BET Protein Inhibitor JQ1 Ameliorates Experimental Peritoneal Damage by Inhibition of Inflammation and Oxidative Stress.

Peritoneal dialysis (PD) is a current replacement therapy for end-stage kidney diseases (ESKDs). However, long-term exposure to PD fluids may lead to damage of the peritoneal membrane (PM) through mechanisms involving the activation of the inflammatory response and mesothelial-to-mesenchymal transition (MMT), leading to filtration failure. Peritoneal damage depends on a complex interaction among external stimuli, intrinsic properties of the PM, and subsequent activities of the local innate-adaptive immune system. Epigenetic drugs targeting bromodomain and extra-terminal domain (BET) proteins have shown beneficial effects on different experimental preclinical diseases, mainly by inhibiting proliferative and inflammatory responses. However the effect of BET inhibition on peritoneal damage has not been studied. To this aim, we have evaluated the effects of treatment with the BET inhibitor JQ1 in a mouse model of peritoneal damage induced by chlorhexidine gluconate (CHX). We found that JQ1 ameliorated the CHX-induced PM thickness and inflammatory cell infiltration. Moreover, JQ1 decreased gene overexpression of proinflammatory and profibrotic markers, together with an inhibition of the nuclear factor-κB (NF-κB) pathway. Additionally, JQ1 blocked the activation of nuclear factor erythroid 2-related factor 2 (NRF2) and restored changes in the mRNA expression levels of NADPH oxidases (NOX1 and NOX4) and NRF2/target antioxidant response genes. To corroborate the in vivo findings, we evaluated the effects of the BET inhibitor JQ1 on PD patients' effluent-derived primary mesothelial cells and on the MeT-5A cell line. JQ1 inhibited tumor necrosis factor-α (TNF-α)-induced proinflammatory gene upregulation and restored MMT phenotype changes, together with the downmodulation of oxidative stress. Taken together, these results suggest that BET inhibitors may be a potential therapeutic option to ameliorate peritoneal damage.

Regulated cell death pathways in kidney disease.

Disorders of cell number that result from an imbalance between the death of parenchymal cells and the proliferation or recruitment of maladaptive cells contributes to the pathogenesis of kidney disease. Acute kidney injury can result from an acute loss of kidney epithelial cells. In chronic kidney disease, loss of kidney epithelial cells leads to glomerulosclerosis and tubular atrophy, whereas interstitial inflammation and fibrosis result from an excess of leukocytes and myofibroblasts. Other conditions, such as acquired cystic disease and kidney cancer, are characterized by excess numbers of cyst wall and malignant cells, respectively. Cell death modalities act to clear unwanted cells, but disproportionate responses can contribute to the detrimental loss of kidney cells. Indeed, pathways of regulated cell death - including apoptosis and necrosis - have emerged as central events in the pathogenesis of various kidney diseases that may be amenable to therapeutic intervention. Modes of regulated necrosis, such as ferroptosis, necroptosis and pyroptosis may cause kidney injury directly or through the recruitment of immune cells and stimulation of inflammatory responses. Importantly, multiple layers of interconnections exist between different modalities of regulated cell death, including shared triggers, molecular components and protective mechanisms.

Gain-of-function TLR7 and loss-of-function A20 gene variants identify a novel pathway for Mendelian lupus and lupus nephritis.

Systemic lupus erythematosus (SLE) is a chronic and inflammatory autoimmune disease of unknown origin that may cause kidney disease, i.e. lupus nephritis (LN). Within a wider trend towards an expanding field of genetic causes of kidney disease, two recent reports have emphasized the role of Mendelian autoimmune disorders in causing LN both in children and in young adults. Loss-of-function (LOF) variants of tumor necrosis factor alpha-induced protein 3 (TNFAIP3) and gain of function (GOF) variants of Toll-like receptor 7 (TLR7) cause SLE and LN, respectively. Interestingly, both genes regulate the same signaling route, as A20, the protein encoded by TNFAIP3, inhibits nuclear factor ĸB (NF-ĸB) activation while TLR7 promoted NF-ĸB activation. Moreover, TNFAIP3 and TLR7 variants are relatively frequent, potentially contributing to polygenic risk for LN. Finally, they both may be expressed by kidney cells, potentially contributing to the severity of kidney injury in persons who have already developed autoimmunity. The fact that both genes regulate the same pathway may lead to novel therapeutic approaches targeting the shared molecular pathway.

Effective Nephroprotection Against Acute Kidney Injury with a Star-Shaped Polyglutamate-Curcuminoid Conjugate.

The lack of effective pharmacological treatments for acute kidney injury (AKI) remains a significant public health problem. Given the involvement of apoptosis and regulated necrosis in the initiation and progression of AKI, the inhibition of cell death may contribute to AKI prevention/recovery. Curcuminoids are a family of plant polyphenols that exhibit attractive biological properties that make them potentially suitable for AKI treatment. Now, in cultured tubular cells, we demonstrated that a crosslinked self-assembled star-shaped polyglutamate (PGA) conjugate of bisdemethoxycurcumin (St-PGA-CL-BDMC) inhibits apoptosis and necroptosis induced by Tweak/TNFα/IFNγ alone or concomitant to caspase inhibition. St-PGA-CL-BDMC also reduced NF-κB activation and subsequent gene transcription. In vivo, St-PGA-CL-BDMC prevented renal cell loss and preserved renal function in mice with folic acid-induced AKI. Mechanistically, St-PGA-CL-BDMC inhibited AKI-induced apoptosis and expression of ferroptosis markers and also decreased the kidney expression of genes involved in tubular damage and inflammation, while preserving the kidney expression of the protective factor, Klotho. Thus, due to renal accumulation and attractive pharmacological properties, the application of PGA-based therapeutics may improve nephroprotective properties of current AKI treatments.

Cell death-based approaches in treatment of the urinary tract-associated diseases: a fight for survival in the killing fields.

Urinary tract-associated diseases comprise a complex set of disorders with a variety of etiologic agents and therapeutic approaches and a huge global burden of disease, estimated at around 1 million deaths per year. These diseases include cancer (mainly prostate, renal, and bladder), urinary tract infections, and urolithiasis. Cell death plays a key role in the pathogenesis and therapy of these conditions. During urinary tract infections, invading bacteria may either promote or prevent host cell death by interfering with cell death pathways. This has been studied in detail for uropathogenic E. coli (UPEC). Inhibition of host cell death may allow intracellular persistence of live bacteria, while promoting host cell death causes tissue damage and releases the microbes. Both crystals and urinary tract obstruction lead to tubular cell death and kidney injury. Among the pathomechanisms, apoptosis, necroptosis, and autophagy represent key processes. With respect to malignant disorders, traditional therapeutic efforts have focused on directly promoting cancer cell death. This may exploit tumor-specific characteristics, such as targeting Vascular Endothelial Growth Factor (VEGF) signaling and mammalian Target of Rapamycin (mTOR) activity in renal cancer and inducing survival factor deprivation by targeting androgen signaling in prostate cancer. An area of intense research is the use of immune checkpoint inhibitors, aiming at unleashing the full potential of immune cells to kill cancer cells. In the future, this may be combined with additional approaches exploiting intrinsic sensitivities to specific modes of cell death such as necroptosis and ferroptosis. Here, we review the contribution of diverse cell death mechanisms to the pathogenesis of urinary tract-associated diseases as well as the potential for novel therapeutic approaches based on an improved molecular understanding of these mechanisms.

Translational science in chronic kidney disease.

The KDIGO definition of chronic kidney disease (CKD) allowed a more detailed characterization of CKD causes, epidemiology and consequences. The picture that has emerged is worrisome from the point of view of translation. CKD was among the fastest growing causes of death in the past 20 years in age-adjusted terms. The gap between recent advances and the growing worldwide mortality appears to result from sequential roadblocks that limit the flow from basic research to clinical development (translational research type 1, T1), from clinical development to clinical practice (translational research T2) and result in deficient widespread worldwide implementation of already available medical advances (translational research T3). We now review recent advances and novel concepts that have the potential to change the practice of nephrology in order to improve the outcomes of the maximal number of individuals in the shortest possible interval. These include: (i) updating the CKD concept, shifting the emphasis to the identification, risk stratification and care of early CKD and redefining the concept of aging-associated 'physiological' decline of renal function; (ii) advances in the characterization of aetiological factors, including challenging the concept of hypertensive nephropathy, the better definition of the genetic contribution to CKD progression, assessing the role of the liquid biopsy in aetiological diagnosis and characterizing the role of drugs that may be applied to the earliest stages of injury, such as SGLT2 inhibitors in diabetic kidney disease (DKD); (iii) embracing the complexity of CKD as a network disease and (iv) exploring ways to optimize implementation of existing knowledge.

Out of the TWEAKlight: Elucidating the Role of Fn14 and TWEAK in Acute Kidney Injury.

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a tumor necrosis factor superfamily cytokine that activates the fibroblast growth factor-inducible-14 (Fn14) receptor. Functional studies have established a role of TWEAK/Fn14 in experimental acute kidney injury (AKI) and the AKI to chronic kidney disease transition through actions on tubular cells and renal fibroblasts. The renal cell expression of TWEAK and Fn14 is increased in human and experimental AKI and targeting TWEAK or Fn14 by genetic means or neutralizing antibodies was protective in kidney injury induced by folic acid overdose, ischemia-reperfusion, or unilateral ureteral obstruction. TWEAK/Fn14 targeting preserved renal function, and reduced tubular cell injury and death, nuclear factor-κB activation, chemokine expression, inflammatory cell infiltration by macrophages and T cells, myofibroblast numbers, and extracellular matrix deposition, while preserving the expression of the anti-aging factor klotho and the mitochondrial regulator Peroxisome proliferator-activated receptor gamma coactivator 1-alpha(PGC1α), as well as of PGC1α-dependent genes. The beneficial in vivo effects of TWEAK/Fn14 targeting are consistent with known actions of TWEAK on kidney cells. We review the literature on TWEAK and AKI and propose further avenues of research to unravel the contribution of TWEAK to kidney injury. Although a randomized clinical trial of neutralizing anti-TWEAK antibodies for lupus nephritis recently was terminated for futility, AKI represents a potential target for clinical development because it is potentially lethal and, as opposed to severe lupus nephritis, is very common, lacks effective therapy, and is not autoimmune in nature.

TNF-related weak inducer of apoptosis (TWEAK) regulates junctional proteins in tubular epithelial cells via canonical NF-κB pathway and ERK activation.

The tubular epithelium may be intrinsically involved in promoting kidney injury by junctional instability, epithelial-mesenchymal transition (EMT) and extracellular matrix remodelling. In this work, we investigated whether the pleiotropic and proinflammatory cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK), could be able to disturb junctional protein expression and to induce EMT of tubular cells. In cultured murine proximal tubular cells TWEAK induced phenotypic changes that were accompanied by F-actin redistribution, loss of epithelial adherent (E-cadherin, Cadherin-16, ß-catenin) and tight junction (ZO-1) proteins, and re-expression of the mesenchymal protein Vimentin. The transcriptional repressors Snail and HNF1ß were also modulated by TWEAK. In a murine model of obstructive renal pathology, TWEAK expression correlated with the appearance of the mesenchymal marker αSMA in kidney tubular cells. Mechanistically, the epithelial changes induced by TWEAK, including loss of epithelial integrity and EMT, via Fn14 were TGF-ß1 independent, but mediated by several intracellular signaling systems, including the canonical NF-κB, ERK activation and the vitamin D receptor modulation. These results highlight potential contributions of TWEAK-induced inflammatory mechanisms that could unveil new pathogenic effects of TWEAK starting tubulointerstitial damage and fibrosis.

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and kidney disease.

PURPOSE OF REVIEW: The tumor necrosis factor-like weak inducer of apoptosis (TWEAK) cytokine has been linked to kidney injury by functional studies in experimental animals, and has biomarker potential in kidney disease. RECENT FINDINGS: TWEAK was known to promote tubular cell injury and kidney inflammation. Recent studies have expanded these observations, identifying additional targets of TWEAK relevant to kidney injury. Thus, TWEAK upregulates the chemokine and cholesterol scavenger receptor CXCL16 and downregulates the antiaging and antifibrotic molecule Klotho in tubular cells. Furthermore, fibrogenic TWEAK actions on renal fibroblasts were described. TWEAK or factor-inducible molecule 14 targeting decreased the kidney fibrosis resulting from immune and nonimmune kidney injury induced by transient tubular or glomerular insults or by persistent urinary tract obstruction. TWEAK might also contribute to the link between chronic kidney disease and kidney cancer, as suggested by its role in other genitourinary cancers. Progress has also been made in TWEAK targeting. A phase I clinical trial showed that TWEAK targeting is well tolerated in humans, and an ongoing trial is exploring efficacy in lupus nephritis. Nanomolecules and inhibitors of epidermal growth factor receptor pathway may also protect from the adverse effects of TWEAK in the kidney. SUMMARY: These findings suggest that TWEAK targeting has clinical potential in kidney injury of immune and nonimmune origin.

Acute kidney injury transcriptomics unveils a relationship between inflammation and ageing.

There are no pathophysiolgical therapeutic approaches to acute kidney injury (AKI) and the mortality remains high. In addition chronic kidney disease (CKD) predisposes to AKI and AKI contributes to progression of CKD. Recently a transcriptomics approach unveiled a relationship between AKI, inflammation and the regulation of ageing. A transcriptomics analysis of experimental AKI revealed increased kidney expression of Fn14 and transmembrane chemokine CXCL16, as well as a decreased expression of the kidney-secreted anti-ageing hormone Klotho. Fn14 is the receptor for tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the TNF superfamily. In AKI kidneys there was a positive correlation between Fn14 and CXCL16 mRNA expression and an inverse correlation between Fn14 and Klotho mRNA. Tubular cells were the site of Fn14, CXCL16 and Klotho expression in vivo. Research on the relationships between these three molecules disclosed that TWEAK activation of Fn14 promoted inflammation through secretion of chemokines such as CXL16 in tubular cells in culture and in vivo. Furthermore, TWEAK activation of Fn14 decreased expression of Klotho mRNA and protein in culture and in vivo. Interestingly, both TWEAK activation of CXCL16 mRNA transcription and suppression of Klotho mRNA transcription were mediated by the NFκB transcription factor. In conclusion, TWEAK engagement of Fn14 is a central event promoting NFκB-mediated activation of inflammation pathways and suppression of anti-inflammatory/anti-ageing pathways. This information may influence future therapeutic approaches to AKI and inflammation/aging.

TNF superfamily: a growing saga of kidney injury modulators.

Members of the TNF superfamily participate in kidney disease. Tumor necrosis factor (TNF) and Fas ligand regulate renal cell survival and inflammation, and therapeutic targeting improves the outcome of experimental renal injury. TNF-related apoptosis-inducing ligand (TRAIL and its potential decoy receptor osteoprotegerin are the two most upregulated death-related genes in human diabetic nephropathy. TRAIL activates NF-kappaB in tubular cells and promotes apoptosis in tubular cells and podocytes, especially in a high-glucose environment. By contrast, osteoprotegerin plays a protective role against TRAIL-induced apoptosis. Another family member, TNF-like weak inducer of apoptosis (TWEAK induces inflammation and tubular cell death or proliferation, depending on the microenvironment. While TNF only activates canonical NF-kappaB signaling, TWEAK promotes both canonical and noncanonical NF-kappaB activation in tubular cells, regulating different inflammatory responses. TWEAK promotes the secretion of MCP-1 and RANTES through NF-kappaB RelA-containing complexes and upregulates CCl21 and CCL19 expression through NF-kappaB inducing kinase (NIK-) dependent RelB/NF-kappaB2 complexes. In vivo TWEAK promotes postnephrectomy compensatory renal cell proliferation in a noninflammatory milieu. However, in the inflammatory milieu of acute kidney injury, TWEAK promotes tubular cell death and inflammation. Therapeutic targeting of TNF superfamily cytokines, including multipronged approaches targeting several cytokines should be further explored.

Quercetin decreases intracellular GSH content and potentiates the apoptotic action of the antileukemic drug arsenic trioxide in human leukemia cell lines.

Arsenic trioxide (ATO) is an effective therapeutic agent for the treatment of acute promyelocytic leukemia, but successful application of this agent may occasionally require the use of sensitizing strategies. The present work demonstrates that the flavonoids quercetin and chrysin cooperate with ATO to induce apoptosis in U937 promonocytes and other human leukemia cell lines (THP-1, HL-60). Co-treatment with ATO plus quercetin caused mitochondrial transmembrane potential dissipation, stimulated the mitochondrial apoptotic pathway, as indicated by cytochrome c and Omi/Htra2 release, XIAP and Bcl-X(L) down-regulation, and Bax activation, and caused caspase-8/Bid activation. Bcl-2 over-expression abrogated cytochrome c release and apoptosis, and also blocked caspase-8 activation. Quercetin and chrysin, alone or with ATO, decreased Akt phosphorylation as well as intracellular GSH content. GSH depletion was regulated at the level of L-buthionine-(S,R)-sulfoximine (BSO)-sensitive enzyme activity, and N-acetyl-L-cysteine failed both to restore GSH content and to prevent apoptosis. Treatment with BSO caused GSH depletion and potentiated ATO-provoked apoptosis, but did not affect apoptosis induction by ara-C and cisplatin. As an exception, ATO plus quercetin failed to elicit Akt de-phosphorylation and GSH depletion in NB4 acute promyelocytic leukemia cells, and correspondingly exhibited low cooperative effect in inducing apoptosis in this cell line. It is concluded that GSH depletion explains at least in part the selective potentiation of ATO toxicity by quercetin, and that this flavonoid might be used to increase the clinical efficacy of the antileukemic drug.

Arsenic trioxide sensitizes promonocytic leukemia cells to TNFalpha-induced apoptosis via p38-MAPK-regulated activation of both receptor-mediated and mitochondrial pathways.

Treatment with the anti-leukemic drug arsenic trioxide (As(2)O(3), 1-4 microM) sensitizes U937 promonocytes and other human myeloid leukemia cell lines (HL60, NB4) to apoptosis induction by TNFalpha. As(2)O(3) plus TNFalpha increases TNF receptor type 1 (TNF-R1) expression, decreases c-FLIP(L) expression, and causes caspase-8 and Bid activation, and apoptosis is reduced by anti-TNF-R1 neutralizing antibody and caspase-8 inhibitor. The treatment also causes Bax translocation to mitochondria, cytochrome c and Omi/HtrA2 release from mitochondria, XIAP down-regulation, and caspase-9 and caspase-3 activation. Bcl-2 over-expression inhibits cytochrome c release and apoptosis, and also prevents c-FLIP(L) down-regulation and caspase-8 activation, but not TNF-R1 over-expression. As(2)O(3) does not affect Akt phosphorylation/activation or intracellular GSH content, nor prevents the TNFalpha-provoked stimulation of p65-NF-kappaB translocation to the nucleus and the increase in NF-kappaB binding activity. Treatments with TNFalpha alone or with As(2)O(3) plus TNFalpha cause TNF-R1-mediated p38-MAPK phosphorylation/activation. P38-MAPK-specific inhibitors attenuate the As(2)O(3) plus TNFalpha-provoked activation of caspase-8/Bid, Bax translocation, cytochrome c release, and apoptosis induction. In conclusion, the sensitization by As(2)O(3) to TNFalpha-induced apoptosis in promonocytic leukemia cells is an Akt/NF-kappaB-independent, p38-MAPK-regulated process, which involves the interplay of both the receptor-mediated and mitochondrial executioner pathways.

Pharmacologic inhibitors of extracellular signal-regulated kinase (ERKs) and c-Jun NH(2)-terminal kinase (JNK) decrease glutathione content and sensitize human promonocytic leukemia cells to arsenic trioxide-induced apoptosis.

Treatment with 1-4 microM As(2)O(3) slightly induced apoptosis in U-937 human promonocitic leukemia cells. This effect was potentiated by co-treatment with MEK/ERK (PD98059, U0126) and JNK (SP600125, AS601245) inhibitors, but not with p38 (SB203580, SB220025) inhibitors. However, no potentiation was obtained using lonidamine, doxorubicin, or cisplatin instead of As(2)O(3). Apoptosis potentiation by mitogen-activated protein kinase (MAPK) inhibitors involved both the intrinsic and extrinsic executionary pathways, as demonstrated by Bax activation and cytochrome c release from mitochondria, and by caspase-8 activation and Bid cleavage, respectively; and the activation of both pathways was prevented by Bcl-2 over-expression. Treatment with MEK/ERK and JNK inhibitors, but not with p38 inhibitors, caused intracellular glutathione (GSH) depletion, which was differentially regulated. Thus, while it was prevented by N-acetyl-L-cysteine (NAC) in the case of U0126, it behaved as a NAC-insensitive process, regulated at the level of DL-buthionine-(S,R)-sulfoximine (BSO)-sensitive enzyme activity, in the case of SP600125. The MEK/ERK inhibitor also potentiated apoptosis and decreased GSH content in As(2)O(3)-treated NB4 human acute promyelocytic leukemia (APL) cells, but none of these effects were produced by the JNK inhibitor. MEK/ERK and JNK inhibitors did not apparently affect As(2)O(3) transport activity, as measured by intracellular arsenic accumulation. SP600126 greatly induced reactive oxygen species (ROS) accumulation, while BSO and U0126 had little or null effects. These results, which indicate that glutathione is a target of MAP kinases in myeloid leukemia cells, might be exploited to improve the antitumor properties of As(2)O(3), and provide a rationale for the use of kinase inhibitors as therapeutic agents.

Pharmacological inhibitors of extracellular signal-regulated protein kinases attenuate the apoptotic action of cisplatin in human myeloid leukemia cells via glutathione-independent reduction in intracellular drug accumulation.

It has been reported that inhibition of extracellular signal-regulated protein kinases (ERKs) attenuates the toxicity cisplatin (cis-platinum (II)-diammine dichloride) in some cell types. This response was here investigated using human myeloid leukemia cells. Cisplatin stimulated ERK1/2 phosphorylation and caused apoptosis in U-937 promonocytic cells, an effect which was attenuated by the MEK/ERK inhibitors PD98059 and U0126. While ERK1/2 activation was a general phenomenon, irrespective of the used cell type or antitumour drug, the MEK/ERK inhibitors only reduced cisplatin toxicity in human myeloid cells (THP-1, HL-60 and NB-4), but not in RAW 264.7 mouse macrophages and NRK-52E rat renal tubular cells; and failed to reduce the toxicity etoposide, camptothecin, melphalan and arsenic trioxide, in U-937 cells. U0126 attenuated cisplatin-DNA binding and intracellular peroxide accumulation, which are important regulators of cisplatin toxicity. Although cisplatin decreased the intracellular glutathione (GSH) content, which was restored by U0126, treatments with GSH-ethyl ester and dl-buthionine-(S,R)-sulfoximine revealed that GSH does not regulate cisplatin toxicity in the present experimental conditions. In spite of it, PD98059 and U0126 reduced the intracellular accumulation of cisplatin. These results suggest that GSH-independent modulation of drug transport is a major mechanism explaining the anti-apoptotic action of MEK/ERK inhibitors in cisplatin-treated myeloid cells.

Pharmacologic inhibitors of PI3K/Akt potentiate the apoptotic action of the antileukemic drug arsenic trioxide via glutathione depletion and increased peroxide accumulation in myeloid leukemia cells.

Treatment for 14 to 24 hours with low concentrations of arsenic trioxide (As2O3, 1-4 microM) caused apoptosis in U-937 promonocytes and other human myeloid leukemia cell lines (HL-60, NB4). This effect was potentiated by cotreatment with the phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 and wortmannin, and the Akt inhibitor Akt(i)5. However, the inhibitors did not increase the toxicity of the mitochondria-targeting drug lonidamine, and the DNA-specific drugs camptothecin and cisplatin, when used under similar experimental conditions as As2O3. The potentiation of As2O3-provoked apoptosis involved the increased disruption of mitochondrial transmembrane potential, increased caspase-3 activation and cytochrome c release from mitochondria, increased Bax and Bid activation, and attenuation of 27-kDa heat shock protein (HSP27) expression; the potentiation was prevented by Bcl-2 overexpression. The PI3K/Akt inhibitors decreased the intracellular glutathione content, and caused intracellular oxidation, as measured by peroxide accumulation. Cotreatment with subcytotoxic concentrations of hydrogen peroxide increased apoptosis induction by As2O3. On the other hand, the treatments did not significantly affect glutathione S-transferase pi expression and activity. These results, which indicate that glutathione is a target of PI3K/Akt in myeloid leukemia cells, may partially explain the selective increase of As2O3 toxicity by PI3K/Akt inhibitors, and may provide a rationale to improve the efficacy of these inhibitors as therapeutic agents.

12-O-tetradecanoylphorbol-13-acetate may both potentiate and decrease the generation of apoptosis by the antileukemic agent arsenic trioxide in human promonocytic cells. Regulation by extracellular signal-regulated protein kinases and glutathione.

Arsenic trioxide (As(2)O(3)) caused apoptosis in U-937 human promonocytic cells. This effect was potentiated by the simultaneous addition of the glutathione (GSH) synthesis inhibitor DL-buthionine-(R,S)-sulfoximine or the protein kinase C activators 12-O-tetradecanoylphorbol-13-acetate (TPA) and bryostatin 1. In addition TPA decreased the intracellular GSH content, caused ERK activation, and potentiated the As(2)O(3)-provoked activation of p38 and JNK. The addition of N-acetyl-L-cysteine, the PKC inhibitor GF109203X, and the MEK/ERK inhibitors PD98059 and U0126 attenuated both apoptosis induction and GSH decrease, whereas the p38 inhibitor SB203580 and the JNK inhibitor SP600125 were ineffective. TPA also potentiated ERK activation and GSH depletion when added simultaneously to cadmium chloride (CdCl(2)) and doxorubicin. However, TPA only enhanced apoptosis in the case of CdCl(2), which is a GSH-sensitive agent, whereas it reduced the toxicity of doxorubicin and other DNA-specific drugs. Finally, preincubation for 14-24 h with TPA did not potentiate but, instead, attenuated the As(2)O(3)- and CdCl(2)-provoked apoptosis. The same result was obtained by preincubation with bryostatin 1 and other differentiation inducers. It is concluded that TPA increases the apoptotic action of As(2)O(3), an effect mediated by ERK activation and GSH depletion. However, the increase in apoptosis is only effective in non-differentiated cells.

Trypanosoma cruzi: cruzipain and membrane-bound cysteine proteinase isoform(s) interacts with human alpha(2)-macroglobulin and pregnancy zone protein.

Plasmatic levels of pregnancy zone protein (PZP) increase in children with acute Chagas disease. PZP, as well as alpha2-macroglobulin (alpha2-M), are able to interact with Trypanosoma cruzi proteinases. The interaction of alpha2-M and PZP with cruzipain, the major cysteine proteinase of T. cruzi, was investigated. Several molecular changes on both alpha-M inhibitors under reaction with cruzipain were found. PAGE analysis showed: (i) formation of complexes of intermediate mobility and tetramerization of native alpha2-M and PZP, respectively; (ii) limited proteolysis of bait region in alpha2-M and PZP, and (iii) covalent binding of cruzipain to PZP and alpha2-M. Conformational and structural changes experimented by alpha-Ms correlate with modifications of the enzyme electrophoretic mobility and activity. Cruzipain-alpha-M complexes were also detected by gelatin SDS-PAGE and immunoblotting using polyclonal anti-cruzipain antibodies. Concomitantly, alpha2-M and PZP impaired the activity of cruzipain towards Bz-Pro-Phe-Arg-pNA substrate. In addition, alpha-Ms were able to form covalent complexes with membrane isoforms of cysteine proteinases cross-reacting with cruzipain. The present study suggests that both human alpha-macroglobulin inhibitors could prevent or minimize harmful action of cruzipain on host's molecules and hypothetically regulate parasite functions controlled by cruzipain.