The cytotoxic concentration of rosmarinic acid increases MG132-induced cytotoxicity, proteasome inhibition, autophagy, cellular stresses, and apoptosis in HepG2 cells.

Rosmarinic acid (RA) is a natural polyphenolic compound derived from many common herbal plants. Although it is known that RA has many important biological activities, its effect on proteasome inhibitor-induced changes in cancer treatment or its effects on any experimental proteasome inhibition model is unknown. The aim of the study was to investigate the effect of RA on MG132-induced cytotoxicity, proteasome inhibition, autophagy, cellular stresses, and apoptosis in HepG2 cells. HepG2 cells were treated with 10, 100, and 1000 µM RA in the presence of MG132 for 24 h; 10 and 100 µM RA did not affect but 1000 µM RA decreased cell viability in HepG2 cells. MG132 caused a significant decrease in cell viability and phosphorylation of mammalian target of rapamycin and a significant increase in levels of polyubiquitinated protein, microtubule-associated proteins 1A/1B light chain 3B-II (LC3B-II), heat shock protein 70 (HSP70), binding immunoglobulin protein (BiP), activating transcription factor 4 (ATF4), protein carbonyl, and cleaved poly(adenosine diphosphate-ribose) polymerase 1 (PARP1); 10 and 100 µM RA did not significantly change these effects of MG132 in HepG2 cells; 1000 µM RA caused a significant decrease in cell viability and a significant increase in polyubiquitinated protein, LC3B-II, HSP70, BiP, ATF4, protein carbonyl, and cleaved PARP1 levels in MG132-treated cells. Our study showed that only 1000 µM RA increased MG132-induced cytotoxicity, proteasome inhibition, autophagy, cellular stresses, and apoptosis in HepG2 cells. According to our results, cytotoxic concentration of RA can potentiate the effects of MG132 in hepatocellular carcinoma treatment.

Autophagosome immunoisolation from GFP-LC3B mouse tissue.

We describe a protocol for rapid and efficient enrichment of autophagosomes from various tissues of the GFP-LC3 mouse. In order to increase the number of autophagosomes, we block autophagy flux in the GFP-LC3 mouse tissue with a single intraperitoneal injection of leupeptin 4-5 h before tissue harvesting. We homogenize dissected tissue samples using a Dounce homogenizer followed by passing the slurry through needles of different sizes to dissociate the cells and disrupt their outer membranes. The post-nuclear supernatant fraction of the cell lysate is further centrifuged and the supernatant fraction is discarded to remove residual cytosolic GFP-LC3 that is not associated with autophagosomes. The pellet fraction is resuspended and incubated with magnetic microbeads coated with anti-GFP antibodies for 1 h on ice. The lysate-bead mixture is then applied to a column that is placed in a magnetic separator. After washes, the autophagosome fraction is eluted from the column for morphological and protein analysis. Abbreviations: EDTA: ethylenediaminetetraacetic acid; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GFP: green fluorescent protein; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; LC3: microtubule-associated protein 1 light chain 3 beta; MES: 4-morpholineethanesulfonic acid (MES); SQSTM1: sequestosome 1; TEM: transmission electron microscopy.

The polyphenol quercetin induces cell death in leukemia by targeting epigenetic regulators of pro-apoptotic genes.

BACKGROUND: In the present study, we investigated the molecular mechanisms underlying the pro-apoptotic effects of quercetin (Qu) by evaluating the effect of Qu treatment on DNA methylation and posttranslational histone modifications of genes related to the apoptosis pathway. This study was performed in vivo in two human xenograft acute myeloid leukemia (AML) models and in vitro using HL60 and U937 cell lines. RESULTS: Qu treatment almost eliminates DNMT1 and DNMT3a expression, and this regulation was in part STAT-3 dependent. The treatment also downregulated class I HDACs. Furthermore, treatment of the cell lines with the proteasome inhibitor, MG132, together with Qu prevented degradation of class I HDACs compared to cells treated with Qu alone, indicating increased proteasome degradation of class I HDACS by Qu. Qu induced demethylation of the pro-apoptotic BCL2L11, DAPK1 genes, in a dose- and time-dependent manner. Moreover, Qu (50 µmol/L) treatment of cell lines for 48 h caused accumulation of acetylated histone 3 and histone 4, resulting in three- to ten fold increases in the promoter region of DAPK1, BCL2L11, BAX, APAF1, BNIP3, and BNIP3L. In addition, Qu treatment significantly increased the mRNA levels of all these genes, when compared to cells treated with vehicle only (control cells) (*p < 0.05). CONCLUSIONS: In summary, our results showed that enhanced apoptosis, induced by Qu, might be caused in part by its DNA demethylating activity, by HDAC inhibition, and by the enrichment of H3ac and H4ac in the promoter regions of genes involved in the apoptosis pathway, leading to their transcription activation.

Sulforaphane attenuates postnatal proteasome inhibition and improves spatial learning in adult mice.

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-ß5. 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-ß5 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.

Proteasome Inhibitor MG132 Enhances Cisplatin-Induced Apoptosis in Osteosarcoma Cells and Inhibits Tumor Growth.

Although cisplatin has been shown to be an integral part of chemotherapy regimen in osteosarcoma (OS) treatment, toxicity issues and chemoresistance have hindered therapeutic development for OS. Exploring novel combination therapy methods is needed to circumvent the limitations of cisplatin alone. The proteasome inhibitor MG132 has shown antitumor effects in many solid tumors. However, little is known about its effects in combination with cisplatin in OS cells. In this study, we examined the effects of MG132 in combination with cisplatin in human OS cells (MG-63 and HOS). MG132 and cisplatin were applied to OS cells, respectively or jointly. The results demonstrated that MG132 markedly inhibited cell viability in a dose- and time-dependent manner, whereas viability of osteoblast cells was not affected, suggesting a selective toxicity of MG132 to cancerous cells. Mechanistically, MG132 arrested cells in the G2/M phase in association with increased p21waf1 and induced cell apoptosis, which was accompanied by cleaved PARP. In addition to its apoptotic effect alone, MG132 significantly enhanced cisplatin-induced apoptosis in OS cells. Furthermore, cell viability of the combined application of 10 µM MG132 and 5 µg/ml cisplatin was markedly inhibited compared to that of the individual application. These events were accompanied by the downregulation of NF-κB, mitochondrial antiapoptotic protein Bcl-xL, and PI3K/Akt, which play a key role in cell survival. Finally, combination treatment of MG132 and cisplatin showed more antiproliferative effect than the single treatment in OS xenograft models. In summary, we concluded that MG132 interacted synergistically with cisplatin, which raised the possibility that combining the two drugs may represent a novel strategy in OS.

TSS-Seq analysis of low pH-induced gene expression in intercalated cells in the renal collecting duct.

Metabolic acidosis often results from chronic kidney disease; in turn, metabolic acidosis accelerates the progression of kidney injury. The mechanisms for how acidosis facilitates kidney injury are not fully understood. To investigate whether low pH directly affects the expression of genes controlling local homeostasis in renal tubules, we performed transcription start site sequencing (TSS-Seq) using IN-IC cells, a cell line derived from rat renal collecting duct intercalated cells, with acid loading for 24 h. Peak calling identified 651 up-regulated and 128 down-regulated TSSs at pH 7.0 compared with those at pH 7.4. Among them, 424 and 38 TSSs were ≥ 1.0 and ≤ -1.0 in Log2 fold change, which were annotated to 193 up-regulated and 34 down-regulated genes, respectively. We used gene ontology analysis and manual curation to profile the up-regulated genes. The analysis revealed that many up-regulated genes are involved in renal fibrosis, implying potential molecular mechanisms induced by metabolic acidosis. To verify the activity of the ubiquitin-proteasome system (UPS), a candidate pathway activated by acidosis, we examined the expression of proteins from cells treated with a proteasome inhibitor, MG132. The expression of ubiquitinated proteins was greater at pH 7.0 than at pH 7.4, suggesting that low pH activates the UPS. The in vivo study demonstrated that acid loading increased the expression of ubiquitin proteins in the collecting duct cells in mouse kidneys. Motif analysis revealed Egr1, the mRNA expression of which was increased at low pH, as a candidate factor that possibly stimulates gene expression in response to low pH. In conclusion, metabolic acidosis can facilitate renal injury and fibrosis during kidney disease by locally activating various pathways in the renal tubules.

Protein kinase C-eta regulates Mcl-1 level via ERK1.

Protein kinase C (PKC)-eta (PKCη) is a member of the novel category of PKC family. It is overexpressed in breast cancer and was shown to inhibit apoptosis and contribute to chemoresistance. Since the anti-apoptotic Bcl-2 family protein myeloid cell leukemia-1 (Mcl-1) plays an important role in breast cancer cell survival and chemoresistance, we investigated if PKCη regulates Mcl-1 level. Silencing of PKCη decreased Mcl-1 in several breast cancer cells, including MCF-7 and T47D cells. PKCη depletion had no effect on MCL1 mRNA but the decrease in Mcl-1 by PKCη knockdown was blocked by proteasomal inhibitors, such as MG132 and lactacystin. Moreover, knockdown of Mule (Mcl-1 ubiquitin ligase) prevented Mcl-1 downregulation caused by PKCη deficiency. Overexpression of catalytically-active Akt or knockdown of glycogen synthase kinase-3 (GSK3)-ß, a substrate for Akt, had little effect on Mcl-1 downregulation caused by PKCη silencing. However, knockdown of PKCη but not PKCα, -δ or -ε caused a significant decrease in ERK (extracellular signal-regulated kinase) phosphorylation. Knockdown of ERK1 but not ERK2 decreased Mcl-1 level, and the decrease in Mcl-1 caused by PKCη knockdown was restored by ERK1 overexpression. These results suggest that PKCη utilizes the ERK signaling pathway to protect against ubiquitin-mediated proteasomal degradation of Mcl-1.

Epigallocatechin-3-gallate promotes all-trans retinoic acid-induced maturation of acute promyelocytic leukemia cells via PTEN.

Acute promyelocytic leukemia (APL) is a distinctive subtype of acute myeloid leukemia (AML) in which the hybrid protein promyelocytic leukemia protein/retinoic acid receptor α (PML/RARα) acts as a transcriptional repressor impairing the expression of genes that are critical to myeloid cell mutation. We aimed at explaining the molecular mechanism of green tea polyphenol epigallocatechin-3-gallate (EGCG) enhancement of ATRA-induced APL cell line differentiation. Tumor suppressor phosphatase and tensin homolog (PTEN) was found downregulated in NB4 cells and rescued by proteases inhibitor MG132. A significant increase of PTEN levels was found in NB4, HL-60 and THP-1 cells upon ATRA combined with EGCG treatment, paralleled by increased myeloid differentiation marker CD11b. EGCG in synergy with ATRA promote degradation of PML/RARα and restores PML expression, and increase the level of nuclear PTEN. Pretreatment of PTEN inhibitor SF1670 enhances the PI3K signaling pathway and represses NB4 cell differentiation. Moreover, the induction of PTEN attenuated the Akt phosphorylation levels, pretreatment of PI3K inhibitor LY294002 in NB4 cells, significantly augmented the cell differentiation and increased the expression of PTEN. These results therefore indicate that EGCG targets PML/RARα oncoprotein for degradation and potentiates differentiation of promyelocytic leukemia cells in combination with ATRA via PTEN.

MG132-induced progerin clearance is mediated by autophagy activation and splicing regulation.

Hutchinson-Gilford progeria syndrome (HGPS) is a lethal premature and accelerated aging disease caused by a de novo point mutation in LMNA encoding A-type lamins. Progerin, a truncated and toxic prelamin A issued from aberrant splicing, accumulates in HGPS cells' nuclei and is a hallmark of the disease. Small amounts of progerin are also produced during normal aging. We show that progerin is sequestered into abnormally shaped promyelocytic nuclear bodies, identified as novel biomarkers in late passage HGPS cell lines. We found that the proteasome inhibitor MG132 induces progerin degradation through macroautophagy and strongly reduces progerin production through downregulation of SRSF-1 and SRSF-5 accumulation, controlling prelamin A mRNA aberrant splicing. MG132 treatment improves cellular HGPS phenotypes. MG132 injection in skeletal muscle of LmnaG609G/G609G mice locally reduces SRSF-1 expression and progerin levels. Altogether, we demonstrate progerin reduction based on MG132 dual action and shed light on a promising class of molecules toward a potential therapy for children with HGPS.

Proteotoxic Stress Desensitizes TGF-beta Signaling Through Receptor Downregulation in Retinal Pigment Epithelial Cells.

BACKGROUND: Proteotoxic stress and transforming growth factor (TGFß)- induced epithelial-mesenchymal transition (EMT) are two main contributors of intraocular fibrotic disorders, including proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). However, how these two factors communicate with each other is not well-characterized. OBJECTIVE: The aim was to investigate the regulatory role of proteotoxic stress on TGFß signaling in retinal pigment epithelium. METHODS: ARPE-19 cells and primary human retinal pigment epithelial (RPE) cells were treated with proteasome inhibitor MG132 and TGFß. Cell proliferation was analyzed by CCK-8 assay. The levels of mesenchymal markers α-SMA, fibronectin, and vimentin were analyzed by real-time polymerase chain reaction (PCR), western blot, and immunofluorescence. Cell migration was analyzed by scratch wound assay. The levels of p-Smad2, total Smad2, p-extracellular signal-regulated kinase 1/2 (ERK1/2), total ERK1/2, p-focal adhesion kinase (FAK), and total FAK were analyzed by western blot. The mRNA and protein levels of TGFß receptor-II (TGFßR-II) were measured by realtime PCR and western blot, respectively. RESULTS: MG132-induced proteotoxic stress resulted in reduced cell proliferation. MG132 significantly suppressed TGFß-induced upregulation of α-SMA, fibronectin, and vimentin, as well as TGFß-induced cell migration. The phosphorylation levels of Smad2, ERK1/2, and FAK were also suppressed by MG132. Additionally, the mRNA level and protein level of TGFßR-II decreased upon MG132 treatment. CONCLUSION: Proteotoxic stress suppressed TGFß-induced EMT through downregulation of TGFßR-II and subsequent blockade of Smad2, ERK1/2, and FAK activation.

Proteasome Inhibitor-Loaded Micelles Enhance Antitumor Activity Through Macrophage Reprogramming by NF-κB Inhibition.

Macrophage reprogramming toward a tumor-attacking phenotype is a promising treatment strategy, yet such strategies are scarce and it is not clear how to combine them with cytotoxic therapies that are often used to treat solid tumors. Here, we evaluate whether a micelle-encapsulated proteasome inhibitor, that is, the peptide aldehyde drug MG132, which is cytotoxic to cancer cells, can reprogram macrophages to attack the tumor. Through in vitro studies, we demonstrated that the proteasome inhibition reduces nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling-a known promoter of tumor-supporting macrophages and chemoresistance-in both cancer cells and macrophages. In in vivo studies, we showed that, although free MG132 did not affect the macrophage phenotype in tumors even at its maximum tolerated dose, the micellar formulation of MG132 safely achieved simultaneous cancer cell killing and macrophage reprogramming, thereby enhancing the antitumor efficacy in a syngeneic, orthotopic breast cancer model.

Effect of an MG132-Sustained Drug Delivery Capsular Ring on the Inhibition of Posterior Capsule Opacification in a Rabbit Model.

PURPOSE: To design an MG132-sustained drug delivery capsular ring (SDDCR) and investigate its effect on the inhibition of posterior capsule opacification (PCO) in a rabbit model. METHODS: The SDDCRs were prepared by forming a slice of film made by the mixture of poly lactic-co-glycolic acid (PLGA) and MG132 on the surface of capsular tension rings (CTRs). The drug-loading capacity, entrapment efficiency, and in vitro release of the drug-containing film were detected. Eighteen New Zealand white rabbits were operated with phacoemulsification and MG132-SDDCRs/PLGA-CTRs/CTRs implantation in the single eye. The images of the anterior segments were acquired at certain days, and the epithelial-mesenchymal transition (EMT) markers were detected by western blot and immunofluorescence. RESULTS: The drug-loading capacity and entrapment efficiency of MG132-SDDCRs were 1.15% ± 0.04% and 66.16% ± 0.027%, respectively, and the drug released well within a month. The PCO degree of the MG132-SDDCR group was significantly lower than the other groups. The expression of alpha-smooth muscle actin, fibronectin, vimentin, and collagen-I was lower, and the expression of E-cadherin (E-cad) was higher in the MG132-SDDCR group than the other groups. CONCLUSIONS: MG132-SDDCRs could be established successfully. The PCO process was prevented, and the expression of EMT markers was inhibited by the implantation of MG132-SDDCRs, indicating that this could be a potential treatment against PCO.

Using CRISPR/Cas9-Mediated GLA Gene Knockout as an In Vitro Drug Screening Model for Fabry Disease.

The CRISPR/Cas9 Genome-editing system has revealed promising potential for generating gene mutation, deletion, and correction in human cells. Application of this powerful tool in Fabry disease (FD), however, still needs to be explored. Enzyme replacement therapy (ERT), a regular administration of recombinant human α Gal A (rhα-GLA), is a currently available and effective treatment to clear the accumulated Gb3 in FD patients. However, the short half-life of rhα-GLA in human body limits its application. Moreover, lack of an appropriate in vitro disease model restricted the high-throughput screening of drugs for improving ERT efficacy. Therefore, it is worth establishing a large-expanded in vitro FD model for screening potential candidates, which can enhance and prolong ERT potency. Using CRISPR/Cas9-mediated gene knockout of GLA in HEK-293T cells, we generated GLA-null cells to investigate rhα-GLA cellular pharmacokinetics. The half-life of administrated rhα-GLA was around 24 h in GLA-null cells; co-administration of proteasome inhibitor MG132 and rhα-GLA significantly restored the GLA enzyme activity by two-fold compared with rhα-GLA alone. Furthermore, co-treatment of rhα-GLA/MG132 in patient-derived fibroblasts increased Gb3 clearance by 30%, compared with rhα-GLA treatment alone. Collectively, the CRISPR/Cas9-mediated GLA-knockout HEK-293T cells provide an in vitro FD model for evaluating the intracellular pharmacokinetics of the rhα-GLA as well as for screening candidates to prolong rhα-GLA potency. Using this model, we demonstrated that MG132 prolongs rhα-GLA half-life and enhanced Gb3 clearance, shedding light on the direction of enhancing ERT efficacy in FD treatment.

Inhibition of calpain on oxygen glucose deprivation-induced RGC-5 necroptosis.

The purpose of this study was to investigate the effect of inhibition of calpain on retinal ganglion cell-5 (RGC-5) necroptosis following oxygen glucose deprivation (OGD). RGC-5 cells were cultured in Dulbecco's-modified essential medium and necroptosis was induced by 8-h OGD. PI staining and flow cytometry were performed to detect RGC-5 necrosis. The calpain expression was detected by Western blotting and immunofluorescence staining. The calpain activity was tested by activity detection kit. Flow cytometry was used to detect the effect of calpain on RGC-5 necroptosis following OGD with or without N-acetyl-leucyl-leucyl-norleucinal (ALLN) pre-treatment. Western blot was used to detect the protein level of truncated apoptosis inducing factor (tAIF) in RGC-5 cells following OGD. The results showed that there was an up-regulation of the calpain expression and activity following OGD. Upon adding ALLN, the calpain activity was inhibited and tAIF was reduced following OGD along with the decreased number of RGC-5 necroptosis. In conclusion, calpain was involved in OGD-induced RGC-5 necroptosis with the increased expression of its downstream molecule tAIF.

Combination treatment with proteasome inhibitors and antiestrogens has a synergistic effect mediated by p21WAF1 in estrogen receptor-positive breast cancer.

Although antiestrogens significantly improve the survival of patients with ER-positive breast cancer, therapeutic resistance remains a major limitation. The combinatorial use of antiestrogen with other therapies was proposed to increase their efficiency and more importantly, to prevent or delay the resistance phenomenon. In the present study, we addressed their combined effects with proteasome inhibitors (PIs). The effects of antiestrogens (hydroxyl-tamoxifen, raloxifen and fulvestrant) currently used in endocrine therapy were tested in combination with PIs, bortezomib or MG132, on the growth of three ER-positive breast cancer cell lines and in two cellular models of acquired antiestrogen resistance. When compared to single treatments, these combined treatments were significantly more effective in preventing the growth of the cell lines. The regulation of key cell cycle proteins, the cyclin-dependent kinase inhibitors, p21WAF1 and p27KIP1, were also studied. Bortezomib and MG132 drastically increased p21WAF1 expression through elevation of its mRNA concentration. Notably, p27KIP1 regulation was quite different from that of p21WAF1. Furthermore, the effect of bortezomib in combination with antiestrogen was evaluated on antiestrogen-resistant cell lines. The growth of two antiestrogen-resistant cell lines appeared responsive to proteasome inhibition and was strongly decreased by a combined therapy with an antiestrogen. Collectively, these findings provide new perspectives for the use of PIs in combination with endocrine therapies for breast cancer and possibly to overcome acquired hormonal resistance.

Cardiomyocyte GTP Cyclohydrolase 1 Protects the Heart Against Diabetic Cardiomyopathy.

Diabetic cardiomyopathy increases the risk of heart failure and death. At present, there are no effective approaches to preventing its development in the clinic. Here we report that reduction of cardiac GTP cyclohydrolase 1 (GCH1) degradation by genetic and pharmacological approaches protects the heart against diabetic cardiomyopathy. Diabetic cardiomyopathy was induced in C57BL/6 wild-type mice and transgenic mice with cardiomyocyte-specific overexpression of GCH1 with streptozotocin, and control animals were given citrate buffer. We found that diabetes-induced degradation of cardiac GCH1 proteins contributed to adverse cardiac remodeling and dysfunction in C57BL/6 mice, concomitant with decreases in tetrahydrobiopterin, dimeric and phosphorylated neuronal nitric oxide synthase, sarcoplasmic reticulum Ca(2+) handling proteins, intracellular [Ca(2+)]i, and sarcoplasmic reticulum Ca(2+) content and increases in phosphorylated p-38 mitogen-activated protein kinase and superoxide production. Interestingly, GCH-1 overexpression abrogated these detrimental effects of diabetes. Furthermore, we found that MG 132, an inhibitor for 26S proteasome, preserved cardiac GCH1 proteins and ameliorated cardiac remodeling and dysfunction during diabetes. This study deepens our understanding of impaired cardiac function in diabetes, identifies GCH1 as a modulator of cardiac remodeling and function, and reveals a new therapeutic target for diabetic cardiomyopathy.

Carnosic acid promotes degradation of the androgen receptor and is regulated by the unfolded protein response pathway in vitro and in vivo.

Androgen deprivation therapy in prostate cancer is extremely effective; however, due to the continuous expression and/or mutagenesis of androgen receptor (AR), the resistance to antihormonal therapy is a natural progression. Consequently, targeting the AR for degradation offers an alternate approach to overcome this resistance in prostate cancer. In this study, we demonstrate that carnosic acid, a benzenediol diterpene, binds the ligand-binding domain of the AR and degrades the AR via endoplasmic reticulum (ER) stress-mediated proteasomal degradative pathway. In vitro, carnosic acid treatment induced degradation of AR and decreased expression of prostate-specific antigen in human prostate cancer cell lines LNCaP and 22Rv1. Carnosic acid also promoted the expression of ER proteins including BiP and CHOP in a dose-dependent manner. Downregulation of CHOP by small interfering RNA somewhat restored expression of AR suggesting that AR degradation is dependent on ER stress pathway. Future studies will need to evaluate other aspects of the unfolded protein response pathway to characterize the regulation of AR degradation. Furthermore, cotreating cells individually with carnosic acid and proteasome inhibitor (MG-132) and carnosic acid and an ER stress modulator (salubrinal) restored protein levels of AR, suggesting that AR degradation is mediated by ER stress-dependent proteasomal degradation pathway. Degradation of AR and induction of CHOP protein were also evident in vivo along with a 53% reduction in growth of xenograft prostate cancer tumors. In addition, carnosic acid-induced ER stress in prostate cancer cells but not in normal prostate epithelial cells procured from patient biopsies. In conclusion, these data suggest that molecules such as carnosic acid could be further evaluated and optimized as a potential therapeutic alternative to target AR in prostate cancer.

Proteasome Inhibitor MG132 Enhances Sensitivity to Cisplatin on Ovarian Carcinoma Cells In Vitro and In Vivo.

BACKGROUND: Platinum-based combination chemotherapy after surgery is considered a standard treatment; therefore, any recent drug development should be new, effective, and low toxic, and should have a synergistic effect with platinum. This study aimed to observe the growth of SKOV3 cells after treatment with cisplatin by combining with carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132) and to investigate the effect of the relationship between MG132 and cisplatin combination. MATERIALS AND METHODS: Cell growth was detected by methyl thiazolyl tetrazolium assay after treatment with MG132 at 0.5, 1.5, 2.5, 3.5, and 5.0 µg/mL concentrations for 24, 48, and 72 hours; with cisplatin at 1.0, 2.0, 3.0, 4.0, and 5.0 µg/mL concentrations; and with combination with MG132 at 1.5 µg/mL for 24 hours. The apoptotic rates of cells were detected by a flow cytometer after cisplatin treatment at 1.0, 2.0, 3.0, and 4.0 µg/mL concentrations and that combined with MG132 at 1.5 µg/mL concentration for 12, 24, and 36 hours. A total of 20 BALB/c (nu/nu) female nude mice (age, 4-6 weeks; body weight, 17-19 g) were divided into 4 groups: control, MG132, cisplatin, and combination groups. The expression of Caspase3 and Beclin1 was detected by Western blot analysis and reverse transcription-polymerase chain reaction after treatment with 3.0 µg/mL of the cisplatin group and combined treatment with 1.5 µg/mL of MG132 group for 24 hours, respectively. RESULTS: Methyl thiazolyl tetrazolium assay demonstrated the inhibitory rates, and the flow cytometery showed that the apoptotic rates in the combination group were higher than those in the cisplatin group (P < 0.01). Western blot analysis and reverse transcription-polymerase chain reaction detected that Caspase3 and Beclin1 at a relative quantity in the combination group were higher than those in the cisplatin group (P < 0.05). CONCLUSIONS: MG132 has a synergistic antitumor effect by combining with cisplatin, and it is expected to be an effective antitumor drug for platinum-resistant refractory ovarian cancer.

Enhanced efficacy against cervical carcinomas through polymeric micelles physically incorporating the proteasome inhibitor MG132.

Treatment of recurrent or advanced cervical cancer is still limited, and new therapeutic choices are needed for improving prognosis and quality of life of patients. Because human papilloma virus (HPV) infection is critical in cervical carcinogenesis, with the E6 and E7 oncogenes of HPV degrading tumor suppressor proteins through the ubiquitin proteasome system, the inhibition of the ubiquitin proteasome system appears to be an ideal target to suppress the growth of cervical tumors. Herein, we focused on the ubiquitin proteasome inhibitor MG132 (carbobenzoxy-Leu-Leu-leucinal) as an anticancer agent against cervical cancer cells, and physically incorporated it into micellar nanomedicines for achieving selective delivery to solid tumors and improving its in vivo efficacy. These MG132-loaded polymeric micelles (MG132/m) showed strong tumor inhibitory in vivo effect against HPV-positive tumors from HeLa and CaSki cells, and even in HPV-negative tumors from C33A cells. Repeated injection of MG132/m showed no significant toxicity to mice under analysis by weight change or histopathology. Moreover, the tumors treated with MG132/m showed higher levels of tumor suppressing proteins, hScrib and p53, as well as apoptotic degree, than tumors treated with free MG132. This enhanced efficacy of MG132/m was attributed to their prolonged circulation in the bloodstream, which allowed their gradual extravasation and penetration within the tumor tissue, as determined by intravital microscopy. These results support the use of MG132 incorporated into polymeric micelles as a safe and effective therapeutic strategy against cervical tumors.

The downregulation of SnoN expression in human renal proximal tubule epithelial cells under high-glucose conditions is mediated by an increase in Smurf2 expression through TGF-ß1 signaling.

Transforming growth factor (TGF)-ß1 is a profibrotic cytokine that plays a critical role in the progression of diabetic nephropathy (DN). Previous studies have demonstrated that the Smad transcriptional co-repressor, Ski-related novel protein N (SnoN), an antagonizer of TGF-ß1/Smad signaling, is downregulated in the kidneys of diabetic rats; however, the underlying molecular mechanisms remain elusive. In the present study, we demonstrated that the upregulation of Smad ubiquitination regulatory factor-2 (Smurf2), through TGF-ß1/Smad signaling, contributes to the downregulation of SnoN under high-glucose conditions in primary human renal proximal tubule epithelial cells (hRPTECs). The hRPTECs were cultured in high-glucose (30 mmol/l D-glucose) medium in the presence or absence of either the proteasome inhibitor, MG132, or the TGF-ß type I receptor kinase inhibitor, SB-431542. Small interfering RNA (siRNA) was used to silence Smurf2. The expression levels of SnoN, Smurf2, Smad2 and phosphorylated (p-)Smad2 were measured by western blot analysis and RT-qPCR. The protein levels of SnoN were markedly downregulated, while its mRNA levels were increased in the hRPTECs cultured under high-glucose conditions. The protein and mRNA levels of Smurf2 were significantly increased under high-glucose conditions. The knockdown of Smurf2 increased SnoN expression in the hRPTECs cultured in high-glucose medium. Moreover, MG132 partially inhibited SnoN degradation in the hRPTECs under high-glucose conditions and SB-431542 decreased the phosphorylation of Smad2 and the expression of Smurf2 induced under high-glucose conditions. Taken together, the findings of this study demonstrate that the downregulation of SnoN expression in hRPTECs under high-glucose conditions is mediated by the increased expression of Smurf2 through the TGF-ß1/Smad signaling pathway.