VEGF induces Mcl-1 up-regulation and protects multiple myeloma cells against apoptosis

Interleukin-6 (IL-6) triggers multiple myeloma (MM) cell proliferation and protects against apoptosis by up-regulating myeloid cell leukemia 1 (Mcl-1). Vascular endothelial growth factor (VEGF) induces modest proliferation of MM cells and induces IL-6 secretion in a paracrine loop involving MM cells and bone marrow stromal cells. Using murine embryonic fibroblast cell lines as a model (Mcl-1(wt/wt) and Mcl-1(Delta/null) MEFs), we here demonstrate that deletion of Mcl-1 reduces fetal bovine serum (FBS)-, VEGF-, and IL-6-induced proliferation. We also show that VEGF up-regulates Mcl-1 expression in a time- and dose-dependent manner in 3 human MM cell lines and MM patient cells. Importantly, we demonstrate that the pan-VEGF inhibitor, GW654652, inhibits VEGF-induced up-regulation of Mcl-1 and, as with Mcl-1 siRNA, is associated with decreased proliferation and induction of apoptosis. Finally, we show that VEGF protects MM patient cells against FBS starvation-induced apoptosis. Our studies therefore demonstrate that VEGF-induced MM cell proliferation and survival are mediated via Mcl-1, providing the preclinical framework for novel therapeutics targeting Mcl-1 and/or VEGF to improve patient outcome in MM.     

The incidence and possible relevance of Bence-Jones protein in the sera of patients with multiple myeloma

We present results showing that the detectable incidence of Bence-Jones protein in the sera of patients with multiple myeloma is equivalent to the highest detectable incidence of Bence-Jones protein in highly concentrated urine when tested by immuno-isoelectric focusing. In a series of 25 multiple myeloma patients, immuno-isoelectric focusing showed the incidence of Bence-Jones proteinemia to be 68% compared with a detectable incidence of Bence-Jones proteinuria of 32-64% by immunoelectrophoresis and immuno-isoelectric focusing depending upon the urine concentration factor. In view of the variability of factors governing urinary Bence-Jones protein levels, such as renal catabolism, reabsorption and losses during concentration, we suggest that monitoring of serum Bence-Jones protein by immuno-isoelectric focusing should be carried out in conjunction with measurements of urinary Bence-Jones protein in multiple myeloma.     

Interleukin-1 induces time-dependent potentiation in isolated rat islets: possible involvement of phosphoinositide hydrolysis

Prior exposure of isolated perfused rat islets to the monokine interleukin-1 (IL-1) amplifies their subsequent insulin secretory response to 10 mM glucose. This potentiating effect of the monokine is dose dependent, lasts for at least 45 min after IL-1 removal from the medium, and is not confined to glucose; IL-1 also potentiates the insulin secretory responses to tolbutamide and glyceraldehyde. IL-1 exposure of islets incubated with myo-[2-3H]inositol to label their phosphoinositides (PI) results in an increase in [3H]inositol efflux, an event that persists long after removal of IL-1 from the medium. Direct measurements of labeled inositol phosphate accumulation substantiate the concept that this sustained [3H]inositol efflux response is the direct result of a sustained increase in PI hydrolysis. These results expand the list of compounds that induce time-dependent potentiation in islets to include IL-1. This action of the monokine, mediated at least in part by PI-derived second messenger molecules, may contribute to its postulated effects on insulin and glucose homeostasis.     

多发性骨髓瘤患者肾损害临床与病理特征——附24例分析

目的　了解多发性骨髓瘤 (MM)肾损害患者临床病理特征。方法　回顾性分析经临床、病理明确诊断的 2 4例MM肾损害患者的临床病理特征。结果　MM肾损害的临床症候群以肾功能不全 (血肌酐 >177μmol/L)最为常见 (83.3% ) ,其次为肾病综合征 (12 .5 % )、无症状尿检异常 (4.17% )。病理改变以管型肾病最为常见(6 2 .0 % ,13/ 2 1例 ) ,慢性间质性肾炎、轻链沉积病、肾小球淀粉样变性和肾小球系膜增生性病变的发生率分别为14 .3% ,9.5 2 % ,9.5 2 %和 4 .76 %。血清轻链阳性率为 6 8.4 % (13/ 19例 ) ,尿中轻链阳性率为 70 .0 % (14 / 2 0例 ) ,以λ链为主。肾组织κ、λ轻链阳性检出率为 82 .3% (14 / 17例 )。管型肾病 (13例 )较非管型肾病患者 (8例 )肾功能不全更为常见 (10 0 %vs 6 2 .5 % ,P <0 .0 5 )、本 周氏蛋白阳性率更高 (5 3.8%vs 13.5 % ,P <0 .0 5 )、小管间质病变更重 (重度小管间质病变发生率 76 .9%vs 2 5 .0 % ,P <0 .0 5 )。结论　MM伴肾损害患者临床症候群以肾功能不全多见 ,病理主要表现为管型肾病。血清与尿液中轻链以λ为主。MM伴肾损害、管型肾病患者其临床表现与病理改变均有一定的特点     

Thioredoxin-1 protects against hyperoxia-induced apoptosis in cells of the alveolar walls

BACKGROUND: The mechanisms of hyperoxia-induced lung injury remain poorly defined. Thioredoxin-1 (TRX-1) is a small ubiquitous protein that acts as an important radical scavenger. We investigated the effect of TRX-1 on apoptosis in hyperoxia-induced lung injury. METHODS: Mice were exposed to 98% O(2) to produce a model of hyperoxia-induced lung injury. Using transgenic mice overexpressing human TRX-1 (hTRX-1), we assessed lung structure (n=4 per group), immunohistochemical staining for 8-hydroxy-deoxyguanosine (n=4 per group), TUNEL staining (n=5 per group), cytokine (n=5 per group) of IL-1beta and IL-6, and protein (n=6 per group) and m-RNA levels (n=4 per group) (or both) of cytochrome c, Bcl-2, Bax, p21, and p53 in the lungs. RESULTS: After exposure to hyperoxia, hTRX-1 transgenic mice had significantly decreased alveolar damage. The apoptotic index was significantly lower in hTRX-1 transgenic mice than in wild-type (WT) mice after exposure to hyperoxia. Protein expression of cytochrome c in the lung was significantly lower in hTRX-1 transgenic mice than in WT mice after exposure to hyperoxia. Protein expression and m-RNA levels of Bcl-2 in the lung were significantly higher in hTRX-1 transgenic mice than in WT mice after exposure to hyperoxia. TRX-1 had no effect on the protein and m-RNA levels of Bax and p21. The protein and m-RNA levels of p53 was unaffected by hyperoxia in hTRX-1 transgenic mice. The cytokine level of IL-6 was significantly higher in hTRX-1 transgenic mice than in WT mice after exposure to hyperoxia. TRX-1 had no effect on cytokine level of IL-1beta. CONCLUSIONS: These findings suggest that overexpression of hTRX-1 protects against hyperoxia-induced apoptosis in cells of the alveolar walls. The up-regulating Bcl-2 protein is considered to be one of antiapoptotic effects of TRX-1 in hyperoxia-induced lung injury.     

Assessment of bone involvement in patients with multiple myeloma using bone densitometry

Abstract: To evaluate the use of dual energy X-ray absorptiometry (DXA) in multiple myeloma (MM) we performed a prospective study of 34 patients with newly diagnosed MM. Most patients had advanced disease and all but two patients had osteolytic bone destructions and/or pathological fractures. Bone mineral content (BMC) and bone mineral density (BMD) of the lumbar spine (L₁–L₄) and hip were measured using a Hologic QDR-1000 scanner. Collapsed vertebrae were not excluded from analysis. Data from 289 healthy Danish volunteers aged 21–79 yr were used for calculation of Z-scores. Lumbar spine BMC (Z-score –0.46±0.23, p=0.05) and lumbar spine BMD (Z-score –0.56±0.23, p=0.02) were significantly reduced in MM patients, whereas no reduction was seen in hip BMC or BMD. Collapsed vertebrae had marked reduced BMD (Z-score –1.34±0.22, p<0.001), as had non-fractured vertebrae in the same individuals (Z-score –1.42±0.25, p<0.001). Lumbar spine BMD correlated with radiologically assessed bone morbidity (r –0.37, p=0.03) and stronger with the incidence of vertebral fractures (r –0.64, p<0.001). Thus, osteopenia of the back is common in multiple myeloma and correlates with an increased incidence of fractures. DXA may identify subjects with increased risk of vertebral fractures for more intensive chemotherapeutic or anti-resorptive treatment.     

Prostaglandin E1 protects hepatocytes against endoplasmic reticulum stress-induced apoptosis via protein kinase A-dependent induction of glucose-regulated protein 78 expression

AIM To investigate the protective effect of prostaglandin E1(PGE1) against endoplasmic reticulum(ER) stressinduced hepatocyte apoptosis, and to explore its underlying mechanisms.METHODS Thapsigargin(TG) was used to induce ER stress in the human hepatic cell line L02 and hepatocarcinomaderived cell line Hep G2. To evaluate the effects of PGE1 on TG-induced apoptosis, PGE1 was used an hour prior to TG treatment. Activation of unfolded protein response signaling pathways were detected by western blotting and quantitative real-time RTPCR. Apoptotic index and cell viability of L02 cells and Hep G2 cells were determined with flow cytometry and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium] assay. RESULTS Pretreatment with 1 μmol/L PGE1 protected against TG-induced apoptosis in both L02 cells and Hep G2 cells. PGE1 enhanced the TG-induced expression of C/EBP homologous protein(CHOP), glucose-regulated protein(GRP) 78 and spliced X box-binding protein 1 at 6 h. However, it attenuated their expressions after 24 h. PGE1 alone induced protein and m RNA expressions of GRP78; PGE1 also induced protein expression of DNA damage-inducible gene 34 and inhibited the expressions of phospho-PKR-like ER kinase, phosphoeukaryotic initiation factor 2α and CHOP. Treatment with protein kinase A(PKA)-inhibitor H89 or KT5720 blocked PGE1-induced up-regulation of GRP78. Further, the cytoprotective effect of PGE1 on hepatocytes was not observed after blockade of GRP78 expression by H89 or small interfering RNA specifically targeted against human GRP78.CONCLUSION Our study demonstrates that PGE1 protects against ER stress-induced hepatocyte apoptosis via PKA pathwaydependent induction of GRP78 expression.     

A role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy

We demonstrate a role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy. In particular, transient increased expression of Sirt1 is sufficient to stimulate basal rates of autophagy. In addition, we show that Sirt1(-/-) mouse embryonic fibroblasts do not fully activate autophagy under starved conditions. Reconstitution with wild-type but not a deacetylase-inactive mutant of Sirt1 restores autophagy in these cells. We further demonstrate that Sirt1 can form a molecular complex with several essential components of the autophagy machinery, including autophagy genes (Atg)5, Atg7, and Atg8. In vitro, Sirt1 can, in an NAD-dependent fashion, directly deacetylate these components. The absence of Sirt1 leads to markedly elevated acetylation of proteins known to be required for autophagy in both cultured cells and in embryonic and neonatal tissues. Finally, we show that Sirt1(-/-) mice partially resemble Atg5(-/-) mice, including the accumulation of damaged organelles, disruption of energy homeostasis, and early perinatal mortality. Furthermore, the in utero delivery of the metabolic substrate pyruvate extends the survival of Sirt1(-/-) pups. These results suggest that the Sirt1 deacetylase is an important in vivo regulator of autophagy and provide a link between sirtuin function and the overall cellular response to limited nutrients.     

Endothelin‐1 receptor blockade as new possible therapeutic approach in multiple myeloma

New effective treatments are needed to improve outcomes for multiple myeloma (MM) patients. Receptors with restricted expression on plasma cells (PCs) represent attractive new therapeutic targets. The endothelin‐1 (EDN1) axis, consisting of EDN1 acting through EDN‐receptor A (EDNRA) and B (EDNRB), was previously shown to be overexpressed in several tumours, including MM. However, there is incomplete understanding of how EDN1 axis regulates MM growth and response to therapy. Besides EDNRA, the majority of MM cell lines and primary malignant PCs express high levels of EDNRB and release EDN1. Similarly, bone‐marrow microenvironment cells also secrete EDN1. Investigating the extent of epigenetic dysregulation of EDNRB gene in MM, we found that hypermethylation of EDNRB promoter and subsequent down‐regulation of EDNRB gene was observed in PCs or B lymphocytes from healthy donors compared to EDNRB‐expressing malignant PCs. Pharmacological blockade with the dual EDN1 receptor antagonist bosentan decreased cell viability and MAPK activation of U266 and RPMI‐8226 cells. Interestingly, the combination of bosentan and the proteasome inhibitor bortezomib, currently approved for MM treatment, resulted in synergistic cytotoxic effects. Overall, our data has uncovered EDN1‐mediated autocrine and paracrine mechanisms that regulate malignant PCs growth and drug response, and support EDN1 receptors as new therapeutic targets in MM.     

Pamidronate causes apoptosis of plasma cells in vivo in patients with multiple myeloma

Anti-resorptive bisphosphonates, such as pamidronate, are an effective treatment for osteolytic disease and hypercalcaemia in patients with multiple myeloma, but have also been shown to cause apoptosis of myeloma cell lines in vitro. In this study, we found that a single infusion of pamidronate, in 16 newly diagnosed patients with multiple myeloma, caused a marked increase in apoptosis of plasma cells in vivo in 10 patients and a minimal increase in four patients (P < 0.05). The nitrogen-containing bisphosphonates pamidronate and zoledronic acid also induced apoptosis of authentic, human bone marrow-derived plasma cells in vitro. Apoptosis of plasma cells in vitro was probably caused by inhibition of the mevalonate pathway and loss of prenylated small GTPases, as even low concentrations (>or= 1 micro mol/l) of zoledronic acid caused accumulation of unprenylated Rap1A in cultures of bone marrow mononuclear cells in vitro. GGTI-298, a specific inhibitor of geranylgeranyl transferase I, also induced apoptosis in human plasma cells in vitro, suggesting that geranylgeranylated proteins play a role in signalling pathways that prevent plasma cell death. Our results suggest that pamidronate may have direct and/or indirect anti-tumour effects in patients with multiple myeloma, which has important implications for the further development of the more potent nitrogen-containing bisphosphonates, such as zoledronic acid, in the treatment of myeloma.     

HTLV-1 Tax protects against CD95-mediated apoptosis by induction of the cellular FLICE-inhibitory protein (c-FLIP)

The HTLV-1 transactivator protein Tax is essential for malignant transformation of CD4 T cells, ultimately leading to adult T-cell leukemia/lymphoma (ATL). Malignant transformation may involve development of apoptosis resistance. In this study we investigated the molecular mechanisms by which HTLV-1 Tax confers resistance toward CD95-mediated apoptosis. We show that Tax-expressing T-cell lines derived from HTLV-1-infected patients express elevated levels of c-FLIP(L) and c-FLIP(S). The levels of c-FLIP correlated with resistance toward CD95-mediated apoptosis. Using an inducible system we demonstrated that both resistance toward CD95-mediated apoptosis and induction of c-FLIP are dependent on Tax. In addition, analysis of early cleavage of the BH3-only Bcl-2 family member Bid, a direct caspase-8 substrate, revealed that apoptosis is inhibited at a CD95 death receptor proximal level in Tax-expressing cells. Finally, using siRNA we directly showed that c-FLIP confers Tax-mediated resistance toward CD95-mediated apoptosis. In conclusion, our data suggest an important mechanism by which expression of HTLV-1 Tax may lead to immune escape of infected T cells and, thus, to persistent infection and transformation.     

骨髓瘤肾病患者自体外周血干细胞移植治疗临床分析

目的:探讨骨髓瘤肾脏累及患者进行自体外周血干细胞移植(APBSCT)治疗的疗效和安全性。方法:首诊为多发性骨髓瘤(MM)肾病的男性患者4例,年龄35~53岁,进行常规血生化,血、尿免疫蛋白电泳和骨髓穿刺检查,3例行肾脏活组织检查(活检),常规化学治疗(化疗)后均行大剂量化疗(HDT)联合APBSCT。结果:4例患者中IgG型2例,IgD及无分泌型各1例;ⅡA期2例,ⅢA期1例,ⅢB期1例;确诊时均有蛋白尿(1.51～5.70 g),急性肾损伤(AKI)2例,慢性肾脏病(CKD)Ⅱ期、CKDⅤ期各1例。肾穿刺3例中1例弥漫间质炎细胞浸润,1例为轻链沉淀肾病(LCDD),另1例为管型肾病;1例未肾活检,临床推测MM管型肾病可能。移植前常规化疗4~6疗程。3例轻、中度肾功能受累者APBSCT治疗后1例再次发生AKI,但短时间恢复,长期随访尿蛋白基本转阴性,肾功能稳定无进展;1例移植后1年转变为浆细胞白血病死亡,2例MM完全缓解(CR)。1例严重肾功能受累(CKDⅤ期)者移植后因感染性休克死亡。结论:HDT联合APBSCT是治疗MM的有效方法。APBSCT治疗对轻、中度肾功能受累MM肾病患者的肾功能无明显影响,疗效满意。严重肾功能衰竭患者中进行该治疗尚需进一步探讨。     

Proteomic evaluation of pathways associated with dexamethasone-mediated apoptosis and resistance in multiple myeloma

We have used global protein expression analysis to characterize the pathways of dexamethasone-mediated apoptosis and resistance in myeloma. Analysis of MM.1S cells by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) identified a series of proteins that were up- and downregulated following dexamethasone treatment. Downregulated proteins included proteins involved in cell survival and proliferation, whereas upregulated proteins were involved in post-translational modification, protein folding and trafficking. A comparison with published gene expression studies identified FK binding protein 5 (FKBP5) (also known as FKBP51), a key regulatory component of the Hsp90-steroid-receptor complex to be increased at the mRNA and protein level postdexamethasone exposure. Quantitative real time polymerase chain reaction and 2D-PAGE analysis of the dexamethasone resistant cell line MM.1R demonstrated no increase in FKBP5, consistent with its association with dexamethasone-mediated apoptosis. Western blot analysis of FKBP5 and other members of the Hsp90-receptor complex showed an increase in FKBP5 whilst FKBP4 (also known as FKBP52) and Hsp90 expression remained constant. No changes were observed in MM.1R. In conclusion, we demonstrated that following steroid receptor signalling, the cell carries out a number of adaptive responses prior to cell death. Interfering with these adaptive responses may enhance the myeloma killing effect of dexamethasone.     

Inhibition of protein geranylgeranylation induces apoptosis in myeloma plasma cells by reducing Mcl-1 protein levels

HMG-CoA reductase is the rate-limiting enzyme of the mevalonate pathway leading to the formation of cholesterol and isoprenoids such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP). The inhibition of HMG-CoA reductase by lovastatin induced apoptosis in plasma cell lines and tumor cells from patients with multiple myeloma. Here we show that cotreatment with mevalonate or geranylgeranyl moieties, but not farnesyl groups, rescued myeloma cells from lovastatin-induced apoptosis. In addition, the inhibition of geranylgeranylation by specific inhibition of geranylgeranyl transferase I (GGTase I) induced the apoptosis of myeloma cells. Apoptosis triggered by the inhibition of geranylgeranylation was associated with reduction of Mcl-1 protein expression, collapse of the mitochondrial transmembrane potential, expression of the mitochondrial membrane protein 7A6, cytochrome c release from mitochondria into the cytosol, and stimulation of caspase-3 activity. These results imply that protein geranylgeranylation is critical for regulating myeloma tumor cell survival, possibly through regulating Mcl-1 expression. Our results show that pharmacologic agents such as lovastatin or GGTase inhibitors may be useful in the treatment of multiple myeloma.