The proteasome inhibitor MG132 induces apoptosis in human pancreatic cancer cells

The ubiquitin-proteasome system plays a critical role in the regulation of programmed cell death. Proteasome inhibitors induce apoptosis in various cancer cells and have antitumor effects in murine tumor models. In the present study, we investigated whether the cell-permeable proteasome inhibitor MG132 (carbobenzoxyl-L-leucyl-L-leucyl-L-leucinal) reduced the growth of a human pancreatic cancer cell line through induction of apoptosis in vitro. The effects of MG132 (0.125-1.000 microM) on the growth of the human pancreatic cancer cell line BxPC-3 were analyzed by cell count and MTT assay. Apoptosis was determined by FACS analysis after annexin V and propidium iodide staining and the enrichment of intracellular nucleosomes. The proteasome inhibitor MG132 decreased cell growth of the human pancreatic cancer cell line BxPC-3 in a dose- and time-dependent manner. This effect was at least in part mediated by the induction of apoptosis. A combination therapy with standard cytotoxic agents and proteasome inhibitors could potentially be a novel therapeutic strategy in treatment of pancreatic cancer.     

BAG2对蛋白酶体抑制剂诱导人甲状腺癌细胞凋亡作用影响的观察

背景与目的：蛋白酶体抑制剂对不同组织来源的肿瘤均有抑制其增长和促进细胞凋亡的作用,其作用机制可能与Bcl-2相关抗凋亡基因2(Bcl-2-associated athanogene 2,BAG2)有关,本研究探讨BAG2在蛋白酶体抑制剂诱导甲状腺癌细胞凋亡中的作用。方法：选取人甲状腺未分化癌细胞系ARO、FRO、KTC1、KTC2、KTC3、8305C和8505C,分别设培养液处理空白对照组、蛋白酶体抑制剂MG132处理组;利用实时定量逆转录聚合酶链反应(quantitative real-time polymerase chain reaction,qRT-PCR)检测各组细胞BAG2 mRNA表达及MG132诱导其表达的时效性;利用蛋白质印迹法(Western blot)检测各组细胞BAG2蛋白表达。结果：MTT结果显示,FRO和KTC2细胞系对蛋白酶体抑制剂最为敏感,与空白对照组相比,MG132能不同程度的增加各种细胞BAG2 mRNA及蛋白的表达水平(P<0.01),在FRO和KTC2细胞中,BAG2 mRNA水平较对照组增加20~25倍,蛋白质的表达水平也显著增加;时间效应实验中,敏感的FRO细胞BAG2 mRNA水平增加快,没有延迟期;并且增加的最高水平显著高于非敏感的ARO细胞(P<0.01)。结论：BAG2是由蛋白酶体抑制作用诱导产生的新型分子,在蛋白酶体抑制剂诱导的甲状腺癌细胞的死亡中起着促凋亡作用。     

Proteasome inhibition by MG132 induces growth inhibition and death of human pulmonary fibroblast cells in a caspase-independent manner

MG132 as a proteasome inhibitor that can induce apoptotic cell death in various cell types including lung cancer cells. We investigated the cellular effects of MG132 on human pulmonary fibroblast (HPF) cells in relation to cell growth inhibition and death, and described the molecular mechanisms of MG132 in HPF cell death. This agent dose-dependently inhibited the growth of HPF cells with an IC50 of approximately 20 μM at 24 h and induced cell death accompanied by the loss of mitochondrial membrane potential (MMP; ?Ψm) and an increase in caspase-3 and -8 activities. MG132 increased intracellular ROS levels and GSH-depleted cell numbers. However, all the tested caspase inhibitors intensified HPF growth inhibition by MG132 and caspase-9 inhibitor also enhanced cell death and MMP (?Ψm) loss. Moreover, the administration of Bcl-2 siRNA augmented HPF cell death by MG132 whereas p53, Bax, caspase-3 and -8 siRNAs did not strongly affect cell death. In addition, each caspase inhibitor and siRNA differently affects ROS levels including O2?- regardless of cell growth inhibition and cell death levels. Caspase-8 and -9 inhibitors increased the number of GSH-depleted cells in MG132-treated HPF cells. In conclusion, MG132 induced growth inhibition and death in HPF cells in a caspase-independent manner. The growth inhibition and death of HPF cells by MG132 and/or each caspase inhibitor or apoptosis-related siRNA were not tightly related to the changes in ROS levels.     

MG132, a proteasome inhibitor, induces human pulmonary fibroblast cell death via increasing ROS levels and GSH depletion

MG132 as a proteasome inhibitor can induce apoptotic cell death in lung cancer cells. However, little is known about the toxicological cellular effects of MG132 on normal primary lung cells. Here, we investigated the effects of N-acetyl cysteine (NAC) and vitamin C (well known antioxidants) or L-buthionine sulfoximine (BSO; an inhibitor of GSH synthesis) on MG132-treated human pulmonary fibroblast (HPF) cells in relation to cell death, reactive oxygen species (ROS) and glutathione (GSH). MG132 induced growth inhibition and death in HPF cells, accompanied by the loss of mitochondrial membrane potential (MMP; ?ψm). MG132 increased ROS levels and GSH-depleted cell numbers in HPF cells. Both antioxidants, NAC and vitamin C, prevented growth inhibition, death and MMP (?ψm) loss in MG132-treated HPF cells and also attenuated ROS levels in these cells. BSO showed a strong increase in ROS levels in MG132-treated HPF cells and slightly enhanced the growth inhibition, cell death, MMP (?ψm) loss and GSH depletion. In addition, NAC decreased anonymous ubiquitinated protein levels in MG132-treated HPF cells. Furthermore, superoxide dismutase (SOD) 2, catalase (CTX) and GSH peroxidase (GPX) siRNAs enhanced HPF cell death by MG132, which was not correlated with ROS and GSH level changes. In conclusion, MG132 induced the growth inhibition and death of HPF cells, which were accompanied by increasing ROS levels and GSH depletion. Both NAC and vitamin C attenuated HPF cell death by MG132, whereas BSO slightly enhanced the death.     

The enhancement of propyl gallate-induced apoptosis in HeLa cells by a proteasome inhibitor MG132

Propyl gallate (PG) used in processed food and medicinal preparations has been shown to induce cell death in normal and cancer cells. The inhibition of proteasome function has emerged as a useful strategy to maneuver apoptosis. Here, we investigated the combined effects of PG and MG132 (a proteasome inhibitor) on HeLa cells in relation to cell growth, cell death, reactive oxygen species (ROS) and glutathione (GSH). PG induced growth inhibition and apoptosis in HeLa cells, accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨm), activation of caspase 3 and PARP cleavage. The levels of ROS and GSH depletion were increased in PG-treated HeLa cells. MG132 intensified apoptosis and PARP cleavage in PG-treated HeLa cells. MG132 also increased ROS levels including mitochondrial O2?-, MMP (ΔΨm) loss and GSH depletion in PG-treated HeLa cells. PG induced a G1 phase arrest of the cell cycle in HeLa cells, which was significantly prevented by MG132. MG132 alone inhibited HeLa cell growth via inducing the cell cycle arrests and triggering apoptosis. Conclusively, the inhibition of proteasome by MG132 plays a role as an enhancement factor in PG-induced apoptosis of HeLa cells via increasing ROS levels and GSH depletion.     

Proteasome inhibitor MG132 induces death receptor 5 through CCAAT/enhancer-binding protein homologous protein

Combined treatment with a proteasome inhibitor and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising strategy for cancer therapy. Proteasome inhibitors induce the expression of death receptor 5 (DR5), a receptor for TRAIL, and sensitize cancer cells to TRAIL-induced apoptosis; however, the molecular mechanism of DR5 up-regulation has not been elucidated. In this study, we report that CCAAT/enhancer-binding protein homologous protein (CHOP) is a regulator of DR5 induction by proteasome inhibitor MG132. MG132 induced DR5 expression at a protein and mRNA level in prostate cancer DU145 cells. Furthermore, MG132 increased DR5 promoter activity. Using a series of deletion mutant plasmids containing DR5 promoters of various sizes, we found that MG132 stimulated the promoter activity via the region of -289 to -253. This region contained a CHOP-binding site. Site-directed mutation of the site abrogated the promoter activity enhanced by MG132. An electrophoretic mobility shift assay showed that CHOP directly bound to the MG132-responsive site on the DR5 promoter. Expression of the CHOP protein was increased with MG132 along with DR5 up-regulation. Furthermore, CHOP small interfering RNA attenuated the DR5 up-regulation due to MG132. These results indicate that the proteasome inhibitor MG132 induces DR5 expression through CHOP up-regulation.     

Protease inhibitors restore radiation-induced apoptosis to Bcl-2-expressing lymphoma cells.

The proteasome pathway is important for the turnover of many regulatory proteins. This pathway has recently become a target for antitumor agents and several research groups have demonstrated that inhibitors with specificities for the proteasome are potent apoptosis-inducing agents. Many mechanisms by which proteasome inhibitors exert their effects have been suggested, including inhibition of NF-kappa B activity and stabilization of the p53 tumor suppressor protein. We investigated the ability of inhibitors with specificities for the proteasome and for another protein degradation enzyme, calpain, to sensitize a murine B-cell lymphoma with constitutive NF-kappa B1 homodimer activity and high expression of Bcl-2 protein to radiation-induced apoptosis. Protease inhibitors tested were calpain inhibitor I, calpain inhibitor II, calpeptin, MG132, and Lactacystin. All five inhibitors induced apoptosis and sensitized cells to radiation despite the maintenance of Bcl-2 protein levels throughout the course of treatment. An electrophoretic migration shift assay for NF-kappa B1 activity provided evidence that reversal of NF-kappa B activity was not required for induction of cell death; however, p53 levels were elevated for all inhibitors tested. HL-60 cells, devoid of p53, could not be sensitized to radiation by MG132 treatment, suggesting that p53 was important for cell death induced by combined treatment with protease inhibitors and radiation. We concluded that protease inhibitors are capable of overcoming the protective effects of Bcl-2 to induce apoptosis and suggest that protease inhibitor treatment, when combined with ionizing radiation, leads to p53-mediated apoptosis.     

Hydrogen peroxide controls Akt activity via ubiquitination/degradation pathways

Akt is a well-established protein that regulates cell growth, survival and anti-apoptotic mechanisms. In this study, we demonstrated that hydrogen peroxide (H2O2)-induced oxidative stress regulates the activity of the anti-apoptotic protein Akt via the ubiquitin-proteasome degradation system. H2O2 induced cytotoxicity in PC12 cells and decreased the cellular protein and phosphorylation levels of Akt in a concentration- and exposure time-dependent manner. This downregulation was blocked by the proteasome inhibitor MG132 and the Akt-specific inhibitor LY294002. In addition, an in?vivo ubiquitination assay revealed that the degradation of Akt was mediated by the ubiquitin-mediated proteasome pathway and further demonstrated that this ubiquitination was dependent on the phosphorylation status of Akt. Furthermore, the exogenously overexpressed active form of Akt, but not its inactive form, induced resistance to H2O2-mediated cell death. These results suggested that H2O2-induced cytotoxicity was mediated by active Akt degradation.     

蛋白酶体抑制剂MG132诱导HL-60细胞凋亡前G2/M期阻滞及机制

背景和目的:蛋白酶体(proteasome)抑制剂能够诱导多种肿瘤细胞凋亡,是一种潜在的有应用前景的抗肿瘤剂.本研究旨在探讨蛋白酶体抑制剂MGl32(Z-Leu-Leu-Leu-CHO)诱导白血病细胞HL-60凋亡和C2/M期阻滞的机制.方法:采用荧光显微镜观察、流式细胞术和免疫印迹研究测定MG132诱导HL-60细胞凋亡和周期阻滞及机制.结果:2μmol/L的MG132能够有效地诱导HL-60细胞凋亡,用药后24 h就显现有细胞凋亡;在MG132诱导HL-60细胞凋亡出现之前有一个明显的G2/M期阻滞,加MG132后12 h时G2/M期时相百分比为63.42±2.02;24 h时加MG132组细胞凋亡为16.67±1.48,与对照组G2/M期时相百分比为7.29±3.01及细胞凋亡为0相比,两者之间有显著性差别(P＜0.01);咖啡因CAF能够减少MG132诱导HL-60细胞出现的G2/M期阻滞,同时也减少凋亡细胞的比例;细胞周期检查点的负调控因子p21waf/cip1蛋白在加MG132处理后3 h有明显的表达,但并未能检测到p53和p27蛋白.结论:MG132诱导HL-60细胞凋亡之前有一个明显的G2/M期阻滞,p21蛋白表达明显上调提示:是p21waf/cip1而不是p53或其同源蛋白参与了其中的调控.     

Antiglioma effects of N6‐isopentenyladenosine,an endogenous isoprenoid end product,through the downregulation of epidermal growth factor receptor

Malignant gliomas are highly dependent on the isoprenoid pathway for the synthesis of lipid moieties critical for cell proliferation. The isoprenoid derivative N6‐isopentenyladenosine (iPA) displays pleiotropic biological effects, including a direct anti‐tumor activity in several tumor models. The antiglioma effects of iPA was then explored in U87MG cells both in vitro and grafted in mice and the related molecular mechanism confirmed in primary derived patients' glioma cells. iPA powerfully inhibited tumor cell growth and induced caspase‐dependent apoptosis through a mechanism involving a marked accumulation of the pro‐apoptotic BIM protein and inhibition of EGFR. Indeed, activating AMPK following conversion into its iPAMP active form, iPA stimulated EGFR phosphorylation and ubiquitination along a proteasome‐mediated pathway which was responsible for receptor degradation and its downstream signaling pathways inhibition, including the STAT3, ERK and AKT cascade. The inhibition of AMPK by compound C prevented iPA‐mediated phosphorylation of EGFR, known to precede receptor loss. As expected the block of EGFR degradation, by exposure to the proteasome inhibitor MG132, significantly reduced iPA‐induced cell death. Given the importance of receptor degradation in iPA‐mediated cytotoxicity, we also documented that the EGFR expression levels in a panel of primary glioma cells confers them a high sensitivity to iPA treatment. In conclusion our study provides the first evidence of iPA antiglioma effect. Indeed, as glioma is driven by aberrant signaling of growth factor receptors, particularly the EGFR, iPA, alone or in association with EGFR targeted therapies, might be a promising therapeutic tool to achieve a potent anti‐tumoral effect.     

蛋白酶体抑制剂通过 Bcl-2 相关抗凋亡蛋白 3 剪切诱导乳腺癌 MCF-7 细胞凋亡

[目的]探讨蛋白酶体抑制剂诱导乳腺癌MCF-7细胞凋亡与BAG3蛋白剪切的相互关系,以及BAG3蛋白剪切是否依赖胱天蛋白酶介导完成。[方法]选取人MCF-7乳腺癌细胞系,设空白对照组、MG132处理组、Z—VAD处理组和MGl32＋Z-VAD联合组。实时定量RT—PCR和WesternBlot分别检测各组细胞中BAG3mRNA和蛋白的表达水平：流式细胞仪检测细胞凋亡。[结果]MGl32诱导乳腺癌细胞凋亡过程中BAG3基因和蛋白表达均增高（P〈0．05）,但BAG3蛋白剪切体也随之增加,流式细胞术结果证明细胞凋亡率增高fP〈0．05）。同时,应用胱天蛋白酶体抑制剂Z-VAD处理细胞后,BAG3蛋白剪切体消失。[结论]蛋白酶体抑制剂可诱导上调乳腺癌MCF-7细胞中BAG3基因和蛋白的表达,但BAG3蛋白裂解增强,可导致细胞凋亡增加,这一效应可能依赖胱天蛋白酶介导完成。     

Beclin 1 在 MG132 抗 OVCAR3 卵巢癌细胞中的变化及作用研究简

目的：研究Beclin1在MG132抗OVCAR3卵巢癌细胞中的变化及作用。方法：OVCAR3细胞经小同浓度MG132处理24h后,MTT法检测细胞存活率,Western blot法检测自噬相关Beclin1蛋白的变化。采用细胞转染技术过表达卵巢癌细胞中Beclin1,MG132处理24h后,MTT法检测细胞存活率;Hoechst33258进行核染色观察染色质浓缩和核碎片等凋亡特征性形态学改变。采用shRNA技术下调OVCAR3细胞的Beclin1表达,MG132处理细胞,AO染色观察酸性自噬泡的形成。结果：与空白对照组比较,1、2、5和10μmol/L浓度的MG132作用24h均能明显抑制OVCAR3细胞的生长（均P〈0．05）,5μmol／L浓度接近半抑制率;而且5μmol／L和10μmol／L浓度的MG132作用24h能显著诱导OVCAR3细胞的Beclin1蛋白水平下降（P=0．001）。与卒质粒转染组对比,过表达Beclin1的卵巢癌细胞在MG132处理后核的凋亡明显增加;细胞存活率明显降低,（P=0．00089）。下调卵巢癌细胞中Beclin1表达后,MG132诱导的酸性囊泡蓄积无改变。结论：MG132使OVCAR3细胞中Beelin1降低,Beclin1具有增强MG132抗OVCAR3的作用,但该怍用与自噬无关。     

Gap junctions sensitize cancer cells to proteasome inhibitor MG132‐induced apoptosis

(Cancer Sci 2010; 101: 713–721) Proteasome inhibition is a promising approach for cancer therapy. However, the mechanisms involved have not been fully elucidated. Gap junctions play important roles in the regulation of tumor cell phenotypes and mediation of the bystander effect in cancer therapy. Because the degradation of gap junction proteins involves the proteasome, we speculated that altered gap junctions might contribute to the antitumor activities of proteasome inhibition. Incubation of Hepa‐1c1c7 cells with the proteasome inhibitor MG132 elevated the levels of gap junction protein connexin 43 (Cx43) and promoted gap junctional intercellular communication. This was associated with a marked accumulation of ubiquitylated Cx43 and a significantly decreased rate of Cx43 degradation. The elevated Cx43 contributed to MG132‐induced cell apoptosis. This is shown by the observations that: (i) overexpression of Cx43 in the gap junction‐deficient LLC‐PK1 cells rendered them vulnerable to MG132‐elicited cell injury; (ii) fibroblasts derived from Cx43‐null mice were more resistant to MG‐132 compared with Cx43 wild‐type control; and (iii) the gap junction inhibitor flufenamic acid significantly attenuated cell damage caused by MG132 in Hepa‐1c1c7 cells. Further studies demonstrated that MG132 activates endoplasmic reticulum stress. Exposure of cells to the endoplasmic reticulum stress inducers thapsigargin and tunicamycin also led to cell apoptosis, which was modulated by Cx43 levels in a way similar to MG132. These results suggested that elevated Cx43 sensitizes cells to MG132‐induced cell apoptosis. Regulation of gap junctions could be an important mechanism behind the antitumor activities of proteasome inhibitors.     

Proteasome inhibitor MG132 reverses multidrug resistance of gastric cancer through enhancing apoptosis and inhibiting P-gp

Multidrug resistance (MDR) is a major impediment to the effective chemotherapy of many human malignancies. Although much effort has been devoted to develop new drugs for overcoming MDR, until now, still no useful method of reversing MDR, suitable for clinical use, has emerged from this large quantity of work. Some researchers have reported that proteasome inhibitors could induce apoptosis in a variety of cancer cells. In the present study, we found that, in vincristine-resistant human gastric cancer cell line SGC7901/VCR, proteasome inhibitor MG132 was an effective inducer of apoptosis, and also had the capacity of downregulating the expression of anti-apoptotic Bcl-2 and MDR1 (P-gp), by which MG132 resensitized tumor cells to the apoptosis induced by anticancer drugs. Data presented by drug sensitivity assay further demonstrated that MG132 could reverse the resistant phenotype of gastric cancer cells effectively through both enhancing drug-induced apoptosis and inhibiting P-gp. The further study of the effectiveness and safety of proteasome inhibitor in vivo may be helpful for developing a new possible strategy to treat gastric cancer MDR.     

蛋白酶体抑制剂对甲状腺未分化癌细胞ATF-4表达影响的研究

目的：探讨蛋白酶体抑制剂对甲状腺未分化癌FRO细胞中活化转录因子4（ATF-4）表达的影响,以及ATF-4在蛋白酶体抑制剂诱导FRO细胞凋亡中的作用。方法：选取人甲状腺未分化癌细胞系FRO,分别设空白对照纽和蛋白酶体抑制剂MG132处理组;利用微小RNA干扰技术,减少ATF-4的表达,实时定量PCR法、蛋白质印迹法分别检测各组细胞中ATF-4、氧调节蛋白150（ORP150）mRNA和蛋白表达;流式细胞仪（FCM）检测细胞凋亡。结果：与空白对照组相比,蛋白酶体抑制剂MG132显著增加FRO细胞系中ATF-4基因表达水平,P〈0．01;与空白对照组和随机序列核酸siRNA组相比,siATF-4处理组中ATF-4、ORP150mRNA及蛋白表达水平显著降低（P〈0．01）,其凋亡率显著增加,P〈0．01。结论：蛋白酶体抑制剂能够上调甲状腺未分化癌FRO细胞系中ATF4基因的表达水平,siATF-4具有降低ATF-4及OPR150基因表达的作用,同时增加蛋白酶体抑制剂诱导甲状腺未分化癌FRO细胞的凋亡作用。     

Inhibition of proteasome function induced apoptosis in gastric cancer

The ubiquitin-proteasome pathway plays a critical role in the degradation of cellular proteins and cell cycle control. Dysregulating the degradation of such proteins should have profound effects on tumor growth and causes cells to undergo apoptosis. The aims of this study are to evaluate the ubiquitin-proteasome pathway in gastric cancer and the potential role of pharmacological inhibition of proteasome on induction of apoptosis in gastric cancer cells. Gastric cancer cell lines AGS (p53 wild-type) and MKN-28 (p53 mutant) were treated with proteasome inhibitor MG132. The results showed that MG132 inhibited cell proliferation in AGS and MKN-28 cells in a time- and dose-dependent manner. The inhibition of cell proliferation was caused by apoptosis which was also time- and dose-dependent. AGS cells were more responsive to MG132 than MKN-28 cells. Induction of apoptosis was preceded by the activation of caspase-3, as measured by a colorimetric caspase-3 cellular activity and Western blotting of the cleavage of caspase-3 and its substrate PARP. Activation of caspase-7 was also exhibited. In addition, z-VAD-fmk, a broad spectrum caspase inhibitor, reversed apoptosis induced by MG132 in AGS and MKN28 cells. Although z-DEVD-fmk, a specific caspase-3 inhibitor, suppressed MG132-induced apoptosis in MKN28 cells, it only partially rescued the apoptotic effect in AGS cells. Caspase-3 activation was the result of release of cytochrome c from mitochondria into the cytosol, as a consequence of upregulation of bax. There were overexpressions of all the proteasome-related proteins p53, p21(waf1) and p27(kip1) at 4 hr after proteasome inhibition which was identified by the accumulation of ubiquitin-tagged proteins. This was accompanied by accumulation of cells at G(1) phase. Our present study suggests that inhibition of proteasome function in gastric cancer cells induces apoptosis and proteasomal inhibitors have potential use as novel anticancer drugs in gastric cancer.     

Apoptosis induced in hepatoblastoma HepG2 cells by the proteasome inhibitor MG132 is associated with hydrogen peroxide production,expression of Bcl-XS and activation of caspase-3

This report is focused on the apoptotic effect induced by MG132, an inhibitor of 26S proteasome, in human hepatoma HepG2 cells. The results were compared with those obtained with non-transformed human Chang liver cells. MG132 reduced the viability of HepG2 cells in a time- and dose-dependent manner. The effect was in tight connection with the induction of apoptosis, as indicated by fluorescence microscopy and cytometric analysis, and was accompanied by a remarkable increase in the production of H2O2 and a reduction in mitochondrial transmembrane potential (Deltapsim). In addition cell death was prevented by antioxidants such as GSH, N-acetylcysteine or catalase. Western blot analysis showed that HepG2 cells contain a very low level of Bcl-2 and a much higher level of Bcl-XL, another antiapoptotic factor of the same family. When the cells were exposed to MG132 the level of Bcl-XL diminished, while a new band, corresponding to the expression of the proapoptotic protein Bcl-XS was detected. MG132 also caused the release of cytochrome c from mitochondria and the activation of caspase-3 with the consequent degradation of poly-ADP ribose polymerase (PARP). The observation that the broad spectrum caspase inhibitor z-VAD markedly reduced the apoptotic effect of the drug clearly demonstrated that caspases play an important role in MG132-induced apoptosis. MG132 exerted a modest effect on the viability of Chang liver cells which primarily depended on the G2/M arrest of cell cycle while only a small percentage of apoptotic cells was found. The remarkable differences in the effects induced by MG132 in Chang liver cells and HepG2 cells made us hypothesise the potential use of proteasome inhibitors in hepatocarcinoma therapy.     

Proteasome inhibitor MG132 upregulates death receptor 5 and cooperates with Apo2L/TRAIL to induce apoptosis in Bax-proficient and -deficient cells

Apo2L/TRAIL (tumor necrosis factor-related apoptosis inducing ligand (TRAIL), also known as Apo2L) is a potentially important anticancer agent awaiting clinical trials. Unfortunately, however, some cancer cells exhibit resistance to Apo2L/TRAIL, which could limit the use of this potentially promising anticancer agent. Although the molecular basis of the inherent or acquired resistance to Apo2L/TRAIL remains unclear, previous studies indicate that Bax deficiency can confer resistance to Apo2L/TRAIL. Proteasome inhibition is also emerging as a promising therapeutic strategy to manage human malignancies. Here, we report that proteasome inhibitor MG132 upregulates Apo2L/TRAIL death receptor 5 expression in both Bax-proficient and -deficient HCT116 cells. MG132 effectively cooperated with Apo2L/TRAIL to induce apoptosis in both Bax-proficient and -deficient cells that was coupled with caspases-8 and -3 activation and Bid cleavage. Although both agents in combination also induced cytochrome c and Smac release from mitochondria into cytosol and activated caspase-9 in Bax-proficient cells, their effects on these events were significantly diminished in Bax-deficient cells. These results suggest that Bax is not absolutely required for death receptor 5-dependent apoptotic signals and MG132 by upregulating DR5 effectively cooperates with Apo2L/TRAIL to overcome Bax deficiency-induced resistance to Apo2L/TRAIL. Our results have important clinical implications in that the use of Apo2L/TRAIL and proteasome inhibitors in combination could prove to be a novel therapeutic strategy to manage the Apo2L/TRAIL-resistant tumors.     

Gambogic acid enhances proteasome inhibitor-induced anticancer activity

Proteasome inhibition has emerged as a novel approach to anticancer therapy. Numerous natural compounds, such as gambogic acid, have been tested in vitro and in vivo as anticancer agents for cancer prevention and therapy. However, whether gambogic acid has chemosensitizing properties when combined with proteasome inhibitors in the treatment of malignant cells is still unknown. In an effort to investigate this effect, human leukemia K562 cells, mouse hepatocarcinoma H22 cells and H22 cell allografts were treated with gambogic acid, a proteasome inhibitor (MG132 or MG262) or the combination of both, followed by measurement of cellular viability, apoptosis induction and tumor growth inhibition. We report, for the first time, that: (i) the combination of natural product gambogic acid and the proteasome inhibitor MG132 or MG262 results in a synergistic inhibitory effect on growth of malignant cells and tumors in allograft animal models and (ii) there was no apparent systemic toxicity observed in the animals treated with the combination. Therefore, the findings presented in this study demonstrate that natural product gambogic acid is a valuable candidate to be used in combination with proteasome inhibitors, thus representing a compelling anticancer strategy.