Merlin is a potent inhibitor of glioma growth

Neurofibromatosis 2 (NF2) is an inherited cancer syndrome in which affected individuals develop nervous system tumors, including schwannomas, meningiomas, and ependymomas. The NF2 protein merlin (or schwannomin) is a member of the Band 4.1 superfamily of proteins, which serve as linkers between transmembrane proteins and the actin cytoskeleton. In addition to mutational inactivation of the NF2 gene in NF2-associated tumors, mutations and loss of merlin expression have also been reported in other types of cancers. In the present study, we show that merlin expression is dramatically reduced in human malignant gliomas and that reexpression of functional merlin dramatically inhibits both subcutaneous and intracranial growth of human glioma cells in mice. We further show that merlin reexpression inhibits glioma cell proliferation and promotes apoptosis in vivo. Using microarray analysis, we identify altered expression of specific molecules that play key roles in cell proliferation, survival, and motility. These merlin-induced changes of gene expression were confirmed by real-time quantitative PCR, Western blotting, and functional assays. These results indicate that reexpression of merlin correlates with activation of mammalian sterile 20-like 1/2-large tumor suppressor 2 signaling pathway and inhibition of canonical and noncanonical Wnt signals. Collectively, our results show that merlin is a potent inhibitor of high-grade human glioma.     

1,4-Naphthoquinone is a potent inhibitor of human cancer cell growth and angiogenesis

Angiogenesis inhibitors are beneficial for the prevention and treatment of angiogenesis-dependent diseases including cancer. Vitamin K2 and K3, which are naphthoquinone derivatives, inhibit angiogenesis. We examined the anti-cancer and anti-angiogenic effects of naphthoquinones and its structurally related compounds. Among these 13 compounds, 1,4-naphthoquinone strongly inhibited both human colon cancer cell (HCT116) growth and angiogenesis. To clarify the anti-angiogenic mechanism, the effects of 1,4-naphthoquinone on human umbilical vein endothelial cell (HUVEC) tube formation, proliferation and chemotaxis were examined. Consequently, 1,4-naphthoquinone inhibited HUVEC functions. These results suggest that 1,4-naphthoquinone may be useful to cancer therapy.     

ABT-737, an inhibitor of Bcl-2 family proteins, is a potent inducer of apoptosis in multiple myeloma cells.

Disruption of pathways leading to programmed cell death plays a major role in most malignancies, including multiple myeloma (MM). ABT-737 is a BH3 mimetic small-molecule inhibitor that binds with high affinity to Bcl-2 and Bcl-xL, preventing the sequestration of proapoptotic molecules and shifting the cell survival/apoptosis balance toward apoptosis induction. In this study, we show that ABT-737 is cytotoxic to MM cell lines, including those resistant to conventional therapies, and primary tumor cells. Flow cytometric analysis of intracellular levels of Bcl-2 family proteins demonstrates a clear inversion of the Bax/Bcl-2 ratio leading to induction of apoptosis. Activation of the mitochondrial apoptosis pathway was indicated by mitochondrial membrane depolarization and caspase cleavage. Additionally, several signaling pathways known to be important for MM cell survival are disrupted following treatment with ABT-737. The impact of ABT-737 on survival could not be overcome by the addition of interleukin-6, vascular endothelial growth factor or insulin-like growth factor, suggesting that ABT-737 may be effective in preventing the growth and survival signals provided by the microenvironment. These data indicate that therapies targeting apoptotic pathways may be effective in MM treatment and warrant clinical evaluation of ABT-737 and similar drugs alone or in combination with other agents in the setting of MM.     

TRAIL is a potent inducer of apoptosis in myeloma cells derived from multiple myeloma patients and is not cytotoxic to hematopoietic stem cells.

TRAIL, the ligand for the newly discovered DR-4 and DR-5 receptor, is a member of the TNF family of death signal transduction proteins with a mechanism of cell death similar to that of Fas and Fas ligand (Fas-L) system. We provide first time evidence that TRAIL is a potent inducer of apoptosis in multiple myeloma (MM) cell lines. TRAIL effectively induced extensive apoptosis in 8226 and ARP-1 MM cells in a time- and dose-dependent manner. Apoptosis with TRAIL reached about 80% within 48 h of treatment with a dose of 160 ng/ml. Furthermore, we provide first time evidence that similar to Fas, TRAIL-induced apoptosis is not blocked by bcl-2 in MM cell lines. Most importantly, TRAIL induced substantial apoptosis in freshly isolated, flow-sorted myeloma cells obtained from different MM patients expressing variable levels of bcl-2. Finally, we demonstrate for the first time that TRAIL is not cytotoxic to purified CD34+/CD45dim hematopoietic stem cells and does not inhibit CFU-GM or BFU-E colony formation in methylcellulose.     

CD40 ligand-induced apoptosis is Fas-independent in human multiple myeloma cells

We and others previously demonstrated that human multiple myeloma (MM) cells express CD40 and have an active CD40-growth regulatory pathway. This study characterizes the growth outcome of soluble (gp39) or membrane-bound recombinant human CD40-ligand (rCD40L) and its relationship with Fas-dependent apoptosis. Contrary to the moderate growth-stimulatory effect of the CD40-MAb G28.5, gp39 inhibited 3H-thymidine uptake of the plasma dyscrasia lines ARH-77, U266, and HS-Sultan in a dose-dependent fashion by up to 82%. By comparison, RPMI 8226 cells were resistant to CD40L-growth modulation, which may be attributable to a single base substitution (TCA-->TTA, serine-->leucine) at the 3rd cysteine-rich extramembrane region of CD40. Gp39 similarly reduced myeloma clonogenic colony (MCC) formation in patient primary bone marrow cultures by 50% (40-76%; n=6). Studies using transfectant L cells that constitutively expressed CD40L showed that membrane-bound CD40L inhibited the growth of ARH-77, U266, and HS-Sultan cells (66%, 63%, and 32%, respectively), whereas untransfected L cells did not. Growth inhibition by gp39 or CD40L+ L cells was neutralized by coincubation with the CD40L antibodies 5c8 or LL48. CD40L-treatment increased apoptotic activity of MM cells, as defined by oligonucleosomal DNA fragmentation and an increased binding to annexin V (16-28%). All three untreated CD40-responsive MM lines expressed the Fas/Apo-1/CD95 antigen (65-92% CD95+). However, only ARH-77 cells responded to the growth inhibitory effect of the CD95-agonistic antibody CH-11. CD95 expression was not affected significantly by gp39 treatment, and growth inhibition by CH-11 was additive to gp39 (from 42% to 64% decrease in 3H-thmidine uptake). Conversely, the CD95 antagonist antibody ZB4 reversed the Fas-dependent growth inhibitory process but did not significantly alter gp39-mediated growth outcome. Gp39 treatment lowered the expression of TNFR-associated factors TRAF4 and TRAF6 by 38% and 32%, respectively, whereas detectable levels of TRAF1,2,3, and 5 levels remained unchanged. Our observations indicate that the CD40L-binding inhibits human MM cell growth and increases its apoptotic activity. This growth inhibitory effect corresponds to lower levels of cytoplasmic TRAF signaling elements, and appears independent of the Fas-signaling pathway. CD40 receptor mutation may lead to unresponsiveness to CD40 growth modulation in multiple myeloma cells.     

1例多发性骨髓瘤治疗期间并发垂体危象

多发性骨髓瘤（myltiple myeloma, MM）是一种老年病,治疗原则以化疗为主。然而化学治疗相关的不良反应与希恩综合征的临床表现相似,对于无明确产后出血史的老年患者,往往容易造成误诊、漏诊。因此,老年多产的女性,在化疗期间出现顽固性低钠血症及消化道反应时,应考虑希恩综合征的可能,早期明确诊断,及时给予个体化治疗。      

Effects of a novel proteasome inhibitor BU-32 on multiple myeloma cells

Proteasome inhibition is associated with substantial antitumor effects in preclinical models of multiple myeloma (MM) as well as in patients. However, results of recent clinical trials to evaluate the effect of the proteasome inhibitor Bortezomib (Velcade^®, also called PS-341) in MM patients have shown limited activity when used as a single agent. This underscores the need to find new efficacious and less toxic proteasome inhibitors. Recently, carfilzomib was approved for the treatment of refractory/relapsed MM and several new agents have been introduced into the clinic, including marizomib and MLN9708, and trials investigating these second-generation proteasome inhibitors have demonstrated promising results. We have recently synthesized a novel proteasome inhibitor, BU-32, and tested its growth inhibitory effects in different human MM cells including RPMI8226, MM.1S, MM.1R, and U266. In this study, we evaluate the efficacy of the novel proteasome inhibitor BU-32 (NSC D750499) using an in vitro MM model. BU-32 exhibits strong cytotoxicity in a panel of MM cell lines—RPMI8226, MM1S, MM1R, and U266. In addition, we demonstrate by proteasome inhibition assay that BU-32 potently inhibits the chymotryptic- and caspase-like activities of the 26S proteasome. We further show from Annexin V-FITC binding studies that BU-32, like Bortezomib, induces apoptosis in a panel of MM cell lines but the effect is more pronounced with BU-32-treated cells. Invasion assay with the MM.1S cell line indicates that BU-32 inhibits the invasiveness of myeloma cells. Results from our studies using real-time PCR array analyses show that BU-32 effectively downregulates an array of angiogenesis and inflammatory markers. Our results suggest that BU-32 might be a potential chemotherapeutic agent with promising antitumor activity for the treatment of MM.     

IL-6 is a key factor in growth inhibition of human myeloma cells induced by pravastatin,an HMG-CoA reductase inhibitor

Although recent developments in initial chemotherapeutic regimens and stem cell transplantation have achieved improvements of initial remission for myeloma patients, relapse and recurrence are still major problems. The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) have been developed for treating hyperlipidemia. Recently, there have been several reports concerning the effects of statins on cancer cells including liver, colon, leukemia, malignant B, stomach, and breast cells. In this study, the in vitro effects of pravastatin on human myeloma cells and the factors closely related to its growth inhibitory effects were examined. Although concentrations were higher than those used clinically, 4 out of 10 myeloma lines showed growth inhibition by pravastatin. The study of factors related to the inhibition indicated IL-6 is important. Indeed, rhIL-6 abolished pravastatin-induced growth inhibition in KMS-21BM cells which did not express IL-6. Statins may be useful in maintenance therapy for myeloma after the screening of IL-6 status.     

A newly developed bisphosphonate, YM529, is a potent apoptosis inducer of human myeloma cells

We examined the effect of YM529, a newly developed third-generation bisphosphonate (BP), on the growth of human myeloma cell lines using the trypan blue dye exclusion test and Alamar blue assay. BPs induced inhibition of proliferation in all cell lines dose-dependently, and YM529 had a most potent growth inhibitory effect, followed by incadronate and pamidronate. Flow cytometric analysis using annexinV and 7AAD showed that YM529 most significantly induced apoptosis of all myeloma cell lines. These observations suggested that YM529 is a potent apoptosis inducer of myeloma cells, and might have some benefit not only on the improvement of bone lesions but also on survival in some myeloma patients.     

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.     

IFN-β is a potent inhibitor of insulin and insulin like growth factor stimulated proliferation and migration in human pancreatic cancer cells

Introduction: Pancreatic cancer is a highly aggressive malignancy with few treatment options. The overexpression of several growth factors, including insulin and insulin-like growth factors (IGFs), can underlie the aggressive nature of this disease. Previous research has demonstrated potent effects of interferon (IFN)-β on pancreatic cancer cell growth, however up till now it is unknown whether IFN-β is able to counteract IGF1, IGF2 and insulin-induced pancreatic cancer cell proliferation and migration. Methods: Expression of IGF- and insulin receptors was determined and the stimulatory effects of IGF1, IGF2 and insulin on cell proliferation and migration, as well as the inhibitory effects of IFN-β were evaluated in 3 human pancreatic adenocarcinoma cell lines. Results: Both the insulin- and the IGF1 receptor were variably expressed in the cell lines. IGF1, IGF2 and insulin were capable of stimulating cell proliferation in all three cell lines, however cell migration was significantly enhanced only in the BxPC-3 cell line. IFN-β significantly inhibited IGF1-, IGF2- and insulin-stimulated proliferation in all three cell lines in a dose and time dependent manner. Furthermore, in the BxPC-3 cell line IFN-β significantly inhibited both basal and IGF1-, IGF2- and insulin-stimulated cell migration. Conclusion: Both IGF1, -2 and insulin were capable of stimulating proliferation and migration in human pancreatic cancer cells irrespective of the type of receptor expressed. This study demonstrates that insulin, in addition to IGF1 and IGF2, may play an important role in the progression of pancreatic cancer. Moreover, IFN-β strongly inhibits growth factor stimulated cell proliferation and migration. Our study supports previous findings which have suggested that IFN-β can be a potential promising anti-cancer agent in pancreatic cancer.     

Growth inhibition of multiple myeloma cells by a novel IkappaB kinase inhibitor.

Involvement of nuclear factor-kappaB (NF-kappaB) in cell survival and proliferation of multiple myeloma has been well established. In this study we observed that NF-kappaB is constitutively activated in all human myeloma cell lines, thus confirming the previous studies. In addition, we found the phosphorylation of p65 subunit of NF-kappaB in addition to the phosphorylation of IkappaBalpha and the activation of NF-kappaB DNA binding and that various target genes of NF-kappaB including bcl-x(L), XIAP, c-IAP1, cyclin D1, and IL-6 are up-regulated. We then examined the effect of a novel IkappaB kinase inhibitor, 2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin-4-yl nicotinonitrile (ACHP). When myeloma cells were treated with ACHP, the cell growth was efficiently inhibited with IC(50) values ranging from 18 to 35 mumol/L concomitantly with inhibition of the phosphorylation of IkappaBalpha/p65 and NF-kappaB DNA-binding, down-regulation of the NF-kappaB target genes, and induction of apoptosis. In addition, we observed the treatment of ACHP augmented the cytotoxic effects of vincristine and melphalan (l-phenylalanine mustard), conventional antimyeloma drugs. These findings indicate that IkappaB kinase inhibitors such as ACHP can sensitize myeloma cells to the cytotoxic effects of chemotherapeutic agents by blocking the antiapoptotic nature of myeloma cells endowed by the constitutive activation of NF-kappaB.     

Proliferation of immature myeloma cells by interleukin-6 is associated with CD45 expression in human multiple myeloma

Multiple myeloma (MM) is a hematologic malignancy of human plasma cells, and myeloma cells can be classified into several subpopulations according to phenotypic differences, such as CD38 MPC-1- CD49e- immature, CD38 MPC-1+ CD49e- intermediate and CD38 MPC-1+ CD49e+ mature myeloma cells. The expression of the CD45 molecule on myeloma cells is quite variable, and the physiological consequence of CD45 on myeloma cells is still unknown. Recently, we have found that a few MPC-1- immature myeloma cells express CD45 antigens while most myeloma cells do not express the CD45. MPC-1- CD45+ CD49e- but not MPC-1- CD45- CD49e- immature cells contain proliferating cells in response to interleukin-6 (IL-6). IL-6 can also induce expression of CD45 on the MPC-1- CD45- subpopulation of immature myeloma cells. In addition, myeloma cell lines responding to IL-6 express CD45, whereas cell lines proliferating independent of IL-6 do not express CD45. In the U266 cell line, IL-6 leads to the induction of CD45 expression and cell proliferation, indicating that IL-6-induced effects are closely linked to CD45 expression. Thus, there is a heterogeneity in human myeloma cells, and among these subpopulations immature myeloma cells expressing the CD45 molecules appear to proliferate in response to IL-6. In this review we propose the involvement of CD45 in MM pathogenesis, and the possible implications of CD45 as both a phenotypic marker and a functional molecule is discussed.     

Interleukin-18 inhibits lodging and subsequent growth of human multiple myeloma cells in the bone marrow

An intravenous injection of ARH-77 cells (human multiple myeloma cell line) into mice with severe combined immunodeficiency disease (SCID mice) results in lodging of tumor cells in the bone marrow of thoracic and lumbar vertebrae, and in their subsequent growth, the cells destroying bone and invading the spinal cord and surrounding tissues, and the mice show hind leg paralysis. Using this model, we investigated the effects of interleukin (IL)-18 on the lodging and subsequent growth of multiple myeloma cells in the bone marrow. Mouse recombinant IL-18 (mIL-18) at 1 microg/mouse was daily injected according to protocols A and B. In protocol A, mIL-18 was injected from day 6 after tumor cell injection to examine the effect of mIL-18 on tumor growth, and in protocol B, it was injected from day 3 prior to tumor cell injection to day 3 after it to examine the effect of mIL-18 on lodging of tumor cells. The spread of a tumor was monitored as to the appearance of hind leg paralysis and the tumor area in a median longitudinal section of the vertebrae with the surrounding tissues. With protocol A, mIL-18 significantly and markedly decreased the cumulative rate of hind leg paralysis and the tumor area. This antitumor effect of mIL-18 was ascribed to its action on the activation of NK cells because mIL-18 exerted no significant effect when anti-asialo GM1 antiserum (a-ASGM1) was simultaneously injected to deplete the NK cell activity. With protocol B, mIL-18 also significantly and markedly decreased the cumulative rate of hind leg paralysis and the tumor area. However, most of this effect was not due to the action of mIL-18 on NK cells because mIL-18 showed a marked and significant effect with the administration of a-ASGM1. The present results indicate that mIL-18 inhibited the lodging and subsequent growth of multiple myeloma cells in the bone marrow, and suggest that IL-18 is worth investigating further as to its usefulness as a therapy for multiple myeloma.     

硼替佐米在多发性骨髓瘤治疗中的应用

硼替佐米通过抑制蛋白酶体的活性而抑制核因子-κB（NF-κB）的功能,具有选择性抗骨髓瘤活性。在新诊断、难治、复发多发性骨髓瘤患者中的应用,显示出了其他药物无可比拟的优势,具有缓解率高,能延长患者生存期等特点。尽管其副作用常见,但是可以控制。