluorescence resonance energy transfer assay. GA functionally inhibited the antiapoptotic Bcl-2 family proteins as shown by experiments using isolated mitochondria in which recombinant purified Bcl-2 family proteins suppress SMAC release in vitro, showing that GA neutralizes their suppressive effects on mitochondria in a concentration-dependent manner. GA killed tumor cell lines via an apoptotic mechanism, whereas analogues of GA with greatly reduced potency at BH3 peptide displacement showed little or no cytotoxic activity. However, GA retained cytotoxic activity against bax-/-bak-/- cells in which antiapoptotic Bcl-2 family proteins lack a cytoprotective phenotype, implying that GA also has additional targets that contribute to its cytotoxic mechanism. Altogether, the findings suggest that suppression of antiapoptotic Bcl-2 family proteins may be among the cytotoxic mechanisms by which GA kills tumor cells.

Comparison of pharmacokinetic and metabolic profiling among gossypol, apogossypol and apogossypol hexaacetate.

PURPOSE: To characterize the stability, pharmacokinetics and metabolism of analogs of gossypol, apogossypol and apogossypol hexaacetate to provide a basis for comparison. METHODS: Gossypol, apogossypol and apogossypol hexaacetate were incubated in plasma or liver microsomes from various species, or administered to mice, respectively, from which the stability, metabolism and pharmacokinetic profiles of these analogs were quantitatively determined using a liquid chromatography-mass spectrometry (LC/MS/MS) method. RESULTS: In various species of plasma, apogossypol and gossypol exhibited similar stability, while 20-40% of apogossypol hexaacetate was converted into apogossypol with concurrent formation of the corresponding di-, tri-, tetra-, and penta-acetates of apogossypol. (+/-)-Gossypol and (-)-gossypol showed comparable pharmacokinetic profile and oral bioavailability (12.2-17.6%) with some variations of clearance and V (ss) following oral and intravenous administration to mice. At the same molar dose, apogossypol showed delayed T (max)(1 h), a slower clearance rate and less distribution after administration to mice. Mono- and di-glucuronide conjugates of apogossypol were readily observed in mouse plasma following administration. Apogossypol formulated in sesame oil appeared to possess larger AUC and thus higher oral bioavailability than that formulated in cremophor EL:ethanol:saline. In contrast, intravenous apogossypol hexaacetate exhibited highest clearance rate partially due to its conversion into apogossypol. Concomitant with disappearance of apogossypol hexaacetate (iv), apogossypol converted from apogossypol hexaacetate was quantitatively detected, and accounted for approximately 30% of total plasma apogossypol hexaacetate. Oral apogossypol hexaacetate showed no bioavailability with little apogossypol occurring in the plasma. In human and mouse liver microsomes, glucuronide conjugates of apogossypol and its acetates were readily identified with the exception of gossypol glucuronidation. Apogossypol appeared more stable in human and mouse liver microsomal preparations than gossypol and apogossypol hexaacetate. CONCLUSIONS: Apogossypol and gossypol show similar oral and intravenous pharmacokinetic profiles and in vitro stability although apogossypol appears to have a slower clearance rate, larger AUC, and better microsomal stability. Apogossypol hexaacetate converts to apogossypol in both in vitro and in vivo settings and lacks any quantifiable oral bioavailability.

Cancer prevention by tea polyphenols is linked to their direct inhibition of antiapoptotic Bcl-2-family proteins.

Epidemiological data and in vitro studies on cancer chemoprevention by tea polyphenols have gained attention recently from the scientific community, nutritionists, the pharmaceutical industry, and the public. Despite the several efforts made recently to elucidate the molecular basis for the anticancer activity of these natural products, little correlation has been found thus far between the putative protein targets of compounds found in tea extracts and levels found in plasma after tea consumption. Here, by using a combination of nuclear magnetic resonance binding assays, fluorescence polarization assay, and computational docking studies, we found that certain green tea catechins and black tea theaflavins are very potent inhibitors (K(i) in the nanomolar range) of the antiapoptotic Bcl-2-family proteins, Bcl-x(L) and Bcl-2. These data suggest a strong link between the anticancer activities of these tea polyphenols and their inhibition of a crucial antiapoptotic pathway, which is implicated in the development of many human malignancies.

Melanoma differentiation associated gene-7, mda-7/interleukin-24, induces apoptosis in prostate cancer cells by promoting mitochondrial dysfunction and inducing reactive oxygen species.

Mda-7/IL-24 (Ad.mda-7) is a novel cytokine gene belonging to the interleukin (IL) 10 gene superfamily. Adenoviral-mediated delivery of mda-7/IL-24 causes growth suppression and apoptosis in a wide spectrum of cancer cells, including prostate, without harming normal cells. We now demonstrate that Ad.mda-7 selectively induces apoptosis in prostate cancer cells by promoting mitochondrial dysfunction and reactive oxygen species (ROS) production. Antioxidants (N-acetyl-L-cysteine and Tiron) and inhibitors of mitochondrial permeability transition (cyclosporine A and bongkrekic acid) inhibit Ad.mda-7-induced mitochondrial dysfunction and apoptosis. Conversely, agents augmenting ROS production (arsenic trioxide, NSC656240, and PK11195) facilitate Ad.mda-7-induced apoptosis. Ectopic expression of Bcl-2 and Bcl-x(L) inhibits mitochondrial changes, ROS production, and apoptosis providing additional support for an association between mitochondrial dysfunction and Ad.mda-7 action. These studies present definitive evidence that changes in mitochondrial function and ROS production are key components associated with selective killing of prostate cancer cells by mda-7/IL-24.

Functional blocks in caspase activation pathways are common in leukemia and predict patient response to induction chemotherapy.

Defects in apoptosis mechanisms contribute to chemoresistance in malignancy. However, correlations of apoptosis-regulating proteins with clinical outcome in cancer patients are variable, presumably reflecting the difficulty of using static tests of gene expression in a scenario influenced by a dynamic interplay of multiple pro- and antiapoptotic molecules. Therefore, we assessed the functional integrity of apoptosis pathways in intact primary leukemia cells and correlated the functional status of these pathways with clinical outcome. Active apoptogenic proteins were introduced into primary leukemia cells by electroporation followed by measurement of active caspases by flow cytometric techniques. Cytochrome c was introduced to activate the intrinsic (mitochondrial) pathway, whereas caspase-8 was introduced to activate the extrinsic (death receptor) pathway. In a series of 24 patients with acute myeloid leukemia, 79% had a block in at least one pathway, indicating that defects in caspase activation mechanisms are common in patients with leukemia. Simultaneous blocks in both pathways correlated with chemoresistant disease (92% of patients with chemoresistant disease versus 33% of patients with chemosensitive disease; P = 0.005) and decreased overall patient survival (35% versus 89% 1-year survival; P = 0.02). Simultaneous blockage of the intrinsic and extrinsic pathways could be explained by a defect located at a point of convergence of the two pathways, probably related to overexpression of endogenous inhibitors of the effector-caspases, rather than decreased levels of these proteases. This study supports the importance of apoptosis pathways in determining response to chemotherapy and suggests that functional defects in caspase activation are prognostic in patients with leukemia.

Dysregulation of apoptosis genes in hematopoietic malignancies.

Ever since the discovery of Bcl-2 and the elucidation of its role in apoptosis, tremendous interest has arisen in prospects for triggering suicide of malignant cells by exploiting knowledge emerging from apoptosis research. In this review, we summarize information about the multiple genetic lesions which have been identified in apoptosis-regulatory genes of hematopoietic and lymphoid neoplasms. Emerging data about the structural and biochemical details of apoptosis proteins and their upstream regulators have reveal novel strategies for therapeutic intervention, some of which are under interrogation in clinical trials currently.

The cancer growth suppressing gene mda-7 induces apoptosis selectively in human melanoma cells.

Human melanoma cells growth arrest irreversibly, lose tumorigenic potential and terminally differentiate after treatment with a combination of fibroblast interferon (IFN-beta) and the protein kinase C activator mezerein (MEZ). Applying subtraction hybridization to this model differentiation system permitted cloning of melanoma differentiation associated gene-7, mda-7. Expression of mda-7 inversely correlates with melanoma development and progression, with elevated expression in normal melanocytes and nevi and increasingly reduced expression in radial growth phase, vertical growth phase and metastatic melanoma. When expressed by means of a replication incompetent adenovirus (Ad.mda-7) growth of melanoma, but not normal early passage or immortal human melanocytes, is dramatically suppressed and cells undergo programmed cell death (apoptosis). Infection of metastatic melanoma cells with Ad.mda-7 results in an increase in cells in the G(2)/M phase of the cell cycle and changes in the ratio of pro-apoptotic (BAX, BAK) to anti-apoptotic (BCL-2, BCL-XL) proteins. Ad.mda-7 infection results in a temporal increase in mda-7 mRNA and intracellular MDA-7 protein in most of the melanocyte/melanoma cell lines and secretion of MDA-7 protein is readily detected following Ad.mda-7 infection of both melanocytes and melanoma cells. The present studies document a differential response of melanocytes versus melanoma cells to ectopic expression of mda-7 and support future applications of mda-7 for the gene-based therapy of metastatic melanoma.

Bcl-2 and Bcl-x(L) differentially protect human prostate cancer cells from induction of apoptosis by melanoma differentiation associated gene-7, mda-7/IL-24.

Subtraction hybridization identified melanoma differentiation associated gene-7, mda-7, in the context of terminally differentiated human melanoma cells. Based on its structure, cytokine-like properties and proposed mode of action, mda-7 has now been classified as IL-24. When expressed by means of a replication-incompetent adenovirus, Ad.mda-7 induces apoptosis in a broad range of cancer cells, without inducing harmful effects in normal fibroblast or epithelial cells. These unique properties of mda-7/IL-24 suggest that this gene will prove beneficial for cancer gene therapy. We now demonstrate that Ad.mda-7 decreases viability by induction of apoptosis in hormone-responsive (LNCaP) and hormone-independent (DU-145 and PC-3) human prostate carcinomas, without altering growth or survival in early-passage normal human prostate epithelial cells (HuPEC). Ad.mda-7 causes G(2)/M arrest and apoptosis in LNCaP (p53-wildtype), DU-145 (p53 mutant, Bax-negative) and PC-3 (p53-negative) prostate carcinomas, but not in HuPEC. Apoptosis induction correlated with changes in the ratio of pro- to antiapoptotic Bcl-2 protein family members. A potential functional role for changes in bcl-2 family gene expression in Ad.mda-7-induced apoptosis was suggested by the finding that forced overexpression of bcl-x(L) or bcl-2 differentially diminished the apoptotic effect of Ad.mda-7 in prostate carcinomas. These results confirm that induction of apoptosis by the mda-7/IL-24 gene in prostate cancer cells is Bax- and p53-independent and is mediated by mitochondrial pathways involving bcl-2 family gene members. The mda-7/IL-24 gene represents a new class of cancer-specific apoptosis-inducing genes with obvious potential for the targeted gene-based therapy of human prostate cancer.

Apoptotic-regulatory and complement-protecting protein expression in chronic lymphocytic leukemia: relationship to in vivo rituximab resistance.

PURPOSE: Rituximab has clinical activity in patients with chronic lymphocytic leukemia (CLL) and has a variety of proposed mechanisms, including apoptosis, complement-dependent cell lysis (CDC), and antibody-dependent cellular cytotoxicity (ADCC). Here we examine pretreatment biologic features that promote resistance to apoptosis and CDC in CLL patients and correlate it with clinical outcome to rituximab-based therapy. PATIENTS AND METHODS: Pretreatment samples from 21 CLL patients treated on a prospective, single-agent rituximab trial were examined for quantitative expression of apoptotic and CDC regulatory proteins, and the level of expression of these proteins was correlated with clinical outcome. RESULTS: Of the 21 patents for whom samples were available, 10 attained a partial response and 11 failed to respond to rituximab therapy. The mean pretreatment expression of Bcl-2, Mcl-1, XIAP, and the ratio of Bcl-2/Bax were higher but not statistically increased in nonresponding patients versus those responding to treatment. In contrast, the pretreatment Mcl-1/Bax ratio was significantly elevated (0.82 +/- 0.28 v 0.39 +/- 0.29, P <.016) in nonresponding patients compared with patients responding to rituximab therapy. Although pretreatment expression of CD55 and CD59 was not associated with response to rituximab therapy, significantly higher levels of CD59 were observed in the CLL cells that were not cleared from the blood at completion of therapy than the level observed at baseline levels (P =.02). CONCLUSION: These data indicate that baseline expression of the Mcl-1/Bax ratio, but not CD55 and CD59, predict for clinical response to rituximab therapy in CLL patients. Further study of disrupted apoptosis in CLL as a potential mechanism of resistance to rituximab appears warranted.

Rational design and real time, in-cell detection of the proapoptotic activity of a novel compound targeting Bcl-X(L).

Antiapoptotic Bcl-2-family proteins Bcl-2 and Bcl-X(L) have been recently validated as drug discovery targets for cancer. Here, by using a combination of molecular modeling, NMR-based structural analysis, fluorescence polarization assays, and cell-based assays, we have designed and characterized a novel proapoptotic compound targeting these proteins. Our compound, Apogossypol, is capable of binding and inhibiting Bcl-2 and Bcl-X(L) with high affinity and induces apoptosis of tumor cell lines. Mechanistic studies on the action of our compound were also performed via confocal microscopy that provided real-time detection of the interaction with Bcl-X(L) in intact cells. Finally, preliminary data on cells freshly isolated from patients affected by chronic lymphocytic leukemia strongly suggest potential applications of Bcl-2 antagonists as chemosensitizers in cancer therapy.

Apoptosis induction by 1alpha,25-dihydroxyvitamin D3 in prostate cancer.

Calcitriol [1alpha,25-dihydroxyvitamin D3] is the natural ligand of the vitamin D receptor (VDR). Using cultured prostate cancer (PC) cell lines, LN-CaP and ALVA-31, we studied the effects of 1alpha,25(OH)2-Vitamin D3 (VD3) on expression of several apoptosis-regulating proteins including: (a) Bcl-2 family proteins (Bcl-2, Bcl-X(L), Mcl-1, Bax, and Bak); (b) the heat shock protein 70-binding protein BAG1L; and (c) IAP family proteins (XIAP, cIAP1, and cIAP2). VD3 induced decreases in levels of antiapoptotic proteins Bcl-2, Bcl-X(L), and Mcl-1, BAG1L, XIAP, cIAP1, and cIAP2 (without altering proapoptotic Bax and Bak) in association with increases in apoptosis. In contrast to VDR-expressing LN-CaP and ALVA-31 cells, VDR-deficient prostate cancer line Du-145 demonstrated no changes in apoptosis protein expression after treatment with VD3. In sensitive PC cell lines, VD3 activates downstream effector protease, caspase-3, and upstream initiator protease caspase-9, the apical protease in the mitochondrial ("intrinsic") pathway for apoptosis, but not caspase-8, an initiator caspase linked to an alternative ("extrinsic") apoptosis pathway triggered by cytokine receptors. VD3 induced declines in antiapoptotic proteins and also stimulated cytochrome c release from mitochondria by a caspase-independent mechanism. Moreover, apoptosis induction by VD3 was suppressed by overexpressing Bcl-2, a known blocker of cytochrome c release, whereas the caspase-8 suppressor CrmA afforded little protection. Thus, VD3 is capable of inhibiting expression of multiple antiapoptotic proteins in VDR-expressing prostate cancer cells, leading to activation of the mitochondrial pathway for apoptosis.

Modulating apoptosis pathways in low-grade B-cell malignancies using biological response modifiers.

Collectively, low-grade B-cell malignancies constitute the fifth most common form of potentially lethal cancer in North America and Europe, with chronic lymphocytic leukemia (CLL) and follicular non-Hodgkin's lymphoma (FL) representing the most prevalent of these disorders. Chronic lymphocytic leukemia and FL represent quintessential examples of human malignancies that are caused primarily by defects in programmed cell death (apoptosis). During the early stages of disease, the mature B lymphocytes that comprise most CLLs and FLs are largely quiescent G(0) phase cells, which accumulate not because they are dividing more rapidly than normal but because they survive longer than their normal counterparts because of defects in the normal pathways for apoptosis. Defects in apoptosis pathways contribute to chemoresistance, rendering tumor cells less sensitive to the cytotoxic actions of currently available anticancer drugs, and can also promote resistance to cellular immune responses. Several biological agents or their synthetic derivatives show promise as apoptosis modulators, having the potential to place neoplastic cells into a more susceptible state or activating latent programs for cell suicide. These biological response modifiers include monoclonal antibodies such as rituximab (Rituxan; Genentech, Inc, South San Francisco, CA, and IDEC Pharmaceuticals, San Diego, CA) that alter signal transduction pathways, cytokines such as TRAIL (Apo2 ligand), ligands for retinoid/steroid family nuclear receptors, and small-molecule compounds that bind and inhibit protein kinases. Knowledge about the mechanisms by which these agents influence apoptosis pathways in neoplastic diseases may suggest strategies for more effective and less toxic therapies for patients suffering from CLL, FL, and other malignancies.

A method for functional evaluation of caspase activation pathways in intact lymphoid cells using electroporation-mediated protein delivery and flow cytometric analysis.

The purpose of the study was to develop a rapid technique for determining the functional status of caspase activation pathways in intact lymphocytes. Proteins known to activate caspase-family cell death proteases (cytochrome c; granzyme-B; caspase-8) were introduced into human leukemia and lymphoma cell lines, as well as freshly isolated lymphocytes and leukemia cells, by electroporation. Fluorochrome-labeled proteins with a wide range of molecular weights (from 15 to 150 kDa) were used to evaluate electroporation efficiency by flow cytometry and to compare the efficiency of protein delivery using various electroporation conditions. Caspase activity was monitored using a cleavable, cell-permeable fluorogenic substrate. Conditions were identified for efficient delivery of proteins of +150 kDa into lymphoid cells. Caspase activation induced by various proteins was compared in normal and leukemic lymphocytic cells, revealing impaired caspase activation pathways in some malignant cells. We conclude that electroporation of apoptotic proteins into intact lymphoid cells can be used to contrast the status of various caspase activation pathways, thereby providing insights into the pathological defects in apoptosis regulation that exist in individual patient specimens.

Discovery, characterization, and structure-activity relationships studies of proapoptotic polyphenols targeting B-cell lymphocyte/leukemia-2 proteins.

Among the most promising chemopreventive agents, certain natural polyphenols have recently received a great deal of attention because of their demonstrated inhibitory activity against tumorigenesis. In view of their anticancer properties, these compounds also hold great promise as potential chemotherapeutic agents. However, to translate these chemopreventive agents into chemotherapeutic compounds, their exact mechanisms of action must be delineated. By using a multidisciplinary approach guided by modern nuclear magnetic resonance spectroscopy techniques, fluorescence polarization displacement assays, and cell-based assays, we have begun to unravel the mechanisms of actions of certain polyphenols such as Gossypol (a compound from cotton seed extracts) and Purpurogallin (a natural compound extracted from Quercus sp. nutgall) and their derivatives. Our findings suggest that these natural products bind and antagonize the antiapoptotic effects of B-cell lymphocyte/leukemia-2 (Bcl-2) family proteins such as Bcl-x(L). Our in vitro and in vivo data not only open a window of opportunities for the development of novel cancer treatments with these compounds but also provide structural information that can be used for the design and development of novel and more effective analogues.

Small molecule-induced mitochondrial disruption directs prostate cancer inhibition via UPR signaling.

We previously identified SMIP004 (N-(4-butyl-2-methyl-phenyl) acetamide) as a novel inducer of cancer-cell selective apoptosis of human prostate cancer cells. SMIP004 decreased the levels of positive cell cycle regulators, upregulated cyclin-dependent kinase inhibitors, and resulted in G1 arrest, inhibition of colony formation in soft agar, and cell death. However, the mechanism of SMIP004-induced cancer cell selective apoptosis remained unknown. Here, we used chemical genomic and proteomic profiling to unravel a SMIP004-induced pro-apoptotic pathway, which initiates with disruption of mitochondrial respiration leading to oxidative stress. This, in turn, activates two pathways, one eliciting cell cycle arrest by rapidly targeting cyclin D1 for proteasomal degradation and driving the transcriptional downregulation of the androgen receptor, and a second pathway that activates pro-apoptotic signaling through MAPK activation downstream of the unfolded protein response (UPR). SMIP004 potently inhibits the growth of prostate and breast cancer xenografts in mice. Our data suggest that SMIP004, by inducing mitochondrial ROS formation, targets specific sensitivities of prostate cancer cells to redox and bioenergetic imbalances that can be exploited in cancer therapy.