Role of MSC-derived galectin 3 in the AML microenvironment.

In acute myeloid leukemia (AML), high Galectin 3 (LGALS3) expression is associated with poor prognosis. The role of LGALS3 derived from mesenchymal stromal cells (MSC) in the AML microenvironment is unclear; however, we have recently found high LGALS3 expression in MSC derived from AML patients is associated with relapse. In this study, we used reverse phase protein analysis (RPPA) to correlate LGALS3 expression in AML MSC with 119 other proteins including variants of these proteins such as phosphorylated forms or cleaved forms to identify biologically relevant pathways. RPPA revealed that LGALS3 protein was positively correlated with expression of thirteen proteins including MYC, phosphorylated beta-Catenin (p-CTNNB1), and AKT2 and negatively correlated with expression of six proteins including integrin beta 3 (ITGB3). String analysis revealed that proteins positively correlated with LGALS3 showed strong interconnectivity. Consistent with the RPPA results, LGALS3 suppression by shRNA in MSC resulted in decreased MYC and AKT expression while ITGB3 was induced. In co-culture, the ability of AML cell to adhere to MSC LGALS3 shRNA transductants was reduced compared to AML cell adhesion to MSC control shRNA transductants. Finally, use of novel specific LGALS3 inhibitor CBP.001 in co-culture of AML cells with MSC reduced viable leukemia cell populations with induced apoptosis and augmented the chemotherapeutic effect of AraC. In summary, the current study demonstrates that MSC-derived LGALS3 may be critical for important biological pathways for MSC homeostasis and for regulating AML cell localization and survival in the leukemia microenvironmental niche.

Distinct protein signatures of acute myeloid leukemia bone marrow-derived stromal cells are prognostic for patient survival.

Mesenchymal stromal cells (MSC) support acute myeloid leukemia (AML) cell survival in the bone marrow (BM) microenvironment. Protein expression profiles of AML-derived MSC are unknown. Reverse phase protein array analysis was performed to compare expression of 151 proteins from AML-MSC (n=106) with MSC from healthy donors (n=71). Protein expression differed significantly between the two groups with 19 proteins over-expressed in leukemia stromal cells and 9 over-expressed in normal stromal cells. Unbiased hierarchical clustering analysis of the samples using these 28 proteins revealed three protein constellations whose variation in expression defined four MSC protein expression signatures: Class 1, Class 2, Class 3, and Class 4. These cell populations appear to have clinical relevance. Specifically, patients with Class 3 cells have longer survival and remission duration compared to other groups. Comparison of leukemia MSC at first diagnosis with those obtained at salvage (i.e. relapse/refractory) showed differential expression of 9 proteins reflecting a shift toward osteogenic differentiation. Leukemia MSC are more senescent compared to their normal counterparts, possibly due to the overexpressed p53/p21 axis as confirmed by high ß-galactosidase staining. In addition, overexpression of BCL-XL in leukemia MSC might give survival advantage under conditions of senescence or stress and overexpressed galectin-3 exerts profound immunosuppression. Together, our findings suggest that the identification of specific populations of MSC in AML patients may be an important determinant of therapeutic response.

IKK inhibition by BMS-345541 suppresses breast tumorigenesis and metastases by targeting GD2+ cancer stem cells.

We have identified that the ganglioside GD2 is a marker for breast cancer stem cells (BCSCs), and that targeting the enzyme GD3 synthase (GD3S, which regulates GD2 biosynthesis) reduces breast tumorigenesis. The pathways regulating GD2 expression, and their anomalous functions in BCSC, are unclear. Proteomic analysis of GD2+ and GD2- cells from breast cancer cell lines revealed the activation of NFκB signaling in GD2+ cells. Dose- and time-dependent suppression of NFκB signaling by the small molecule inhibitor BMS-345541 reduced GD2+ cells by > 90%. Likewise, BMS-345541 inhibited BCSC GD3S expression, mammosphere formation, and cell migration/invasion in vitro. Breast tumor-bearing mice treated with BMS-345541 showed a statistically significant decrease in tumor volume and exhibited prolonged survival compared to control mice, with a median survival of 78 d for the BMS-345541-treated group vs. 58 d for the controls. Moreover, in an experimental metastases model, treatment with BMS-345541 reduced the lung metastases by > 5-fold. These data suggest that GD2 expression and function,and NFκB signaling, are related, and they control BCSCs tumorigenic characteristics. Thus, the suppression of NFκB signaling by BMS-345541 is a potentially important advance in controlling breast cancer growth and metastases.

Bone Marrow Adipocytes Facilitate Fatty Acid Oxidation Activating AMPK and a Transcriptional Network Supporting Survival of Acute Monocytic Leukemia Cells.

Leukemia cells in the bone marrow must meet the biochemical demands of increased cell proliferation and also survive by continually adapting to fluctuations in nutrient and oxygen availability. Thus, targeting metabolic abnormalities in leukemia cells located in the bone marrow is a novel therapeutic approach. In this study, we investigated the metabolic role of bone marrow adipocytes in supporting the growth of leukemic blasts. Prevention of nutrient starvation-induced apoptosis of leukemic cells by bone marrow adipocytes, as well as the metabolic and molecular mechanisms involved in this process, was investigated using various analytic techniques. In acute monocytic leukemia (AMoL) cells, the prevention of spontaneous apoptosis by bone marrow adipocytes was associated with an increase in fatty acid ß-oxidation (FAO) along with the upregulation of PPARγ, FABP4, CD36, and BCL2 genes. In AMoL cells, bone marrow adipocyte coculture increased adiponectin receptor gene expression and its downstream target stress response kinase AMPK, p38 MAPK with autophagy activation, and upregulated antiapoptotic chaperone HSPs. Inhibition of FAO disrupted metabolic homeostasis, increased reactive oxygen species production, and induced the integrated stress response mediator ATF4 and apoptosis in AMoL cells cocultured with bone marrow adipocytes. Our results suggest that bone marrow adipocytes support AMoL cell survival by regulating their metabolic energy balance and that the disruption of FAO in bone marrow adipocytes may be an alternative, novel therapeutic strategy for AMoL therapy. Cancer Res; 77(6); 1453-64. ©2017 AACR.

Atg7 suppression enhances chemotherapeutic agent sensitivity and overcomes stroma-mediated chemoresistance in acute myeloid leukemia.

Autophagy is a cellular adaptive mechanism to stress, including that induced by chemotherapeutic agents. Reverse phase protein array suggested that high expression of the essential autophagy-related protein, Atg7, was associated with shorter remission in newly diagnosed acute myeloid leukemia (AML) patient samples, indicating a role in chemoresistance. Knockdown of Atg7 in AML cells using short hairpin RNA markedly increased apoptosis and DNA damage following treatment with cytarabine and idarubicin. Interestingly, coculture of AML cells with stromal cells increased autophagy and chemoresistance in the AML cells exposed to chemotherapeutic agents, and this was reversed following Atg7 knockdown. This effect was further enhanced by concomitant knockdown of Atg7 in both AML and stromal cells. These findings strongly suggest that Atg7, and likely microenvironment autophagy in general, plays an important role in AML chemoresistance. Mechanistic studies revealed that Atg7 knockdown induced a proapoptotic phenotype in AML cells, which was manifested by an increased NOXA expression at the transcriptional level. Finally, in a mouse model of human leukemia, Atg7 knockdown extended overall survival after chemotherapy. Thus, the inhibition of Atg7 appears to be a valid strategy to enhance chemosensitivity, and it could indeed improve outcomes in AML therapy.

Combination of galectin inhibitor GCS-100 and BH3 mimetics eliminates both p53 wild type and p53 null AML cells.

Galectin 3 (LGALS3) expression is prognostic for poor survival in acute myeloid leukemia (AML) patients. GCS-100 is a novel galectin inhibitor that may prove useful for AML therapy. In this study, we found that GCS-100 induced apoptosis in AML cells. The agent reduced MCL-1 expression suggesting that GCS-100 could be more effective when combined with a BH3 mimetic. Indeed, potent synergistic cytotoxicity was achieved when GCS-100 was combined with ABT-737 or ABT-199. Furthermore, the GCS-100/ABT-199 combination was effective against primary AML blast cells from patients with FLT3 ITD mutations, which is another prognostic factor for poor outcome in AML. This activity may involve wild-type p53 as shRNA knockdown of LGALS3 or galectin 1 (LGALS1) sensitized wild-type p53 OCI-AML3 cells to GCS-100/ABT-737-induced apoptosis to a much greater extent than p53 null THP-1 cells. Suppression of LGALS3 by shRNA inhibited MCL-1 expression in OCI-AML3 cells, but not THP-1 cells, suggesting the induced sensitivity to ABT-737 may involve a MCL-1 mediated mechanism. OCI-AML3 cells with LGALS1 shRNA were also sensitized to ABT-737. However, these cells exhibited increased MCL-1 expression, so MCL-1 reduction is apparently not required in this process. A role for p53 appears important as GCS-100 induces p53 expression and shRNA knockdown of p53 protected OCI-AML3 cells from the cytotoxic effects of the GCS-100/ABT-737 treatment combination. Our results suggest that galectins regulate a survival axis in AML cells, which may be targeted via combined inhibition with drugs such as GCS-100 and ABT-199.

Phosphorylation of GSK3α/ß correlates with activation of AKT and is prognostic for poor overall survival in acute myeloid leukemia patients.

BACKGROUND: Acute myeloid leukemia (AML) patients with highly active AKT tend to do poorly. Cell cycle arrest and apoptosis are tightly regulated by AKT via phosphorylation of GSK3α and ß isoforms which inactivates these kinases. In the current study we examine the prognostic role of AKT mediated GSK3 phosphorylation in AML. METHODS: We analyzed GSK3α/ß phosphorylation by reverse phase protein analysis (RPPA) in a cohort of 511 acute myeloid leukemia (AML) patients. Levels of phosphorylated GSK3 were correlated with patient characteristics including survival and with expression of other proteins important in AML cell survival. RESULTS: High levels of p-GSK3α/ß correlated with adverse overall survival and a lower incidence of complete remission duration in patients with intermediate cytogenetics, but not in those with unfavorable cytogenetics. Intermediate cytogenetic patients with FLT3 mutation also fared better respectively when p-GSK3α/ß levels were lower. Phosphorylated GSK3α/ß expression was compared and contrasted with that of 229 related cell cycle arrest and/or apoptosis proteins. Consistent with p-GSK3α/ß as an indicator of AKT activation, RPPA revealed that p-GSK3α/ß positively correlated with phosphorylation of AKT, BAD, and P70S6K, and negatively correlated with ß-catenin and FOXO3A. PKCδ also positively correlated with p-GSK3α/ß expression, suggesting crosstalk between the AKT and PKC signaling pathways in AML cells. CONCLUSIONS: These findings suggest that AKT-mediated phosphorylation of GSK3α/ß may be beneficial to AML cell survival, and hence detrimental to the overall survival of AML patients. Intrinsically, p-GSK3α/ß may serve as an important adverse prognostic factor for a subset of AML patients.

Expression, function, and targeting of the nuclear exporter chromosome region maintenance 1 (CRM1) protein.

Nucleocytoplasmic trafficking of proteins/RNAs is essential to normal cellular function. Indeed, accumulating evidence suggests that cancer cells escape anti-neoplastic mechanisms and benefit from pro-survival signals via the dysregulation of this system. The nuclear exporter chromosome region maintenance 1 (CRM1) protein is the only protein in the karyopherin-ß protein family that contributes to the trafficking of numerous proteins and RNAs from the nucleus. It is considered to be an oncogenic, anti-apoptotic protein in transformed cells, since it reportedly functions as a gatekeeper for cell survival, including affecting p53 function, and ribosomal biogenesis. Furthermore, abnormally high expression of CRM1 is correlated with poor patient prognosis in various malignancies. Therapeutic targeting of CRM1 has emerged as a novel cancer treatment strategy, starting with a clinical trial with leptomycin B, the original specific inhibitor of CRM1, followed by development of several next-generation small molecules. KPT-330, a novel member of the CRM1-selective inhibitors of nuclear export (SINE) class of compounds, is currently undergoing clinical evaluation for the therapy of various malignancies. Results from these trials suggest that SINE compounds may be particularly useful against hematological malignancies, which often become refractory to standard chemotherapeutic agents.

The protein phosphatase 2A regulatory subunit B55α is a modulator of signaling and microRNA expression in acute myeloid leukemia cells.

We recently discovered that the protein phosphatase 2A (PP2A) B55α subunit (PPP2R2A) is under-expressed in primary blast cells and is unfavorable for remission duration in AML patients. In this study, reverse phase protein analysis (RPPA) of 230 proteins in 511 AML patient samples revealed a strong correlation of B55α with a number of proteins including MYC, PKC α, and SRC. B55α suppression in OCI-AML3 cells by shRNA demonstrated that the B subunit is a PKCα phosphatase. B55α does not target SRC, but rather the kinase suppresses protein expression of the B subunit. Finally, the correlation between B55α and MYC levels reflected a complex stoichiometric competition between B subunits. Loss of B55α in OCI-AML3 cells did not change global PP2A activity and the only isoform that is induced is the one containing B56α. In cells containing B55α shRNA, MYC was suppressed with concomitant induction of the competing B subunit B56α (PPP2R5A). A recent study determined that FTY-720, a drug whose action involves the activation of PP2A, resulted in the induction of B55α In AML cells, and a reduction of the B subunit rendered these cells resistant to FTY-720. Finally, reduction of the B subunit resulted in an increase in the expression of miR-191-5p and a suppression of miR-142-3p. B55α regulation of these miRs was intriguing as high levels of miR-191 portend poor survival in AML, and miR-142-3p is mutated in 2% of AML patient samples. In summary, the suppression of B55α activates signaling pathways that could support leukemia cell survival.

Mitochondrial uncoupling and the reprograming of intermediary metabolism in leukemia cells.

Nearly 60 years ago Otto Warburg proposed, in a seminal publication, that an irreparable defect in the oxidative capacity of normal cells supported the switch to glycolysis for energy generation and the appearance of the malignant phenotype (Warburg, 1956). Curiously, this phenotype was also observed by Warburg in embryonic tissues, and recent research demonstrated that normal stem cells may indeed rely on aerobic glycolysis - fermenting pyruvate to lactate in the presence of ample oxygen - rather than on the complete oxidation of pyruvate in the Krebs cycle - to generate cellular energy (Folmes et al., 2012). However, it remains to be determined whether this phenotype is causative for neoplastic development, or rather the result of malignant transformation. In addition, in light of mounting evidence demonstrating that cancer cells can carry out electron transport and oxidative phosphorylation, although in some cases predominantly using electrons from non-glucose carbon sources (Bloch-Frankenthal et al., 1965), Warburg's hypothesis needs to be revisited. Lastly, recent evidence suggests that the leukemia bone marrow microenvironment promotes the Warburg phenotype adding another layer of complexity to the study of metabolism in hematological malignancies. In this review we will discuss some of the evidence for alterations in the intermediary metabolism of leukemia cells and present evidence for a concept put forth decades ago by lipid biochemist Feodor Lynen, and acknowledged by Warburg himself, that cancer cell mitochondria uncouple ATP synthesis from electron transport and therefore depend on glycolysis to meet their energy demands (Lynen, 1951; Warburg, 1956).

The CXCR4 antagonist AMD3465 regulates oncogenic signaling and invasiveness in vitro and prevents breast cancer growth and metastasis in vivo.

CXCR4, the receptor for stromal-derived factor-1, is reportedly involved in breast carcinogenesis. However, the mechanisms through which CXCR4 contributes to breast cancer cell growth and metastases are poorly understood. In this study, we examined the putative in vitro and in vivo anti-cancer effects of the specific CXCR4 inhibitor AMD3465. Here, we report that AMD3465 triggers a reduction in breast cancer cell invasiveness in vitro, and promotes marked changes in oncogenic signaling proteins including a reduction in STAT3, JAK2, AKT, and CXCR4 phosphorylation and the reduced expression of GSK3 and cMYC. Using three breast cancer cell lines as murine syngeneic immunocompetent breast cancer models, we found that AMD3465 inhibited breast tumor formation and reduced tumor cell metastases to the lung and liver. Furthermore, treatment with AMD3465 significantly reduced the infiltration of myeloid CD11b positive cells at the aforementioned metastatic sites as well as the spleen implying this agent could regulate the formation of the tumor microenvironment and conceivably the premetastatic niche. In conclusion, our studies suggest that AMD3465 inhibits breast cancer growth and metastases by acting on tumor cells as well as immune cells that constitute the tumor microenvironment. This process appears to be regulated, at least in part, through the modulation of oncogenic signaling that includes the STAT3 pathway. Thus, CXCR4 could be a novel target for breast cancer therapy.

Evidence supporting a role for dihydroorotate dehydrogenase, bioenergetics, and p53 in selective teriflunomide-induced apoptosis in transformed versus normal human keratinocytes.

We have demonstrated previously that the dihydroorotate dehydrogenase (DHODH) inhibitor teriflunomide (TFN) encourages apoptosis in transformed human keratinocytes. Here we sought to determine if this cytotoxic effect could be restricted to transformed keratinocytes relative to their normal human epidermal keratinocyte (NHEK) counterparts, and ascertain a potential mechanistic basis for the selectivity. The NHEK cells proliferated much slower than the premalignant HaCaT and malignant COLO 16 keratinocytes, and exogenous uridine added to the culture medium did not affect this growth. Similarly, DHODH expression and the bioenergetic characteristics of the normal cells were markedly dissimilar from those observed in the transformed cells indicating that de novo pyrimidine synthesis was involved with keratinocyte proliferation. Moreover, a short-term exposure to TFN caused a wild-type p53 response in the NHEK cells illustrating that pyrimidine metabolic stress could regulate this tumor suppressor protein in the normal cells. TFN-induced apoptosis occurred primarily in S phase HaCaT cells. This cell death was sensitive to uridine, an antioxidant, and a caspase inhibitor, and the suppression of Bcl-X(L) and the induction of Mn superoxide dismutase preceded it. These events suggested that mitochondrial/redox stress was involved with the cytotoxic effect of TFN. Conversely, a long-term exposure to TFN caused G(0)/G(1) arrest in the NHEK cells, which supported a cytoprotective role for p53 against TFN-induced apoptosis. Together, these results propose that TFN could be useful in the prevention or therapy of non-melanoma skin cancers and possibly other hyperproliferative keratinocytic diseases.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) inhibition by 4-hydroxynonenal leads to increased Akt activation in hepatocytes.

The production of reactive aldehydes such as 4-hydroxynonenal (4-HNE) is proposed to be an important factor in the etiology of alcoholic liver disease. To understand the effects of 4-HNE on homeostatic signaling pathways in hepatocytes, cellular models consisting of the human hepatocellular carcinoma cell line (HepG2) and primary rat hepatocytes were evaluated. Treatment of both HepG2 cells and primary hepatocytes with subcytotoxic concentrations of 4-HNE resulted in the activation of Akt within 30 min as demonstrated by increased phosphorylation of residues Ser473 and Thr308. Quantification and subsequent immunocytochemistry of phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P(3)[rsqb] resulted in a 6-fold increase in total PtdIns(3,4,5)P(3) and increased immunostaining at the plasma membrane after 4-HNE treatment. Cotreatment of HepG2 cells with 4-HNE and the phosphatidylinositol 3-kinase (PI3K) inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (Ly294002) or the protein phosphatase 2A (PP2A) inhibitor okadaic acid revealed that the mechanism of activation of Akt is PI3K-dependent and PP2A-independent. Using biotin hydrazide detection, it was established that the incubation of HepG2 cells with 4-HNE resulted in increased carbonylation of the lipid phosphatase known as "phosphatase and tensin homolog deleted on chromosome 10" (PTEN), a key regulator of Akt activation. Activity assays both in HepG2 cells and recombinant PTEN revealed a decrease in PTEN lipid phosphatase activity after 4-HNE application. Mass spectral analysis of 4-HNE-treated recombinant PTEN detected a single 4-HNE adduct. Subsequent analysis of Akt dependent physiological consequences of 4-HNE in HepG2 cells revealed significant increases in the accumulation of neutral lipids. These results provide a potential mechanism of Akt activation and cellular consequences of 4-HNE in hepatocytes.

The hydroxyl functional group of N-(4-hydroxyphenyl)retinamide mediates cellular uptake and cytotoxicity in premalignant and malignant human epithelial cells.

In a previous study, we demonstrated that the anticancer synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) redox cycles at the mitochondrial enzyme dihydroorotate dehydrogenase to trigger anomalous reactive oxygen species (ROS) production and attendant apoptosis in transformed human epithelial cells. Furthermore, we speculated that the hydroxyl functional group of 4HPR was required for this pro-oxidant property. In this study, we investigated the role of the hydroxyl functional group in the in vitro cytotoxicity of 4HPR. Using 4HPR, its primary in vivo metabolite N-(4-methoxyphenyl)retinamide (4MPR), and the synthetic derivative N-(4-trifluoromethylphenyl)retinamide (4TPR), we examined the pro-oxidant and apoptotic effects, as well as the cellular uptake, of these three N-(4-substituted-phenyl)retinamides in premalignant and malignant human skin, prostate, and breast epithelial cells. Compared to 4HPR, both 4MPR and 4TPR were ineffective in promoting conspicuous cellular ROS production, mitochondrial disruption, or DNA fragmentation in these transformed cells. Interestingly, both 4MPR and 4TPR were not particularly cell permeative relative to 4HPR in skin or breast epithelial cells, which implied an additional role for the hydroxyl functional group in the cellular uptake of 4HPR. Moreover, the short-term uptake of 4HPR was directly proportional to cell size, but this characteristic, in obvious contrast to cellular bioenergetic status and/or dihydroorotate dehydrogenase expression, was not fundamentally influential in the overall sensitivity to the promotion of cellular ROS production and apoptosis induction by this agent. Together, these results strongly implicate the hydroxyl functional group in the cytotoxic effects of 4HPR.

Teriflunomide encourages cytostatic and apoptotic effects in premalignant and malignant cutaneous keratinocytes.

Teriflunomide (TFN) reportedly inhibits de novo pyrimidine synthesis and exhibits anti-inflammatory, disease-modifying activities in vivo. These qualities would suggest that TFN could be useful in skin cancer chemoprevention or therapy. We investigated some mechanistic aspects of this tenet by characterizing the effects of TFN on premalignant and malignant human cutaneous keratinocytes. TFN promoted a dose- and/or time-dependent cytostasis and in these cells, which was followed by apoptosis. These features occurred in the presence of a physiological concentration of uridine in the culture medium. The short-term S phase arrest triggered by TFN was reversible in the malignant keratinocytes, and the indirect apoptosis induction was apparently preceded by mitochondrial disruption and reactive oxygen species production in both the premalignant and malignant keratinocytes. Respiration deficient malignant keratinocytes were resistant to the acute cytostatic and latent apoptotic effects of TFN implicating de novo pyrimidine synthesis and mitochondrial bioenergetics as the primary targets for TFN in the respiring cells. These novel mechanistic findings support a role for TFN in skin cancer chemoprevention and therapy.

Teriflunomide (leflunomide) promotes cytostatic, antioxidant, and apoptotic effects in transformed prostate epithelial cells: evidence supporting a role for teriflunomide in prostate cancer chemoprevention.

Teriflunomide (TFN) is an inhibitor of de novo pyrimidine synthesis and the active metabolite of leflunomide. Leflunomide is prescribed to patients worldwide as an immunomodulatory and anti-inflammatory disease-modifying prodrug. Leflunomide inhibited the growth of human prostate cancer xenographs in mice, and leflunomide or TFN promoted cytostasis and/or apoptosis in cultured cells. These findings suggest that TFN could be useful in prostate cancer chemoprevention. We investigated the possible mechanistic aspects of this tenet by characterizing the effects of TFN using premalignant PWR-1E and malignant DU-145 human prostate epithelial cells. TFN promoted a dose- and time-dependent cytostasis or apoptosis induction in these cells. The cytostatic effects of TFN, which were reversible but not by the presence of excess uridine in the culture medium, included diminished cellular uridine levels, an inhibition in oxygen consumption, a suppression of reactive oxygen species (ROS) generation, S-phase cell cycle arrest, and a conspicuous reduction in the size and number of the nucleoli in the nuclei of these cells. Conversely, TFN's apoptogenic effects were characteristic of catastrophic mitochondrial disruption (i.e., a dissipation of mitochondrial inner transmembrane potential, enhanced ROS production, mitochondrial cytochrome c release, and cytoplasmic vacuolization) and followed by DNA fragmentation. The respiration-deficient derivatives of the DU-145 cells, which are also uridine auxotrophs, were markedly resistant to the cytostatic and apoptotic effects of TFN, implicating de novo pyrimidine synthesis and mitochondrial bioenergetics as the primary targets for TFN in the respiration competent cells. These mechanistic findings advocate a role for TFN and mitochondrial bioenergetics in prostate cancer chemoprevention.

Dihydroorotate dehydrogenase is required for N-(4-hydroxyphenyl)retinamide-induced reactive oxygen species production and apoptosis.

The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) exhibits anticancer activity in vivo and triggers apoptosis in transformed cells in vitro. Thus, apoptosis induction is acknowledged as a mechanistic underpinning for 4HPR's cancer preventive and therapeutic effects. Apoptosis induction by 4HPR is routinely preceded by and dependent on the production of reactive oxygen species (ROS) in transformed cells. Very little evidence exists, outside the possible involvement of the mitochondrial electron transport chain or the plasma membrane NADPH oxidase complex, that would pinpoint the predominant site of 4HPR-induced ROS production in transformed cells. Here, we investigated the role of dihydroorotate dehydrogenase (DHODH; an enzyme associated with the mitochondrial electron transport chain and required for de novo pyrimidine synthesis) in 4HPR-induced ROS production and attendant apoptosis in transformed skin and prostate epithelial cells. In premalignant prostate epithelial cells and malignant cutaneous keratinocytes the suppression of DHODH activity by the chemical inhibitor teriflunomide or the reduction in DHODH protein expression by RNA interference markedly reduced 4HPR-induced ROS generation and apoptosis. Conversely, colon carcinoma cells that lacked DHODH expression were markedly resistant to the pro-oxidant and cytotoxic effects of 4HPR. Together, these results strongly implicate DHODH in 4HPR-induced ROS production and apoptosis.

Lactoferrin protects against acetaminophen-induced liver injury in mice.

UNLABELLED: Acetaminophen-induced liver injury (AILI) is a significant health problem and represents the most frequent cause of drug-induced liver failure in the United States. The development and implementation of successful therapeutic intervention strategies have been demanding, due to significant limitations associated with the current treatment for AILI. Lactoferrin (Lac), a glycoprotein present in milk, has been demonstrated to possess a multitude of biological functions. Our study demonstrated a profound protective effect of Lac in a murine model of AILI, which was not dependent on its iron-binding ability, inhibition of acetaminophen (APAP) metabolism, or a direct cytoprotective effect on hepatocytes. Instead, Lac treatment significantly attenuated APAP-induced liver sinusoidal endothelial cell dysfunction and ameliorated hepatic microcirculation disorder. This protective effect of Lac appeared to be dependent on hepatic resident macrophages (Kupffer cells [KCs]). CONCLUSION: Collectively, our data indicate that Lac, through activation of KCs, inhibited APAP-induced liver sinusoidal endothelial cell damage and improved hepatic congestion, thereby protecting against AILI. These findings reveal the significant therapeutic potential of Lac during AILI and other types of liver diseases.

Selective apoptosis induction by the cancer chemopreventive agent N-(4-hydroxyphenyl)retinamide is achieved by modulating mitochondrial bioenergetics in premalignant and malignant human prostate epithelial cells.

Prostate tumorigenesis is coupled with an early metabolic switch in transformed prostate epithelial cells that effectively increases their mitochondrial bioenergetic capacity. The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) inhibits prostate cancer development in vivo, and triggers reactive oxygen species (ROS)-dependent prostate cancer cell apoptosis in vitro. The possibility that 4HPR-induced ROS production is associated with mitochondrial bioenergetics and required for apoptosis induction in transformed prostate epithelial cells in vitro would advocate a prospective mechanistic basis for 4HPR-mediated prostate cancer chemoprevention in vivo. We investigated this tenet by comparing and contrasting 4HPR's effects on premalignant PWR-1E and malignant DU-145 human prostate epithelial cells. 4HPR promoted a dose- and/or time-dependent apoptosis induction in PWR-1E and DU-145 cells, which was preceded by and dependent on an increase in mitochondrial ROS production. In this regard, the PWR-1E cells were more sensitive than the DU-145 cells, and they consumed roughly twice as much oxygen as the DU-145 cells suggesting oxidative phosphorylation was higher in the premalignant cells. Interestingly, increasing the [Ca(2+)] in the culture medium of the PWR-1E cells attenuated their proliferation as well as their mitochondrial bioenergetic capacity and 4HPR's cytotoxic effects. Correspondingly, the respiration-deficient derivatives (i.e., rho(0) cells lacking mitochondrial DNA) of DU-145 cells were markedly resistant to 4HPR-induced ROS production and apoptosis. Together, these observations implied that the reduction of mitochondrial bioenergetics protected PWR-1E and DU-145 cells against the cytotoxic effects of 4HPR, and support the concept that oxidative phosphorylation is an essential determinant in 4HPR's apoptogenic signaling in transformed human prostate epithelial cells.

Cancer chemoprevention and mitochondria: targeting apoptosis in transformed cells via the disruption of mitochondrial bioenergetics/redox state.

Cancer chemoprevention employs agents that block, hinder, or reverse tumorigenesis to prevent malignancy. Several putative cancer chemopreventive agents promote apoptosis in transformed cells initiated in animal carcinogenesis models or identified in human subjects, and/or in tumor cells cultured in vitro. Consequently, apoptosis induction is increasingly valued as a biologically significant anticancer mechanism in the arena of chemoprevention. In vitro studies suggest that the permeabilization of mitochondrial membranes is an important mechanistic determinant associated with the apoptosis induced by these agents. Mitochondrial membrane permeabilization (MMP) may occur via the control of proapoptotic Bcl-2 family members, and/or by the induction of the mitochondrial permeability transition. Both of these cell death-inducing regulatory mechanisms are ultimately responsive to the bioenergetic status/redox state of mitochondria. Interestingly, in addition to inducing MMP, various chemopreventive agents can directly modulate mitochondrial bioenergetics and/or redox tone in transformed cells. This review will examine prospective mechanisms associated with the disruption of mitochondrial function by chemopreventive agents that affect MMP and apoptosis. In doing so, we will construct a paradigm supporting the notion that the bioenergetic and/or redox characteristics of the mitochondria in transformed cells are important targets in the chemoprevention of cancer.