Hua-Qin Wang

Role of oxidative stress and intracellular glutathione in the sensitivity to apoptosis induced by proteasome inhibitor in thyroid cancer cells

BackgroundThe proteasome inhibitor bortezomib has shown impressive clinical activity alone and in combination with conventional and other novel agents for the treatment of multiple myeloma (MM) and some solid cancers. Although bortezomib is known to be a selective proteasome inhibitor, the downstream mechanisms of cytotoxicity and drug resistance are poorly understood.MethodsProteasome activity, intracellular glutathione (GSH) and ROS levels, as well as activities of GSH synthesis enzymes were measured using spectrophotometric methods. Cell death was analyzed using flow cytometry and caspase activity assay. The expression level of GSH synthesis enzymes were measured using real-time RT-PCR.ResultsAt concentrations that effectively inhibited proteasome activity, bortezomib induced apoptosis in FRO cells, but not in ARO cells. Bortezomib elevated the amount of glutathione (GSH) and the treatment with bortezomib increased the level of mRNA for GCL, a rate-limiting enzyme in glutathione synthesis. Furthermore, depletion of GSH increases apoptosis induced by bortezomib, in contrast, repletion of GSH decreases bortezomib-mediated cell death.ConclusionGSH protects cells from proteasome inhibition-induced oxidative stress and glutathione-dependent redox system might play an important role in the sensitivity to proteasome inhibition-induced apoptosis.

Proteasome Inhibitor MG132 Induces BAG3 Expression Through Activation of Heat Shock Factor 1

BAG3 protein, a member of the BAG co‐chaperones family, sustains cell survival in a variety of normal and neoplastic cell types, via its interaction with a variety of partners, such as the heat shock protein (HSP) 70, Bcl‐2, Raf‐1 and others. Expression of BAG3 is induced by some stressful stimuli, such as heat shock, heavy metal exposure. We have reported that proteasome inhibitors can also induce BAG3 expression at the transcriptional level and the induction of BAG3 compromises proteasome inhibitors‐mediated apoptosis. However, the molecular mechanism of BAG3 upregulation has not been elucidated. In the current study, we provide evidence that heat shock transcription factor 1 (HSF1) is involved in BAG3 induction by proteasome inhibitor MG132. Using a series of varying lengths of 5′‐flanking region of the BAG3 gene into luciferase reporter vectors, we found that MG132 stimulated the promoter activity via the −326/−233 and −825/−689 regions, which contains one putative heat shock‐responsive element (HSE) for HSF1‐binding, respectively. Site‐directed deletion of the sites abrogated the enhanced reporter activity in response to MG132 treatment. Chromatin immunoprecipitation assay demonstrated that HSF1 directly bound to the MG132‐responsive site on the BAG3 promoter. Activation of HSF1 occurred with MG132 along with BAG3 upregulation. Furthermore, knockdown HSF1 by small interfering RNA attenuated the BAG3 upregulation due to MG132.These results indicate that the proteasome inhibitor MG132 induces BAG3 expression through HSF1 activation. J. Cell. Physiol. 218: 631–637, 2009.

Inhibition of the JNK signalling pathway enhances proteasome inhibitor‐induced apoptosis of kidney cancer cells by suppression of BAG3 expression

Background and purpose:  Proteasome inhibitors represent a novel class of anti‐tumour agents that have clinical efficacy against haematological and solid cancers. The anti‐apoptotic protein BAG3 is a member of the Bcl‐2‐associated athanogene family. We have previously shown that BAG3 is up‐regulated after exposure to proteasome inhibitors and that inhibition of BAG3 sensitized cells to apoptosis induced by proteasome inhibition. However, the mechanisms by which proteasome inhibition induced BAG3 expression remained unclear and the present experiments were designed to elucidate these mechanisms.

Implication of unfolded protein response in resveratrol-induced inhibition of K562 cell proliferation

Resveratrol (RES), a natural plant polyphenol, is an effective inducer of cell cycle arrest and apoptosis in a variety of carcinoma cell types. In addition, RES has been reported to inhibit tumorigenesis in several animal models suggesting that it functions as a chemopreventive and anti-tumor agent in vivo. The chemopreventive and chemotherapeutic properties associated with resveratrol offer promise for the design of new chemotherapeutic agents. However, the mechanisms by which RES mediates its effects are not yet fully understood. In this study, we showed that RES caused cell cycle arrest and proliferation inhibition via induction of unfolded protein response (UPR) in human leukemia K562 cell line. Treatment of K562 cells with RES induced a number of signature UPR markers, including transcriptional induction of GRP78 and CHOP, phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha), ER stress-specific XBP-1 splicing, suggesting the induction of UPR by RES. RES inhibited proliferation of K562 in a concentration-dependent manner. Flow cytometric analyses revealed that K562 cells were arrested in G1 phase upon RES treatment. Salubrinal, an eIF2alpha inhibitor, or overexpression of dominant negative mutants of PERK or eIF2alpha, effectively restored RES-induced cell cycle arrest, underscoring the important role of PERK/eIF2alpha branch of UPR in RES-induced inhibition of cell proliferation.

Resveratrol-induced cytotoxicity in human Burkitt's lymphoma cells is coupled to the unfolded protein response.

BackgroundResveratrol (RES), a natural phytoalexin found at high levels in grapes and red wine, has been shown to induce anti-proliferation and apoptosis of human cancer cell lines. However, the underlying molecular mechanisms are at present only partially understood.MethodThe effects of RES on activation of unfolded protein responses (UPR) were evaluated using Western blotting, semi-quantitative and real-time RT-PCR. Cell death was evaluated using Annexin V/PI staining and subsequent FACS.ResultsSimilar as tunicamycin, treatment with RES lead to the activation of all 3 branches of the UPR, with early splicing of XBP-1 indicative of IRE1 activation, phosphorylation of eIF2α consistent with ER resident kinase (PERK) activation, activating transcription factor 6 (ATF6) splicing, and increase in expression levels of the downstream molecules GRP78/BiP, GRP94 and CHOP/GADD153 in human Burkitts lymphoma Raji and Daudi cell lines. RES was shown to induce cell death, which could be attenuated by thwarting upregulation of CHOP.ConclusionsOur data suggest that activation of the apoptotic arm of the UPR and its downstream effector CHOP/GADD153 is involved, at least in part, in RES-induced apoptosis in Burkitts lymphoma cells.

Proteasome Inhibition Induces a p38 MAPK Pathway-Dependent Antiapoptotic Program via Nrf2 in Thyroid Cancer Cells

CONTEXT Our previous data showed that reactive oxygen species generation might be ascribed to the cytotoxic response of thyroid cancer cells to proteasome inhibition and the ability of cancer cells to induce catalytic subunit for glutamate cysteine ligase (GCLC) and subsequent production of glutathione, thereby scavenging reactive oxygen species was partly ascribed to the cytotoxic responses of thyroid cancer cells to proteasome inhibition. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor responsible for transcriptional activation of various cytoprotective genes including GCLC. OBJECTIVE The purpose of this study was to determine the involvement of Nrf2 in GCLC induction and cytotoxicity of thyroid cancer cells mediated by proteasome inhibition. DESIGN The effects of proteasome inhibition on the expression and distribution of Nrf2 were analyzed using immunocytochemistry and Western blot. To ascertain the effect of Nrf2 and p38 MAPK, cells were transfected with Nrf2 plasmid or small interfering RNA against Nrf2 or p38 MAPK. Apoptotic cells, production of glutathione, and induction of GCLC mediated by proteasome inhibition were investigated using flow cytometry, spectrophotometry, and real-time RT-PCR, respectively. RESULTS Proteasome inhibition caused accumulation and nuclear translocation of Nrf2, which compromised the cytotoxic effects of proteasome inhibition, at least in part, via induction of GCLC. In addition, nuclear translocation of Nrf2 was p38 MAPK dependent, and p38 MAPK inhibition augmented the cytotoxic effects of proteasome, at least partly, via suppression of transactivation of Nrf2. CONCLUSIONS These studies support the hypothesis that proteasome inhibitors activate an antiapoptotic survival program through p38 MAPK that involves transcriptional activity of Nrf2.

Caspase-dependent cleavage of BAG3 in proteasome inhibitors-induced apoptosis in thyroid cancer cells

Proteasome inhibitors are emerging as effective drugs for the treatment of relapsed/refractory multiple myeloma and possibly some solid tumors. Bcl-2-associated athanogene 3 (BAG3) is a survival protein that has been shown to be stimulated during cell response to stressful conditions, such as exposure to high temperature, heavy metals. We have recently demonstrated that BAG3 is also induced by proteasome inhibitors at the transcriptional level and the induction of BAG3 by proteasome inhibition is antiapoptotic. Here, we demonstrated that although proteasome inhibitors triggered similar upregulation of BAG3 transcript in sensitive and insensitive thyroid cancer cells, persistent increase of BAG3 protein was detected in insensitive cells, whereas less increase or even decrease was observed in sensitive cells. Notably, decrease of BAG3 protein was associated with the appearance of a BAG3 fragment of approximately 40kDa, which appeared to be caspase-dependent. Therefore, caspase-dependent cleavage of BAG3 might facilitate apoptosis in sensitive cells.

Significance of survivin, caspase-3, and VEGF expression in thyroid carcinoma

To investigate the clinical significance of survivin, caspase-3, and vascular endothelial growth factor expression in a subset of thyroid carcinoma and their correlation with prognosis. Sixty-eight cases of thyroid carcinoma (TC), 12 cases of thyroid adenoma (TA) and 10 cases of normal thyroid tissue (NT) were involved in immunohistochemical and real-time RT-PCR analyses for survivin, caspase-3, and VEGF expression. Statistical analyses were performed for differential expression among NT, TA and TC, correlations of their expression with the clinicopathological parameters of TC including histological typing, clinical staging and lymphnode metastasis, and relationship of survivin with caspase-3 or VEGF in TC. We observed higher mRNA expression and positive immunostaining for survivin and VEGF in TC compared with TA and NT, with a significant positive correlation among them and significant correlations with histological typing, clinical staging and lymphnode metastasis in TC, but we could not find any significance of caspase-3 in TC and its significant relationship with survivin expression. Our results indicate that survivin and VEGF are unfavorable molecules for TC evolution and prognosis, and possess positive correlation in TC.

Antisurvivin oligonucleotides inhibit growth and induce apoptosis in human medullary thyroid carcinoma cells

Suvivin is a novel member of the inhibitor of apoptosis protein (IAP) family, which is known to be over-expressed in various carcinomas and associated with their biologically aggressive characteristics. The aim of this study was to investigate survivin expression in human medullary thyroid carcinoma (MTC) and a MTC cell line TT, correlate suvivin expression with clinicopathologic features of MTC, and test effects of antisurvivin oligonucleotides (ASODNs) on growth and apoptosis of TT cells. Survivin expression was immunohistochemically determined in formalin-fixed and paraffin-embedded specimens obtained from 10 cases of normal thyroid (NT) and 10 cases of MTC, and in TT cells. In TT cells, we confirmed survivin expression and its down-regulation by ASODNs using RT-PCR and Western blot analyses, and investigated effects of ASODNs on viability and growth by MTT assay and apoptosis by apoptotic analyses including DNA laddering assay, acridine orange/ethidium bromide staining and flow cytometric cell cycle analysis. Immunohistochemical analysis showed high survivin expression in MTC and TT cells, whereas no immunoreactivity was detectable in NT. Statistical analyses revealed no significant correlation of survivin expression with the clinicopathologic features of MTC. In TT cells, survivin expression at both mRNA and protein levels was confirmed and could be down-regulated by ASODNs concomitant with decrease in viability and growth, and increase in apoptosis. Our results suggest that survivin plays an important role in MTC independent of the conventional clinicopathologic factors, and ASODNs is a promising survivin-targeted gene therapy for MTC.

Glucosamine induces cell death via proteasome inhibition in human ALVA41 prostate cancer cell

Glucosamine, a naturally occurring amino monosaccharide, has been reported to play a role in the regulation of apoptosis more than half century. However the effect of glucosamine on tumor cells and the involved molecular mechanisms have not been thoroughly investigated. Glucosamine enters the hexosamine biosynthetic pathway (HBP) downstream of the rate-limiting step catalyzed by the GFAT (glutamine:fluctose-6-phosphate amidotransferase), providing UDP-GlcNAc substrates for O-linked β-N-acetylglucosamine (O-GlcNAc) protein modification. Considering that O-GlcNAc modification of proteasome subunits inhibits its activity, we examined whether glucosamine induces growth inhibition via affecting proteasomal activity. In the present study, we found glucosamine inhibited proteasomal activity and the proliferation of ALVA41 prostate cancer cells. The inhibition of proteasomal activity results in the accumulation of ubiquitinated proteins, followed by induction of apoptosis. In addition, we demonstrated that glucosamine downregulated proteasome activator PA28γ and overexpression of PA28γ rescued the proteasomal activity and growth inhibition mediated by glucosamine. We further demonstrated that inhibition of O-GlcNAc abrogated PA28γ suppression induced by glucosamine. These findings suggest that glucosamine may inhibit growth of ALVA41 cancer cells through downregulation of PA28γ and inhibition of proteasomal activity via O-GlcNAc modification.