Dallas Donohoe

Light Chain Amyloid Fibrils Cause Metabolic Dysfunction in Human Cardiomyocytes

Light chain (AL) amyloidosis is the most common form of systemic amyloid disease, and cardiomyopathy is a dire consequence, resulting in an extremely poor prognosis. AL is characterized by the production of monoclonal free light chains that deposit as amyloid fibrils principally in the heart, liver, and kidneys causing organ dysfunction. We have studied the effects of amyloid fibrils, produced from recombinant λ6 light chain variable domains, on metabolic activity of human cardiomyocytes. The data indicate that fibrils at 0.1 μM, but not monomer, significantly decrease the enzymatic activity of cellular NAD(P)H-dependent oxidoreductase, without causing significant cell death. The presence of amyloid fibrils did not affect ATP levels; however, oxygen consumption was increased and reactive oxygen species were detected. Confocal fluorescence microscopy showed that fibrils bound to and remained at the cell surface with little fibril internalization. These data indicate that AL amyloid fibrils severely impair cardiomyocyte metabolism in a dose dependent manner. These data suggest that effective therapeutic intervention for these patients should include methods for removing potentially toxic amyloid fibrils.

IL-1β reciprocally regulates chemokine and insulin secretion in pancreatic β-cells via NF-κB.

Proinflammatory cytokines impact islet β-cell mass and function by altering the transcriptional activity within pancreatic β-cells, producing increases in intracellular nitric oxide abundance and the synthesis and secretion of immunomodulatory proteins such as chemokines. Herein, we report that IL-1β, a major mediator of inflammatory responses associated with diabetes development, coordinately and reciprocally regulates chemokine and insulin secretion. We discovered that NF-κB controls the increase in chemokine transcription and secretion as well as the decrease in both insulin secretion and proliferation in response to IL-1β. Nitric oxide production, which is markedly elevated in pancreatic β-cells exposed to IL-1β, is a negative regulator of both glucose-stimulated insulin secretion and glucose-induced increases in intracellular calcium levels. By contrast, the IL-1β-mediated production of the chemokines CCL2 and CCL20 was not influenced by either nitric oxide levels or glucose concentration. Instead, the synthesis and secretion of CCL2 and CCL20 in response to IL-1β were dependent on NF-κB transcriptional activity. We conclude that IL-1β-induced transcriptional reprogramming via NF-κB reciprocally regulates chemokine and insulin secretion while also negatively regulating β-cell proliferation. These findings are consistent with NF-κB as a major regulatory node controlling inflammation-associated alterations in islet β-cell function and mass.

PPARα inhibition modulates multiple reprogrammed metabolic pathways in kidney cancer and attenuates tumor growth

Kidney cancer [renal cell carcinoma (RCC)] is the sixth-most-common cancer in the United States, and its incidence is increasing. The current progression-free survival for patients with advanced RCC rarely extends beyond 1-2 yr due to the development of therapeutic resistance. We previously identified peroxisome proliferator-activating receptor-α (PPARα) as a potential therapeutic target for this disease and showed that a specific PPARα antagonist, GW6471, induced apoptosis and cell cycle arrest at G0/G1 in RCC cell lines associated with attenuation of cell cycle regulatory proteins. We now extend that work and show that PPARα inhibition attenuates components of RCC metabolic reprogramming, capitalizing on the Warburg effect. The specific PPARα inhibitor GW6471, as well as a siRNA specific to PPARα, attenuates the enhanced fatty acid oxidation and oxidative phosphorylation associated with glycolysis inhibition, and PPARα antagonism also blocks the enhanced glycolysis that has been observed in RCC cells; this effect did not occur in normal human kidney epithelial cells. Such cell type-specific inhibition of glycolysis corresponds with changes in protein levels of the oncogene c-Myc and has promising clinical implications. Furthermore, we show that treatment with GW6471 results in RCC tumor growth attenuation in a xenograft mouse model, with minimal obvious toxicity, a finding associated with the expected on-target effects on c-Myc. These studies demonstrate that several pivotal cancer-relevant metabolic pathways are inhibited by PPARα antagonism. Our data support the concept that targeting PPARα, with or without concurrent inhibition of glycolysis, is a potential novel and effective therapeutic approach for RCC that targets metabolic reprogramming in this tumor.

Cellular Metabolism and Dose Reveal Carnitine‐Dependent and ‐Independent Mechanisms of Butyrate Oxidation in Colorectal Cancer Cells

Dietary fiber has been suggested to suppress colorectal cancer development, although the mechanisms contributing to this beneficial effect remain elusive. Butyrate, a fermentation product of fiber, has been shown to have anti‐proliferative and pro‐apoptotic effects on colorectal cancer cells. The metabolic fate of butyrate in the cell is important in determining whether, it acts as an HDAC inhibitor or is consumed as a short‐chain fatty acid. Non‐cancerous colonocytes utilize butyrate as the primary energy source whereas cancerous colonocytes increase glucose utilization through the Warburg effect. In this study, we show that butyrate oxidation is decreased in cancerous colonocytes compared to non‐cancerous colonocytes. We demonstrate that colorectal cancer cells utilize both a carnitine‐dependent and carnitine‐independent mechanism that contributes to butyrate oxidation. The carnitine‐dependent mechanism is contingent on butyrate concentration. Knockdown of CPT1A in colorectal cancer cells abolishes butyrate oxidation. In terms of selectivity, the carnitine‐dependent mechanism only regulated butyrate oxidation, as acetate and propionate oxidation were carnitine‐independent. Carnitine decreased the action of butyrate as an HDAC inhibitor and suppressed induction of H3 acetylation by butyrate in colorectal cancer cells. Thus, diminished oxidation of butyrate is associated with decreased HDAC inhibition and histone acetylation. In relation to the mechanism, we find that dichloroacetate, which decreases phosphorylation of pyruvate dehydrogenase, increased butyrate oxidation and that this effect was carnitine‐dependent. In conclusion, these data suggest that colorectal cancer cells decrease butyrate oxidation through inhibition of pyruvate dehydrogenase, which is carnitine‐dependent, and provide insight into why butyrate shows selective effects toward colorectal cancer cells. J. Cell. Physiol. 231: 1804–1813, 2016.

Manipulating Glucose Metabolism during Different Stages of Viral Pathogenesis Can Have either Detrimental or Beneficial Effects

This report deals with physiological changes and their implication following ocular infection with HSV. This infection usually results in a blinding inflammatory reaction in the cornea, orchestrated mainly by proinflammatory CD4 T cells and constrained in severity by regulatory T cells. In the present report, we make the unexpected finding that blood glucose levels change significantly during the course of infection. Whereas levels remained normal during the early phase of infection when the virus was actively replicating in the cornea, they increased around 2-fold during the time when inflammatory responses to the virus was occurring. We could show that glucose levels influenced the extent of induction of the inflammatory T cell subset in vitro that mainly drives lesions, but not regulatory T cells. Additionally, if glucose utilization was limited in vivo as a consequence of therapy in the inflammatory phase with the drug 2-deoxy-glucose (2DG), lesions were diminished compared with untreated infected controls. In addition, lesions in 2DG-treated animals contained less proinflammatory effectors. Glucose metabolism also influenced the acute phase of infection when the replicating virus was present in the eye. Thus, therapy with 2DG to limit glucose utilization caused mice to become susceptible to the lethal effects of HSV infection, with the virus spreading to the brain causing encephalitis. Taken together, our results indicate that glucose metabolism changed during the course of HSV infection and that modulating glucose levels can influence the outcome of infection, being detrimental or beneficial according to the stage of viral pathogenesis.

Characterization of the Pro‐inflammatory Cytokine IL‐1β on Butyrate Oxidation in Colorectal Cancer Cells

Cancer, in part, is driven, by alterations in cellular metabolism that promote cell survival and cell proliferation. Identifying factors that influence this shift in cellular metabolism in cancer cells is important. Interleukin‐1β (IL‐1β) is a pro‐inflammatory cytokine that has been reported to be elevated in colorectal cancer patients. While much is known toward the effect of dietary nutrients on regulating inflammation and the inflammatory response, which includes cytokines such as IL‐1β, far less is understood how cytokines impact nutrient fate to alter cancer cell metabolism. Butyrate, a nutrient derived from the fermentation of dietary fiber in the colon, is the preferential exogenous energetic substrate used by non‐cancerous colonocytes, but is used less efficiently by colorectal cancer cells. To test whether IL‐1β alters colonocyte energy metabolism, we measured butyrate oxidation in HCT116 colorectal cancer cells with and without IL‐1β. We hypothesize that IL‐1β will push cancerous colonocytes away from the utilization and oxidation of butyrate. In this study, we demonstrate that pretreatment of colorectal cancer cells with IL‐1β diminished butyrate oxidation and NADH levels. This effect was blocked with the interleukin receptor antagonist A (IL‐1RA). Moreover, IL‐1β suppressed basal mitochondrial respiration and lowered the mitochondrial spare capacity. By using inhibitors to block downstream targets of the interleukin‐1 receptor pathway, we show that p38 is required for the IL‐1β‐mediated decrease in butyrate oxidation. These data provide insight into the metabolic effects induced by IL‐1β in colorectal cancer, and identify relevant targets that may be exploited to block the effects of this cytokine. J. Cell. Biochem. 118: 1614–1621, 2017.

Influence of phosphatidylserine and phosphatidylethanolamine on farnesol tolerance in Candida albicans

Candida albicans is among the most common human fungal pathogens. The ability to undergo the morphological transition from yeast to hyphal growth is critical for its pathogenesis. Farnesol, a precursor in the isoprenoid/sterol pathway, is a quorum‐sensing molecule produced by C. albicans that inhibits hyphal growth in this polymorphic fungus. Interestingly, C. albicans can tolerate farnesol concentrations that are toxic to other fungi. We hypothesized that changes in phospholipid composition are one of the factors contributing to farnesol tolerance in C. albicans. In this study, we found that loss of enzymes that synthesize the phospholipids phosphatidylserine (PS) and/or phosphatidylethanolamine (PE) compromise the tolerance of C. albicans to farnesol. Compared with wild type, the phospholipid mutant cho1∆/∆ (loss of PS and decreased PE synthesis) shows greater inhibition of growth, loss of ATP production, increased consumption of oxygen, and increased formation of reactive oxygen species in the presence of farnesol. The cho1∆/∆ mutant also exhibits decreased sensitivity to mitochondrial ATPase inhibition, suggesting that cells lacking PS and/or downstream PE rely less on mitochondrial function for ATP synthesis. These data reveal that PS and PE play roles in farnesol tolerance and maintaining mitochondrial respiratory function.

Concurrent regulation of LKB1 and CaMKK2 in the activation of AMPK in castrate-resistant prostate cancer by a well-defined polyherbal mixture with anticancer properties

BackgroundZyflamend, a blend of herbal extracts, effectively inhibits tumor growth using preclinical models of castrate-resistant prostate cancer mediated in part by 5′-adenosine monophosphate-activated protein kinase (AMPK), a master energy sensor of the cell. Clinically, treatment with Zyflamend and/or metformin (activators of AMPK) had benefits in castrate-resistant prostate cancer patients who no longer responded to treatment. Two predominant upstream kinases are known to activate AMPK: liver kinase B1 (LKB1), a tumor suppressor, and calcium-calmodulin kinase kinase-2 (CaMKK2), a tumor promotor over-expressed in many cancers. The objective was to interrogate how Zyflamend activates AMPK by determining the roles of LKB1 and CaMKK2.MethodsAMPK activation was determined in CWR22Rv1 cells treated with a variety of inhibitors of LKB1 and CaMKK2 in the presence and absence of Zyflamend, and in LKB1-null HeLa cells that constitutively express CaMKK2, following transfection with wild type LKB1 or catalytically-dead mutants. Upstream regulation by Zyflamend of LKB1 and CaMKK2 was investigated targeting protein kinase C-zeta (PKCζ) and death-associated protein kinase (DAPK), respectively.ResultsZyflamend’s activation of AMPK appears to be LKB1 dependent, while simultaneously inhibiting CaMKK2 activity. Zyflamend failed to rescue the activation of AMPK in the presence of pharmacological and molecular inhibitors of LKB1, an effect not observed in the presence of inhibitors of CaMKK2. Using LKB1-null and catalytically-dead LKB1-transfected HeLa cells that constitutively express CaMKK2, ionomycin (activator of CaMKK2) increased phosphorylation of AMPK, but Zyflamend only had an effect in cells transfected with wild type LKB1. Zyflamend appears to inhibit CaMKK2 by DAPK-mediated phosphorylation of CaMKK2 at Ser511, an effect prevented by a DAPK inhibitor. Alternatively, Zyflamend mediates LKB1 activation via increased phosphorylation of PKCζ, where it induced translocation of PKCζ and LKB1 to their respective active compartments in HeLa cells following treatment. Altering the catalytic activity of LKB1 did not alter this translocation.DiscussionZyflamend’s activation of AMPK is mediated by LKB1, possibly via PKCζ, but independent of CaMKK2 by a mechanism that appears to involve DAPK.ConclusionsTherefore, this is the first evidence that natural products simultaneously and antithetically regulate upstream kinases, known to be involved in cancer, via the activation of AMPK.

Abstract SY04-02: Dietary fiber protects against colorectal tumorigenesis in a microbiota- and butyrate-dependent manner

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA It is controversial whether dietary fiber protects against colorectal cancer because of conflicting results from human epidemiologic studies. These studies have been complicated by the participants’ genetic heterogeneity and differences in the composition of microbiota within their gastrointestinal tracts. To eliminate these confounding variables, we utilized a gnotobiotic mouse model of colorectal cancer. Our experiments were designed to investigate the function of butyrate because it is a short-chain fatty acid produced by bacterial fermentation of fiber in the colon at high (mM) levels and has potent energetic and epigenetic properties in host colonocytes. Here, we report that fiber did, in fact, have a chemoprotective effect but in a microbiota- and butyrate-dependent manner. The incidence, number, size, and histopathologic progression of AOM/DSS-induced colorectal tumors were significantly diminished when BALB/c mice were provided a high-fiber diet only if they were colonized with defined microbiota that included a butyrate-producing bacteria. This chemoprotective effect was attenuated when mice were colonized with the same microbiota except that the wild-type butyrate producer was replaced by a mutant strain with a 0.8-kb deletion in the butyryl-CoA synthesis operon. To confirm that butyrate is a causal factor, the chemoprotective effect was recapitulated in mice without any butyrate-producing bacteria if they were provided a butyrate-fortified diet. Our data support a general mechanism that includes microbial fermentation of fiber rather than fiber exclusively speeding colonic transit to minimize the exposure of colonocytes to ingested carcinogens. Our data also support a molecular mechanism that is metaboloepigenetic. Normal colonocytes utilize butyrate as their preferred energy source, whereas cancerous colonocytes rely on glucose because of the Warburg effect. Due to this metabolic difference, butyrate accumulated in tumors (as measured by LC-MS) and functioned as an HDAC inhibitor to increase histone acetylation levels and apoptosis. To support the applicability of this model to human cancer, we demonstrate that butyrate also accumulates at higher levels in human colorectal tumors than in normal colonic tissue, and this is associated with higher levels of histone acetylation in tumors. These results link diet and microbiota to a common metabolite that influences epigenetics and cancer predisposition. Citation Format: Dallas Donohoe, Darcy Holley, Leonard Collins, Stephanie Montgomery, Alan Whitmore, Andrew Hillhouse, Kaitlin Curry, Sarah Renner, Alicia Greenwalt, Elizabeth Ryan, Virginia Godfrey, Mark Heise, Deborah Threadgill, James Swenberg, David Threadgill, Scott Bultman. Dietary fiber protects against colorectal tumorigenesis in a microbiota- and butyrate-dependent manner. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY04-02. doi:10.1158/1538-7445.AM2014-SY04-02