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.     

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。     

Bik/NBK accumulation correlates with apoptosis-induction by bortezomib (PS-341, Velcade) and other proteasome inhibitors

Proteasome inhibitors have emerged as promising anticancer therapeutic agents. Bortezomib (PS-341), a specific proteasome inhibitor, exhibits antitumor activity against a wide range of malignancies and has been approved by the US Food and Drug Administration for the treatment of relapsed or refractory multiple myeloma. However, the molecular mechanisms of bortezomib-mediated apoptosis remain unclear. To characterize the mechanisms of apoptosis induction by proteasome inhibitors, we examined levels of Bcl-2 protein family members (Bik/NBK, Bax, Bak, Bcl-2, and Bcl-XL), release of cytochrome c, and activation of caspase-9 and -3 in human colon cancer cell lines DLD1, LOVO, SW620, and HCT116; human lung cancer cell line H1299; and human ovarian cancer cell line SKOV3 after they were treated with bortezomib. The result showed that bortezomib induced rapid accumulation of Bik/NBK but not other Bcl-2 family members in all six cell lines. Bortezomib-mediated Bik/NBK accumulation and apoptosis were also observed in human embryonic kidney cells 293 and normal human bronchial epithelial cells. Moreover, dramatic Bik/NBK accumulation and apoptosis induction were observed when cells were treated with proteasome inhibitor MG132 and calpain inhibitor I (ALLN). Furthermore, no detectable changes in IkappaBalpha levels or in NFkappaB functionality were found after treatment with bortezomib. Finally, Bik/NBK accumulation was caused by stabilization of the protein from degradation and was associated with bortezomib cytotoxicity and apoptosis induction. Pretreatment of DLD1 cells with Bik/NBK siRNA reduced bortezomib-mediated Bik/NBK accumulation and cell death. Our results suggested that Bik/NBK is one of the mediators of proteasome inhibitor-induced apoptosis.     

胰腺癌细胞凋亡伴随胱天蛋白酶依赖性BAG3蛋白剪切的研究

目的:前期研究表明,蛋白酶体抑制剂诱导甲状腺癌细胞凋亡时伴随抗凋亡蛋白BAG3的剪切。本研究探讨BAG3蛋白剪切是否为细胞凋亡伴随的普遍现象。方法:选取人胰腺癌细胞SW1990和PANC1,分别设培养液处理空白对照组、蛋白酶体抑制剂MG132单独或与z-VAD联合处理组、星形孢菌素(STS)单独或与z-VAD联合处理组、依托泊苷单独或与z-VAD联合处理组、TNFα单独或与z-VAD联合处理组以及紫外线单独照射或与z-VAD联合处理组;利用蛋白质印迹法检测各组细胞中BAG3蛋白的剪切情况;利用流式细胞仪(FCM)检测细胞凋亡率。结果:与甲状腺癌细胞类似,蛋白酶体抑制剂MG132诱导胰腺癌细胞凋亡的同时,伴随BAG3蛋白的剪切;其他凋亡诱导剂如依托泊苷、星形孢菌素和紫外线照射在诱导胰腺癌细胞凋亡的同时,也可诱导BAG3蛋白剪切体的出现;而广谱胱天蛋白酶抑制剂z-VAD可以完全抑制BAG3蛋白的剪切。结论:胱天蛋白酶依赖性BAG3蛋白的剪切可能是细胞凋亡时的普遍现象,BAG3可能为胱天蛋白酶作用底物。     

Proteasome inhibitors-mediated TRAIL resensitization and Bik accumulation

Proteasome inhibitors can resensitize cells that are resistant to tumor necrosis factor-related apoptotic-inducing ligand (TRAIL)-mediated apoptosis. However, the underlying mechanisms of this effect are unclear. To characterize the mechanisms of interaction between proteasome inhibitors and TRAIL protein, we evaluated the effects of combined treatment with the proteasome inhibitors bortezomib and MG132 and TRAIL protein on two TRAIL-resistant human colon cancer cell lines, DLD1-TRAIL/R and LOVO-TRAIL/R. Both bortezomib and MG132 in combination with TRAIL enhanced apoptotosis induction in these cells, as evidenced by enhanced cleavage of caspases 8, 9, and 3, Bid, poly(ADP-ribose) polymerase and by the release of cytochrome C and Smac. Subsequent studies showed that combined treatment with bortezomib or MG132 resulted in an increase of death receptor (DR) 5 and Bik at protein levels but had no effects on protein levels of DR4, Bax, Bak, Bcl-2, Bcl-XL or Flice-inhibitory protein (FLIP). Moreover, c-Jun N-terminal kinase (JNK) is activated by these proteasome inhibitors. Blocking JNK activation with the JNK inhibitor SP600125 attenuated DR5 increase, but enhancement of apoptosis induction and increase of Bik protein were not affected. However, bortezomib-mediated TRAIL sensitization was partially blocked by using siRNA to knockdown Bik. Thus, our data suggests that accumulation of Bik may be critical for proteasome inhibitor-mediated resensitization of TRAIL.     

Potent antitumor agent proteasome inhibitors: a novel trigger for Bcl2 phosphorylation to induce apoptosis.

The sustained cytotoxicity conferred by proteasome inhibitors against a broad spectrum of human cancer cells is mediated by a delicate mechanism of programmed cell death. Similar to microtubule disarraying agents, the cell death induced by these potent antitumor agents precedes blocking in cell cycle transition at G2-M phase. The microtubule damaging antineoplastic drugs can kill tumor cells by inducing phosphorylation of antiapoptotic proteins such as Bcl2, Bcl-xL or MCL-1. The simultaneous apoptosis with Bcl2 phosphorylation was evident in cancer cells challenged with the proteasome inhibitor, MG132. Our studies suggest that the proteasome inhibitor MG132 induced tumor cell killing is mediated through Bcl2 phosphorylation.     

Proteasome inhibitor MG132 sensitizes HPV-positive human cervical cancer cells to rhTRAIL-induced apoptosis

In cervical carcinogenesis, the p53 tumor suppressor pathway is disrupted by HPV (human papilloma virus) E6 oncogene expression. E6 targets p53 for rapid proteasome-mediated degradation. We therefore investigated whether proteasome inhibition by MG132 could restore wild-type p53 levels and sensitize HPV-positive cervical cancer cell lines to apoptotic stimuli such as rhTRAIL (recombinant human TNF-related apoptosis inducing ligand). In a panel of cervical cancer cell lines, CaSki was highly, HeLa intermediate and SiHa not sensitive to rhTRAIL-induced apoptosis. MG132 strongly sensitized HeLa and SiHa to rhTRAIL-induced apoptosis in a caspase-dependent and time-dependent manner. MG132 massively induced TRAIL receptor DR4 and DR5 membrane expression in HeLa, whereas in SiHa only DR5 membrane expression was upregulated from almost undetectable to high levels. Antagonistic DR4 antibody partially inhibited apoptosis induction by rhTRAIL and MG132 in HeLa but had no effect on apoptosis in SiHa. Inhibition of E6-mediated p53 proteasomal degradation by MG132 resulted in elevated levels of active p53 as demonstrated by p53 small interfering RNA (siRNA) sensitive p21 upregulation. Although p53 siRNA partially inhibited MG132-induced DR5 upregulation in HeLa and SiHa, no effect on rhTRAIL-induced apoptosis was observed. MG132 plus rhTRAIL enhanced caspase 8 and caspase 3 activation and concomitant cleavage of X-linked inhibitor of apoptosis (XIAP), particularly in HeLa. In addition, caspase 9 activation was only observed in HeLa. Downregulation of XIAP using siRNA in combination with rhTRAIL induced high levels of apoptosis in HeLa, whereas MG132 had to be added to the combination of XIAP siRNA plus rhTRAIL to induce apoptosis in SiHa. In conclusion, proteasome inhibition sensitized HPV-positive cervical cancer cell lines to rhTRAIL independent of p53. Our results indicate that not only DR4 and DR5 upregulation but also XIAP inactivation contribute to rhTRAIL sensitization by MG132 in cervical cancer cell lines. Combining proteasome inhibitors with rhTRAIL may be therapeutically useful in cervical cancer treatment.     

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.     

Combination of proteasomal inhibitors lactacystin and MG132 induced synergistic apoptosis in prostate cancer cells

The proteasome inhibitor Velcade (bortezomib/PS-341) has been shown to block the targeted proteolytic degradation of short-lived proteins that are involved in cell maintenance, growth, division, and death, advocating the use of proteasomal inhibitors as therapeutic agents. Although many studies focused on the use of one proteasomal inhibitor for therapy, we hypothesized that the combination of proteasome inhibitors Lactacystin (AG Scientific, Inc., San Diego CA) and MG132 (Biomol International, Plymouth Meeting, PA) may be more effective in inducing apoptosis. Additionally, this regimen would enable the use of sublethal doses of individual drugs, thus reducing adverse effects. Results indicate a significant increase in apoptosis when LNCaP prostate cancer cells were treated with increasing levels of Lactacystin, MG132, or a combination of sublethal doses of these two inhibitors. Furthermore, induction in apoptosis coincided with a significant loss of IKKalpha, IKKbeta, and IKKgamma proteins and NFkappaB activity. In addition to describing effective therapeutic agents, we provide a model system to facilitate the investigation of the mechanism of action of these drugs and their effects on the IKK-NFkappaB axis.     

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 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.     

Influence of p53 and p21Waf1 expression on G2/M phase arrest of colorectal carcinoma HCT116 cells to proteasome inhibitors

Ubiquitin-mediated protein degradation in vertebrates has been implicated in cell cycle control. In this report we explored the effects of proteasome inhibitors (MG132, lactacystin and ALLN) on cell cycle distribution. Colorectal carcinoma HCT116 cells were treated with proteasome inhibitor MG132. The results showed that MG132 inhibited cell proliferation in a dose-dependent manner. MG132 arrested HCT116 cells at G2/M phase, which was associated with drug-induced blockade of p53 degradation and/or induction of p53-related gene expression along with the accumulation of cyclin B, cyclin A and p21. MG132 treated HCT116 (wild-type) had a similar cell cycle distribution as the MG132 treated HCT116 (p53-/-) and HCT116 (p21-/-) cells, suggesting that p53 and p21 may not be essential for MG132-induced G2/M phase arrest. The release experiments from nocodazole-induced mitotic phase cells indicated that MG132 inhibits the proliferation of HCT116 cells via arrest in the G2 phase. In addition, when HCT116 cells were exposed to combination of sodium butyrate and MG132 enhanced cell growth inhibition and induction of apoptosis were observed.     

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 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.     

Proteasomes act in the pre-mitochondrial signal transduction route towards roscovitine-induced apoptosis

The role of the ubiquitin-proteasome pathway during roscovitine induced apoptosis was evaluated in the non-small cell lung carcinoma cell line MR65. To this end specific inhibitors of proteasome activity, MG132 and lactacystin were used. Addition of MG132 or lactacystin, 1 h prior to the addition of the CDK-inhibitor roscovitine to the cell cultures inhibited apoptosis significantly, as measured by PS exposure, cytokeratin 18 cleavage and caspase-3 activation. Furthermore, we show that inhibition of proteasome activation prior to induction of apoptosis by roscovitine prevents loss of mitochondrial inner transmembrane potential (DeltaPsim). In addition we found that MG132 and lactacystin prevent release of cytochrome c from the mitochondrion. In contrast to the above findings we see no effect of proteasome inhibition in Fas-mediated apoptosis. Taken together our data suggest a specific role for proteasomes very early in roscovitine-induced apoptosis, upstream from the caspase cascade and mitochondrion.     

Wnt pathway activation and ABCB1 expression account for attenuation of Proteasome inhibitor-mediated apoptosis in multidrug-resistant cancer cells

Multiple drug resistance (MDR) is a major obstacle to attenuating the effectiveness of chemotherapy to many human malignancies. Proteasome inhibition induces apoptosis in a variety of cancer cells and is recognized as a novel anticancer therapy approach. Despite its success, some multiple myeloma patients are resistant or become refractory to ongoing treatment by bortezomib suggesting that chemoresistant cancer cells may have developed a novel mechanism directed against the proteasome inhibitor. The present study aimed to investigate potential mechanism(s) of attenuation in a MDR cell line, MES-SA/Dx5. We found that compared to the parental human uterus sarcoma cell line MES-SA cells, MES-SA/Dx5 cells highly expressed the ABCB1 was more resistant to MG132 and bortezomib, escaping the proteasome inhibitor-induced apoptosis pathway. The resistance was reversed by co-treatment of MG132 and the ABCB1 inhibitor verapamil. The data indicated that ABCB1 might play a role in the efflux of MG132 from the MES-SA/Dx5 cells to reduce MG132-induced apoptosis. Furthermore, the canonical Wnt pathway was found activated only in the MES-SA/Dx5 cells through active β-catenin and related transactivation activities. Western blot analysis demonstrated that Wnt-targeting genes, including c-Myc and cyclin D1, were upregulated and were relevant in inhibiting the expression of p21 in MES-SA/Dx5 cells. On the other hand, MES-SA cells expressed high levels of p21 and downregulated cyclin D1 and caused cell cycle arrest. Together, our study demonstrated the existence and participation of ABCB1 and the Wnt pathway in an MDR cell line that attenuated proteasome inhibitor-induced apoptosis.     

Proteasome inhibitor sensitizes oral squamous cell carcinoma cells to TRAIL-mediated apoptosis

Oral squamous cell carcinoma (OSCC) cells are relatively resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis during culture. We investigated the role of a proteaosome inhibitor in the survival and apoptosis of these cells. We found that the proteasome inhibitor MG132 markedly accelerated TRAIL-mediated apoptosis in OSCC cell lines HSC-2 and HSC-3. Addition of TRAIL to MG132-treated cells resulted in Bid cleavage. Furthermore, the inhibitors of caspase-3, caspase-8 and caspase-9 reduced the accelerative effect of MG132 on TRAIL-mediated apoptosis. These results suggest that the pro-apoptotic effect of a proteasome inhibitor on TRAIL-mediated apoptosis may contribute to both extrinsic and intrinsic pathways. MG132 enhanced the expression of the TRAIL receptors DR4 and DR5, and neutralization of DR5 receptors showed a marked reduction of TRAIL-mediated apoptosis, whereas that of DR4 was a partial reduction. MG132 also markedly reduced cellular FLICE-inhibitory protein (c-FLIP), cellular inhibitor of apoptosis protein-1 (cIAP-1), X-linked IAP (XIAP) and survivin. Therefore, MG132 provides partial regulation of TRAIL-mediated apoptosis in OSCC cells via modulation of DR5, c-FLIP, cIAP-1, XIAP and survivin. The proteasome inhibitor MG132 may therefore represent a novel strategy for overcoming resistance to TRAIL-mediated apoptosis in OSCC cells.     

Proteasome inhibitors abolish cell death downstream of caspase activation during anti-microtubule drug-induced apoptosis in leukemia cells

PURPOSE: Anti-microtubule drugs and proteasome inhibitors are currently among the most intensively studied anti-tumor agents, however little is known about their pharmacological interactions at the cellular level. MATERIALS AND METHODS: The human promyelocytic leukemia cell line, HL-60, was exposed to nocodazole or etoposide in combination with proteasome or caspase inhibitors. Apoptotic cell death was detected by flow cytometry as sub-G1 population. Caspase and proteasome activities were monitored by the fluorogenic substrates Ac-DEVD-AMC and Suc-LLVY-AMC, respectively, in cell lysate. Heat shock protein 70 (HSP70) expression was determined by Western blotting. RESULTS: Nocodazole, a microtubule inhibitor, induced caspase-dependent apoptosis in the HL-60 cell line. At sub-cytotoxic concentrations, proteasome inhibitors, including MG-132 or clasto-beta-lactone, decreased nocodazole-induced apoptotic DNA fragmentation without affecting the induction of caspase-3 activity. In contrast, MG-132 decreased both DNA fragmentation and caspase activation induced by etoposide, a topoisomerase-II inhibitor. HSP70 had previously been found to inhibit apoptosis independently from caspase activation. In this study, MG-132 up-regulated HSP70 protein expression, both in the presence or absence of nocodazole. CONCLUSION: Proteasome inhibitors decreased anti-microtubule agent-induced apoptotic DNA fragmentation downstream of caspase-3 activation, possibly due to increased HSP70 expression. The results indicate that combination treatment with these novel anti-tumor agents in leukemia requires careful evaluation of their molecular interaction at the level of apoptosis induction.     

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.