Proteasome inhibitor PS-341 induces growth arrest and apoptosis of non-small cell lung cancer cells via the JNK/c-Jun/AP-1 signaling

Proteasome inhibitor PS-341 induces growth arrest and apoptosis of multiple myeloma (MM) cells via inactivation of NF-κB in vitro and has afforded some objective responses in individuals with relapsed, refractory MM. However, the activity of PS-341 against non-hematological malignancies remains to be fully elucidated. In this study, we found that PS-341 induced growth arrest and apoptosis of NCI-H520 and -H460 non-small cell lung cancer (NSCLC) cells in conjunction with markedly up-regulated levels of p21^waf1 and p53, and down-regulation of bcl-2 protein in these cells. Also, PS-341 caused phosphorylation of c-Jun NH₂-terminal kinase (JNK) and c-Jun, and enhanced AP-1/DNA binding activities in these cells as measured by western blotting and enzyme-linked immunosorbent assay (ELISA), respectively. Interestingly, when the JNK/ c-Jun/AP-1 signal pathway was disrupted by the JNK inhibitor SP600125, the ability of PS-341 to inhibit the growth of NSCLC cells and to up-regulate the levels of p21^waf1 in these cells was blunted, but the expression of p53 was sustained at a high level, suggesting that the JNK/c-Jun/AP-1 signal pathway might mediate the anti-lung cancer effects of PS-341, with p21^waf1 playing the central role. Thus, PS-341 might be useful for the treatment of individuals with NSCLC.     

The proteasome inhibitor PS-341 markedly enhances sensitivity of multiple myeloma tumor cells to chemotherapeutic agents.

Increased nuclear factor kappaB (NF-kappaB) activity is associated with increased tumor cell survival in multiple myeloma. The function of NF-kappaB is inhibited through binding to its inhibitor, IkappaB. Release of activated NF-kappaB follows proteasome-mediated degradation of IkappaB resulting from phosphorylation of the inhibitor and, finally, conjugation with ubiquitin. We report that myeloma cells have enhanced IkappaBalpha phosphorylation and increased NF-kappaB activity compared with normal hematopoietic cells. The proteasome inhibitor PS-341 blocked nuclear translocation of NF-kappaB, blocked NF-kappaB DNA binding, and demonstrated consistent antitumor activity against chemoresistant and chemosensitive myeloma cells. The sensitivity of chemoresistant myeloma cells to chemotherapeutic agents was markedly increased (100,000-1,000,000-fold) when combined with a noncytotoxic dose of PS-341 without affecting normal hematopoietic cells. Similar effects were observed using a dominant negative super-repressor for IkappaBalpha. Thus, these results suggest that inhibition of NF-kappaB with PS-341 may overcome chemoresistance and allow doses of chemotherapeutic agents to be markedly reduced with antitumor effects without significant toxicity.     

Blockade of ubiquitin-conjugating enzyme CDC34 enhances anti-myeloma activity of Bortezomib/Proteasome inhibitor PS-341

The ubiquitin-conjugating enzyme CDC34 (UBC3) is linked to cell cycle progression in diverse cell types; however, its role in multiple myeloma (MM) pathogenesis is unclear. Here, we show that CDC34 is highly expressed in patient MM cells and MM cell lines versus normal cells. Blocking CDC34 using a dominant-negative strategy enhances the anti-MM activity of Bortezomib/Proteasome inhibitor PS-341, dexamethasone (Dex) and 2-Methoxyestradiol (2ME2). The expression of wild-type CDC34 reduces Dex-induced cytotoxicity in MM cells. Moreover, inhibition of CDC34 enzymatic activity abrogates interleukin-6-induced protection against Dex-induced apoptosis. Together, these findings provide evidence that (1) CDC34 expression is associated with growth and survival of MM cells and (2) blocking CDC34 activity not only enhances anti-MM activity of Bortezomib and 2ME2 but also overcomes IL-6-triggered Dex-resistance.     

蛋白酶体抑制剂Bortezomib及其在多发性骨髓瘤中的研究进展

 蛋蛋白酶体抑制剂Bortezomib通过抑制蛋白酶体及NF-κB使骨髓瘤细胞凋亡,从Borte-zomib作用分子机制、药理作用、临床前研究、临床疗效评价、耐药及联合用药等方面进行了阐述。     

The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple myeloma cells

Human multiple myeloma (MM) is a presently incurable hematological malignancy, and novel biologically based therapies are urgently needed. Proteasome inhibitors represent a novel potential anticancer therapy. In this study, we demonstrate that the proteasome inhibitor PS-341 directly inhibits proliferation and induces apoptosis of human MM cell lines and freshly isolated patient MM cells; inhibits mitogen-activated protein kinase growth signaling in MM cells; induces apoptosis despite induction of p21 and p27 in both p53 wild-type and p53 mutant MM cells; overcomes drug resistance; adds to the anti-MM activity of dexamethasone; and overcomes the resistance to apoptosis in MM cells conferred by interleukin-6. PS-341 also inhibits the paracrine growth of human MM cells by decreasing their adherence to bone marrow stromal cells (BMSCs) and related nuclear factor kappaB-dependent induction of interleukin-6 secretion in BMSCs, as well as inhibiting proliferation and growth signaling of residual adherent MM cells. These data, therefore, demonstrate that PS-341 both acts directly on MM cells and alters cellular interactions and cytokine secretion in the BM millieu to inhibit tumor cell growth, induce apoptosis, and overcome drug resistance. Given the acceptable animal and human toxicity profile of PS-341, these studies provide the framework for clinical evaluation of PS-341 to improve outcome for patients with this universally fatal hematological malignancy.     

Effects of PS-341 on the activity and composition of proteasomes in multiple myeloma cells

Multiple myeloma is a B-cell malignancy for which no curative therapies exist to date, despite enormous research efforts. The remarkable activity of the proteasome inhibitor bortezomib (PS-341, Velcade) observed in clinical trials of patients with relapsed refractory myeloma has led to investigations of the role of the ubiquitin-proteasome pathway in the pathogenesis of myeloma. Here we report a biochemical analysis of proteasome activity and composition in myeloma cells exposed to PS-341 in the presence or absence of cytokines present in the bone marrow milieu. We observed that the myeloma cell lines MM1.S, RPMI8226, and U266 contain active immunoproteasomes, the amount of which is enhanced by IFN-gamma and tumor necrosis factor-alpha. Using a radiolabeled active site-directed probe specific for proteasome catalytic subunits, we show that PS-341 targets the beta5 and beta1 subunits in a concentration-dependent manner. Furthermore, PS-341 also targeted the corresponding catalytic subunits of the immunoproteasome, beta5i and beta1i, respectively. These data suggest that PS-341 targets both normal and immunoproteasome species to a similar extent in myeloma cells.     

Analysis of IL-6-mediated growth control of myeloma cells using a gp130 chimeric receptor approach.

Interleukin 6 (IL-6) has been shown to be a key growth factor for myeloma cells. To study IL-6 signal transduction in multiple myeloma (MM), we employed chimeric receptors composed of the epidermal growth factor receptor (EGFR) extracellular domain, gp130 transmembrane domain, and full-length or truncated gp130 cytoplasmic domains lacking regions previously shown to be necessary for MAPK, STAT1, and STAT3 activation. The IL-6-dependent KAS-6/1 MM cell line was transfected with various chimeric receptor constructs and assayed for EGF responsiveness. EGF stimulation surprisingly stimulated DNA synthesis in all transfectants, regardless of receptor length. When cell proliferation was assayed instead, only transfectants capable of inducing high levels of STAT3 activation proliferated in response to EGF. Additional studies revealed that EGF stimulation resulted in tyrosine phosphorylation of endogenous gp130 in cells expressing the chimeric receptor. Replacing the gp130 transmembrane region with the EGFR transmembrane domain diminished but did not disrupt this interaction. This receptor interaction was also observed in the IL-6-dependent MM cell line ANBL-6. In summary, although our results suggest that STAT activation is crucial in gp130-mediated proliferation of myeloma cells, these results must be interpreted with caution given our demonstration of the interaction between chimeric and endogenous receptors in myeloma cells. Importantly, this interaction has not been noted in studies utilizing the same gp130 chimeric receptor system in non-MM cells.     

Characterization of a R115777-resistant human multiple myeloma cell line with cross-resistance to PS-341.

The farnesyl transferase inhibitor R115777 has been found to have clinical activity in diverse hematopoietic tumors. Clinical efficacy, however, does not correlate with Ras mutation status or inhibition of farnesyl transferase. To further elucidate the mechanisms by which R115777 induces apoptosis and to investigate drug resistance, we have identified and characterized a R115777-resistant human myeloma cell line. 8226/R5 cells were found to be at least 50 times more resistant to R115777 compared with the parent cell line 8226/S. K-Ras remained prenylated in both resistant and sensitive cells after R115777 treatment; however, HDJ-2 farnesylation was inhibited in both lines, implying that farnesyl transferase (the drug target) has not been mutated. Whereas many 8226 lines that acquire drug resistance have elevated expression of P-glycoprotein, we found that P-glycoprotein expression is not increased in the 8226/R5 line and intracellular accumulation of R115777 was not reduced. In fact, 8226/R5 cells were insensitive to a diverse group of antitumor agents including PS-341, and multidrug resistance did not correlate with the expression of heat shock proteins. Comparison of gene expression profiles between resistant and sensitive cells revealed expression changes in several genes involved in myeloma survival and drug resistance. Future experiments will attempt to identify genes that are directly linked to the resistant phenotype. Identification of molecules associated with R115777 and PS-341 resistance is clinically relevant because both compounds are being tested in solid tumors and hematopoietic malignancies.     

Expression of IL-6, IL-6 Receptor and its Signal Transducer gp130 mRNAs in Megakaryocytic Cell Lines

IL-6, its receptor and its transducer protein gp130 mRNAs were analyzed in three cell lines with megakaryocytic properties including CMK, K562, and HEL. Their colony proliferation was also assayed in the methylcellulose medium with or without IL-6. CMK had IL-6, IL-6 receptor and gp130 mRNAs, while the other two cell lines lacked IL-6 receptor mRNA expression. K562 responded to IL-6 indicating that it has an alternative receptor to the so called “IL-6 receptor”. HEL was not sensitive to IL-6 suggesting that it proliferates with growth factors other than IL-6 via gp130.     

Inhibition of IL-6-dependent growth of myeloma cells by an acidic peptide repressing the gp130-mediated activation of Src family kinases

An acidic domain (AD) of gp130 was previously found to interact with the Src family kinase (SFK) Hck. Here, the influence of myristoylated peptides derived from this AD was assessed in the mouse myeloma cell line, 7TD1. The IL-6-dependent growth of 7TD1 cells was reduced by approximately 75%, if 100 microM of myristoylated 18mer peptide (18AD) was included in the growth medium, but was unaffected by a control peptide with scrambled sequence (18sc). A similar differential inhibition by peptides 18AD and 18sc was observed for the erythropoietin-dependent growth of BaF-EH cells expressing chimeric erythropoietin receptor-gp130 and human Hck and for the human myeloma cell line INA-6. While the peptide 18AD concentration inhibiting 50% was approximately 30 microM in 7TD1 and BaF-EH cells, peptide 18AD did not significantly inhibit growth of IL-6-independent MM1.S myeloma and OKT1 hybridoma cells or of BaF-EH cells supplied with IL-3. Treatment with 100 microM peptide 18AD caused the same degree or 60% of apoptosis induction as IL-6 deprivation in 7TD1 or INA-6 cells, respectively. Co-immunoprecipitation experiments revealed that peptide 18AD interfered with the association of Hck and gp130 in 7TD1 lysates in a concentration-dependent manner. IL-6-treatment of INA-6 cells induced the kinase activities of Fyn, Lyn and Hck, but not Src, and the IL-6-induced SFK activities were inhibited by peptide 18AD. Expression in 7TD1 cells of a kinase-inactive Hck mutant (K269R) elicited a dominant-negative effect on cell number increases providing further evidence that SFKs are required for gp130 signalling in myeloma cells.     

蛋白酶体抑制剂PS-341诱导U266细胞凋亡与胞浆内[Ca2+]变化的关系

目的探讨蛋白酶体抑制剂PS-341(Bortezomib)诱导骨髓瘤细胞株U266细胞凋亡时对其胞浆内[Ca2 ]([Ca2 ]i)的影响。方法以浓度梯度的PS-341干预U266细胞4h后收集,荧光显微镜观察细胞凋亡,AnnexinV-FITC/PI双参数流式细胞术(FCM)检测细胞凋亡,Fluo-3/AM荧光素标记FCM检测[Ca2 ]i。结果(1)PS-341作用U266细胞4h后荧光显微镜观察到凋亡细胞数随浓度增加而增多;(2)FCM检测凋亡细胞的比例分别为0.56%、7.71%、19.84%、31.10%、40.72%,与PS-341浓度成正比;(3)PS-341作用后U266细胞[Ca2 ]i均值分别为403.65nmol/L、418.20nmol/L、378.65nmol/L、356.36nmol/L、349.21nmol/L;(4)PS-341诱导U266细胞凋亡时伴随[Ca2 ]i的改变,浓度>50nmol/L时诱导的细胞凋亡伴随[Ca2 ]i下降。结论蛋白酶体抑制剂PS-341诱导骨髓瘤细胞凋亡呈浓度依赖性;PS-341浓度>50nmol/L时下调[Ca2 ]i,并由此发挥抗骨髓瘤细胞作用。     

Blockade of Hsp27 overcomes Bortezomib/proteasome inhibitor PS-341 resistance in lymphoma cells

Bortezomib (PS-341), a selective inhibitor of proteasome, induces apoptosis in various tumor cells, but its mechanism of action is unclear. Treatment with PS-341 induces apoptosis in SUDHL6 (DHL6), but not SUDHL4 (DHL4), lymphoma cells. Microarray analysis shows high RNA levels of heat shock protein-27 (Hsp27) in DHL4 versus DHL6 cells, which correlates with Hsp27 protein expression. Blocking Hsp27 using an antisense strategy restores the apoptotic response to PS-341 in DHL4 cells; conversely, ectopic expression of wild-type Hsp27 renders PS-341-sensitive DHL6 cells resistant to PS-341. These findings provide the first evidence that Hsp27 confers PS-341 resistance.