leupeptins and sulforaphane

Diabetic nephropathy (DN) is manifested as increased urinary protein level, decreased glomerular filtration rate, and final renal dysfunction. DN is the leading cause of end-stage renal disease worldwide and causes a huge societal healthcare burden. Since satisfied treatments are still limited, exploring new strategies for the treatment of this disease is urgently needed. Oxidative stress takes part in the initiation and development of DN. In addition, nuclear factor erythroid 2-related factor 2 (Nrf2) plays a key role in the cellular response to oxidative stress. Thus, activation of Nrf2 seems to be a new choice for the treatment of DN. In current review, we discussed and summarized the therapeutic effects of Nrf2 activation on DN from both basic and clinical studies.

Topics: Animals; Anticarcinogenic Agents; Antioxidant Response Elements; Antioxidants; Curcumin; Cysteine Proteinase Inhibitors; Diabetic Nephropathies; Enzyme Inhibitors; Humans; Isothiocyanates; Leupeptins; Molecular Targeted Therapy; NF-E2-Related Factor 2; Oxidative Stress; Resveratrol; Rutin; Signal Transduction; Stilbenes; Sulfoxides; Trace Elements; Zinc

Diabetes is increasing at an alarming rate and, despite anti-hypertensive and insulin therapies, diabetic patients are still at risk of developing complications such as chronic kidney disease, cardiovascular disease, and retinopathy. There is therefore an urgent need for more effective therapies to prevent the development and progression of diabetic complications. Oxidative stress is a major player in the aetiology of diabetic complications. However, results from clinical trials thus far using general antioxidants have been disappointing. Mechanism-based antioxidants have gained considerable attention due to their more targeted approach at reducing oxidative stress and associated complications in diabetes. The transcription factor, NFE2-related factor 2 (Nrf2), is a master regulator of redox homeostasis and the cellular detoxification response. Instead of relying on a single antioxidant, activation of Nrf2 results in the concerted upregulation of several antioxidant enzymes and cytoprotective genes, making it an attractive therapeutic target for diabetic complications. Several Nrf2 activators have been discovered and have proven effective at activating Nrf2 signalling through different mechanisms in both in vitro and in vivo models of diabetes. This review will address some of the most promising and well-known Nrf2 activators and their roles in preventing the development and progression of diabetic complications. Challenges facing the advancement of this drug class into the clinic will be discussed, as will be the future of Nrf2 activation as a therapeutic strategy in preventing the development of diabetic complications.

Topics: Animals; Diabetes Complications; Humans; Intracellular Signaling Peptides and Proteins; Isothiocyanates; Kelch-Like ECH-Associated Protein 1; Leupeptins; Mice; Mice, Knockout; Molecular Targeted Therapy; NF-E2-Related Factor 2; Oleanolic Acid; Oxidative Stress; Resveratrol; Stilbenes; Sulfoxides

Primary effusion lymphoma (PEL) is a rare subtype of non-Hodgkin's B-cell lymphoma and is caused by Kaposi's sarcoma-associated herpesvirus (KSHV) in immunosuppressed patients. PEL is an aggressive lymphoma and is frequently resistant to conventional chemotherapies. Sulforaphane (SFN), a natural compound found in cruciferous vegetables and broccoli sprouts, modulates signaling pathways and epigenetic gene expression. However, the anti-proliferative effects of SFN on PEL cells and the underlying mechanisms have not been identified. Here, we found that SFN decreased the viability of KSHV-infected PEL cells compared to KSHV-uninfected B-lymphoma cells. The anti-proliferative effects of SFN on PEL cells were mediated by apoptosis with activating caspases. In addition, SFN inhibited the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and AKT in PEL cells. We also showed that p38MAPK and AKT inhibitors reduced PEL cell growth. The constitutive and/or transient activation of p38MAPK and AKT signaling are necessary for the survival and proliferation of PEL cells. Our data and previous literature indicate that SFN represses the phosphorylation of p38MAPK and AKT, which results in PEL cell apoptosis. Moreover, we investigated whether MG132 or sangivamycin (Sangi) in combination with SFN potentiated the cytotoxic effects of SFN on PEL cells. Compared to treatment with SFN alone, the addition of MG132 or Sangi enhanced the cytotoxic activity of SFN in a synergistic manner. In conclusion, the anti-proliferative effects of SFN indicate its potential as a new substance for the treatment of PEL.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Survival; Drug Synergism; Humans; Isothiocyanates; Leupeptins; Lymphoma, Primary Effusion; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Pyrimidine Nucleosides; Sulfoxides

Proteasomes are known to degrade proteins involved in various processes like metabolism, signal transduction, cell-cycle regulation, inflammation, and apoptosis. Evidence showed that protein degradation has a strong influence on developing neurons as well as synaptic plasticity. Here, we have shown that sulforaphane (SFN) could prevent the deleterious effects of postnatal proteasomal inhibition on spatial reference and working memory of adult mice. One day old Balb/c mice received intracerebroventricular injections of MG132 and SFN. Sham received an equal volume of aCSF. We observed that SFN pre-administration could attenuate MG132 mediated decrease in proteasome and calpain activities. In vitro findings revealed that SFN could induce proteasomal activity by enhancing the expression of catalytic subunit-β5. SFN pre-administration prevented the hippocampus based spatial memory impairments during adulthood, mediated by postnatal MG132 exposure. Histological examination showed deleterious effects of MG132 on pyramidal neurons and granule cell neurons in DG and CA3 sub-regions respectively. Furthermore, SFN pre-administration has shown to attenuate the effect of MG132 on proteasome subunit-β5 expression and also induce the Nrf2 nuclear translocation. In addition, SFN pre-administered mice have also shown to induce expression of pCaMKII, pCreb, and mature/pro-Bdnf, molecules which play a crucial role in spatial learning and memory consolidation. Our findings have shown that proteasomes play an important role in hippocampal synaptic plasticity during the early postnatal period and SFN pre-administration could enhance the proteasomal activity as well as improve spatial learning and memory consolidation.

Topics: Animals; Animals, Newborn; CA3 Region, Hippocampal; Cell Line, Tumor; Dentate Gyrus; Gene Expression Regulation, Developmental; Hippocampus; Humans; Injections, Intraventricular; Isothiocyanates; Leupeptins; Memory Disorders; Mice, Inbred BALB C; Nerve Tissue Proteins; Neuronal Plasticity; Neurons; Neuroprotective Agents; Neurotoxicity Syndromes; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Pyramidal Cells; Spatial Learning; Sulfoxides

Aldo-keto reductases (AKRs) play central roles in the reductive metabolism of endogenous signaling molecules and in the detoxification of xenobiotics. AKRC1-1C3, AKR1B1 and AKR1B10 have been shown to be regulated via nuclear factor-erythroid 2 related factor 2 (Nrf2), a transcription factor that is activated upon oxidative stress. Proteasome inhibitors bortezomib and MG-132 produce mild oxidative stress that activates Nrf2-mediated gene expression that in turn may have cytoprotective effects. Bortezomib is clinically approved to treat haematological malignancies and it has also proven activity in solid tumors such as colon cancer. The present study investigated the effect of bortezomib and MG-132 on the expression of AKR1C1-1C4, AKR1B1, and AKR1B10 in colon cancer cell lines HT-29 and SW-480. Human cancer cell lines derived from different organs (lung, colon, pancreas, skin, liver, ovary) were initially assayed for the expression of the AKRs, showing a very unequal distribution. Even among the colon cell lines HT-29, Caco-2, HCT116 and SW-480, the AKRs were expressed quite non-uniformly. HT-29 cells expressed all AKRs on the mRNA level including liver-specific AKR1C4, but AKR1B1 was almost undetectable. In SW-480 cells, treatment with bortezomib (50 nM, 48 h) dramatically increased mRNA levels of AKR1B10 (32-fold), AKR1B1 (5.5-fold), and, to a lesser extent, AKR1C1 and AKR1C3. Drug-efflux transporter MRP2 (ABCC2) and Cox-2 were induced as well. AKR1C2 mRNA was down-regulated in SW-480 but induced in HT-29 cells. MG-132 increased mRNA amounts of AKR1C1, 1C3, 1B1, and 1B10 in a concentration-dependent manner. AKR1B10 and AKR1B1 protein expression was inducible by bortezomib in HT-29 cells, but not detectable in SW-480 cells. In conclusion, treatment with proteasome inhibitors increased the expression of several AKRs as well as of MRP2. It remains to be investigated whether this enzyme induction may contribute to enhanced cell survival and thereby supporting the phenomenon of multidrug resistance upon cancer chemotherapy.

Topics: 20-Hydroxysteroid Dehydrogenases; 3-Hydroxysteroid Dehydrogenases; Alcohol Oxidoreductases; Aldehyde Reductase; Aldo-Keto Reductase Family 1 Member C3; Aldo-Keto Reductases; Boronic Acids; Bortezomib; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Induction; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Hydroxyprostaglandin Dehydrogenases; Isothiocyanates; Leupeptins; Maleates; Multidrug Resistance-Associated Protein 2; NF-E2-Related Factor 2; Protease Inhibitors; Proteasome Inhibitors; Pyrazines; RNA, Messenger; Sulfoxides; Thiocyanates; Time Factors

It is conceivable that stimulating proteasome activity for rapid removal of misfolded and oxidized proteins is a promising strategy to prevent and alleviate aging-related diseases. Sulforaphane (SFN), an effective cancer preventive agent derived from cruciferous vegetables, has been shown to enhance proteasome activities in mammalian cells and to reduce the level of oxidized proteins and amyloid β-induced cytotoxicity. Here, we report that SFN activates heat shock transcription factor 1-mediated heat shock response. Specifically, SFN-induced expression of heat shock protein 27 (Hsp27) underlies SFN-stimulated proteasome activity. SFN-induced proteasome activity was significantly enhanced in Hsp27-overexpressing cells but absent in Hsp27-silenced cells. The role of Hsp27 in regulating proteasome activity was further confirmed in isogenic REG cells, in which SFN-induced proteasome activation was only observed in cells stably overexpressing Hsp27, but not in the Hsp27-free parental cells. Finally, we demonstrated that phosphorylation of Hsp27 is irrelevant to SFN-induced proteasome activation. This study provides a novel mechanism underlying SFN-induced proteasome activity. This is the first report to show that heat shock response by SFN, in addition to the antioxidant response mediated by the Keap1-Nrf2 pathway, may contribute to cytoprotection.

Topics: Animals; Anticarcinogenic Agents; Cell Line, Tumor; Chlorocebus aethiops; COS Cells; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Dose-Response Relationship, Drug; Enzyme Activation; Heat Shock Transcription Factors; Heat-Shock Response; HeLa Cells; Hot Temperature; HSP27 Heat-Shock Proteins; Humans; Immunoblotting; Isothiocyanates; Leupeptins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Biosynthesis; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Sulfoxides; Thiocyanates; Transcription Factors; Up-Regulation

Sulforaphane [SUL, 1-isothiocyanato-4-(methylsulfinyl)butane] is an isothiocyanate derived from glucoraphanin present in cruciferous vegetables, and it has a variety of potential chemopreventive actions. We analyzed the effects of SUL on the proliferation of human breast cancer cells and on the expression of estrogen receptor alpha (ERalpha) protein and mRNA in MCF-7 cells. Sulforaphane inhibited cell proliferation with IC(50) values at 24 and 48 h of 12.5 and 7.5 muM doses, respectively, and decreased ERalpha protein expression at concentrations between 2.5 and 30 muM. Inhibition of ERalpha protein expression was also accompanied by decreased progesterone receptor expression. MCF-7 ERalpha mRNA expression was inhibited by SUL at a dose of 30 muM, but not at lower SUL concentrations. At SUL doses <30 muM, the SUL-induced suppression of ERalpha protein was reversed by preincubation with the proteasome inhibitor MG132 and was accompanied by an increase in protein levels of the 20S catalytic core subunit PSMB5. Therefore, SUL can inhibit the expression of ERalpha protein in MCF-7 cells in part by inhibition of ERalpha mRNA transcription as well as by a mechanism that may involve increased proteasome-mediated degradation. These data provide new insights into mechanisms by which SUL inhibits proliferation of and down-regulates hormone receptor expression in MCF-7 cells.

Topics: Breast; Breast Neoplasms; Cell Line; Cell Proliferation; Down-Regulation; Estrogen Receptor alpha; Humans; Isothiocyanates; Leupeptins; RNA, Messenger; Sulfoxides; Thiocyanates; Tumor Cells, Cultured

The 26S proteasome plays a major role in degradation of abnormal proteins within the cell. The indirect antioxidant including sulforaphane (SFN) protects cells from oxidative damage by increasing the expression of Nrf2-target genes. It has been observed that the expression of multiple subunits of the proteasome was up-regulated by indirect antioxidants through the Nrf2 pathway. In the current study, the role of SFN in amyloid beta(1-42) (Abeta(1-42))-induced cytotoxicity has been investigated in murine neuroblastoma cells. Treatment with SFN protected cells from Abeta(1-42)-mediated cell death in Neuro2A and N1E 115 cells. Inhibition of proteasome activities by MG132 could abolish the protective effect of SFN against Abeta(1-42). Neuro2A cells, which were stably overexpressing the catalytic subunit of the proteasome PSMB5, showed an elevated resistance toward Abeta(1-42) toxicity compared to control cells. Furthermore, the in vitro assay demonstrated that the Abeta(1-42) peptide is degraded by the proteasome fraction. These results suggest that proteasome-inducing indirect antioxidants may facilitate the removal of the Abeta(1-42) peptide and lead to the amelioration of abnormal protein-associated etiologies.

Topics: Amyloid beta-Peptides; Animals; Antioxidants; Cell Line, Tumor; Cell Survival; Cysteine Proteinase Inhibitors; Cytoprotection; Dose-Response Relationship, Drug; Isothiocyanates; Leupeptins; Mice; Neuroblastoma; Neurons; Peptide Fragments; Promoter Regions, Genetic; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Sulfoxides; Thiocyanates; Transfection; Up-Regulation