Constitutive hyperexpression of p21(WAF1) in human U266 myeloma cells blocks the lethal signaling induced by oxidative stress but not by Fas.

p21(WAF1/CIP1) is expressed in a majority of myeloma cells. To investigate the role of p21 in myeloma cell death, comparative studies using two clones of myeloma cells, Fas-sensitive RPMI8226, and Fas-resistant U266 were performed. These latter cells were also resistant to H(2)O(2) up to 100 microM, whereas the former cells were not. SAPK/JNK was found to be a common mediator of RPMI8226 cell death induced by both H(2)O(2) and Fas. Interestingly, the concentrations of H(2)O(2) which activated SAPK/JNK in RPMI8226 cells failed to do so in U266 cells. In contrast, Fas ligation activated SAPK/JNK in both cells almost equally. U266 cells expressed p21 to levels much higher than in RPMI8226 cells. When the p21 levels were reduced using its antisense, H(2)O(2) killed U266 cells by activating SAPK/JNK. However, the reduction in p21 levels neither rendered the U266 cells susceptible to Fas-mediated cell death, nor significantly influenced Fas-induced SAPK/JNK activation. Overall, our data suggest that the p21 hyperexpression in U266 cells blocks the lethal signaling that is induced by H(2)O(2), but not by Fas. The mechanism whereby U266 cells resist Fas-mediated cell death is discussed.     

FLIP is Constitutively Hyperexpressed in Fas-resistant U266 Myeloma Cells, but Is Not Induced by IL-6 in Fas-sensitive RPMI8226 Cells

Despite the expression of Fas, some clones of myeloma cells are resistant to Fas-mediated apoptosis. To define a cellular factor involved in the resistance, we performed a comparative study using two clones of myeloma cells, RPMI8226 and U266. These cells were reported to express cell surface Fas at similar levels, but only RPMI8226 cells lost their viability upon anti-Fas treatment. The resistance of U266 cells to anti-Fas did not appear to reflect dysregulation of Bcl-2, Bcl-X^L, and Bax, because these proteins were expressed in both RPMI8226 and U266 cells to similar levels. Moreover, levels of those proteins were not significantly altered by treating RPMI8226 cells with IL-6, a cytokine which suppresses the Fas-mediated death of RPMI8226 cells. Interestingly, mRNA levels of FLIP^L, an endogenous inhibitor of Fas signaling, were constitutively elevated in U266 cells. Consistent with this observation, U266 cells expressed both FLIP^L protein and its truncated 43 kDa product which is seen in FLIP^L-overexpressing cells. The truncated form of FLIP^L protein was not detected in RPMI8226. Moreover, the levels of truncated FLIP^L in U266 cells were considerably higher than those of pro-FLIP^L in RPMI8226. The overall data indicate that FLIP^L is constitutively hyperexpressed in U266 cells. However, IL-6 failed to enhance the protein levels of FLIP molecules in either of the tested cells. It appears, therefore, that FLIP^L plays a role in the intrinsic resistance of U266 cells to the apoptotic action of Fas, but is not involved in the protective action of IL-6.     

Nitrogen-containing bisphosphonates induce S-phase cell cycle arrest and apoptosis of myeloma cells by activating MAPK pathway and inhibiting mevalonate pathway

Bisphosphonates have been used for the treatment of hypercalcemia associated with malignancies and osteoporosis. It was previously reported that the mevalonate pathway is involved in nitrogen-containing bisphosphonate-induced apoptosis in osteoclasts and myeloma cells. The aim of this study was to determine the effects of two bisphosphonates, incadronate, and newly developed bisphosphonate YM529 on human myeloma cells, U266, RPMI-8226, and HS-Sultan. Both incadronate and YM529 induced S-phase cell cycle arrest and apoptosis in these myeloma cells. Treatment of the myeloma cells with cell-permeable substrates for mevalonate pathways, geranylgeraniol, and farnesol prevented bisphosphonate-mediated growth suppression. Checkpoint kinases, Chk1/2, and MAPK became phosphorylated after stimulation with bisphosphonates in the myeloma cells. Bisphosphonate-induced apoptosis was partially prevented by the pretreatment with MAPK inhibitor. These results demonstrate that incadronate and YM529 suppress the proliferation of myeloma cells through mevalonate pathway and MAPK pathway.     

Xanthohumol exhibits anti-myeloma activity in vitro through inhibition of cell proliferation,induction of apoptosis via the ERK and JNK-dependent mechanism,and suppression of sIL-6R and VEGF production

BackgroundXanthohumol (XN, a hop-derived prenylflavonoid) was found to exert anticancer effects on various cancer types. However, the mechanisms by which XN affects the survival of multiple myeloma cells (MM) are little known. Therefore, our study was undertaken to address this issue.MethodsAnti-proliferative activity of XN towards two phenotypically distinct MM cell lines U266 and RPMI8226 was evaluated with the MTT and BrdU assays. Cytotoxicity was determined with the LDH method, whereas apoptosis was assessed by flow cytometry and fluorescence staining. The expression of cell cycle- and apoptosis-related proteins and the activation status of signaling pathways were estimated by immunoblotting and ELISA assays.ResultsXN reduced the viability of RPMI8226 cells more potently than in U266 cells. It blocked cell cycle progression through downregulation of cyclin D1 and increased p21 expression. The marked apoptosis induction in the XN-treated RPMI8226 cells was related to initiation of mitochondrial and extrinsic pathways, as indicated by the altered p53, Bax, and Bcl-2 protein expression, cleavage of procaspase 8 and 9, and elevated caspase-3 activity. The apoptotic process was probably mediated via ROS overproduction and MAPK (ERK and JNK) activation as N-acetylcysteine, or specific inhibitors of these kinases prevented the XN-induced caspase-3 activity and, hence, apoptosis. Moreover, XN decreased sIL-6R and VEGF production in the studied cells.ConclusionsERK and JNK signaling pathways are involved in XN-induced cytotoxicity against MM cells.General Significance: The advanced understanding of the molecular mechanisms of XN action can be useful in developing therapeutic strategies to treat multiple myeloma.     

Molecular mechanisms underlying antitumor activity of camel whey protein against multiple myeloma cells

Treating drug-resistant cancer cells is a clinical challenge and it is also vital to screen for new cancer drugs. Multiple myeloma (MM) is a plasma cell clonal cancer that, despite many experimental therapeutics, remains incurable. In this study, two MM cell line lines U266 and RPMI 8226 were used to determine the impact of camel whey protein (CWP). The CWP IC₅₀ was calculated by MTT examination, while the flow cytometry analysis was used to investigate the chemotaxis responses of MM cells in relation to CXCL12 and the pro-apoptotic effect of CHP. MM cells were treated with CWP and Western blot analysis was used to determine the underlying molecular mechanisms. Dose and time based on the impact of CWP on the cell viability of MM cells with IC₅₀ of 50 μg/ml, without affecting the viability of normal healthy PBMCs. CWP reduced chemotaxis of MM cells significantly from the CXC chemokine ligand 12 (CXCL12). Using Western blot analysis, we found that CWP decreased the activation of AKT, mTOR, PLCβ3, NFαB and ERK, which was mechanistically mediated by CXCL12/CXCR4. In both U266 and RPMI 8226, CWP induced apoptosis by upregulating cytochrome C expression. In addition, CWP mediated the growth arrest of MM cells by robustly decreasing the expression of the anti-apoptotic Bcl-2 family members Bcl-2, Bcl-XL and Mcl-1. Conversely, the expression of pro-apoptotic Bcl-2 family members Bak, Bax and Bim was increased after treatment with CWP. Our data indicates CWP's therapeutic potential for MM cells.     

Human Vdelta1 gamma-delta T cells exert potent specific cytotoxicity against primary multiple myeloma cells

Abstract Background aims. Human gamma-delta (γδ) T cells are potent effector lymphocytes of innate immunity involved in anti-tumor immune surveillance. However, the Vδ1 γδ T-cell subset targeting multiple myeloma (MM) has not previously been investigated. Methods. Vδ1 T cells were purified from peripheral blood mononuclear cells of healthy donors and patients with MM by immunomagnetic sorting and expanded with phytohemagglutinin (PHA) together with interleukin (IL)-2 in the presence of allogeneic feeders. Vδ1 T cells were phenotyped by flow cytometry and used in a 4-h flow cytometric cytotoxicity assay. Cytokine release and blocking studies were performed. Primary myeloma cells were purified from MM patients' bone marrow aspirates. Results. Vδ1 T cells expanded from healthy donors displayed prominent cytotoxicity by specific lysis against patients' CD38 (+) CD138 (+) bone marrow-derived plasma cells. Vδ1 T cells isolated from MM patients showed equally significant killing of myeloma cells as Vδ1 T cells from normal donors. Vδ1 T cells showed similarly potent cytotoxicity against myeloma cell lines U266 and RPMI8226 and plasma cell leukemia ARH77 in a dose-dependent manner. The interferon (IFN)-γ secretion and Vδ1 T-cell cytotoxicity against myeloma cells was mediated in part through the T-cell receptor (TCR) in addition to involvement of Natural killer-G2D molecule (NKG2D), DNAX accessory molecule-1 (DNAM-1), intracellular cell adhesion molecule (ICAM)-1, CD3 and CD2 receptors. In addition, Vδ1 T cells were shown to exert anti-myeloma activity equal to that of Vδ2 T cells. Conclusions. We have shown for the first time that Vδ1 T cells are highly myeloma-reactive and have therefore established Vδ1 γδ T cells as a potential candidate for a novel tumor immunotherapy.     

In vitro sensitization of human lymphocytes to a myeloma cell-related antigen

Peripheral blood lymphocytes from normal human donors were cocultivated with cells from two established human multiple myeloma cell lines, RPMI 8226 and K-737, and with lymphoblastoid cells from a third B cell line, RAMM. After a comparison of three methods of lymphocyte sensitization, a 6-day incubation protocol with equal numbers of normal lymphocytes and mitomycin C-treated tumor cells was selected. Cells from the RPMI 8226 myeloma line stimulated the differentiation of lymphocytes into cytotoxic effector cells as measured by ⁵¹Cr release from labeled target cells. The RPMI 8226-sensitized lymphocytes were cytotoxic for myeloma cells (RPMI 8226 and K-737) and for lymphoblastoid cells (RAMM) but not for cells from human lung tumor lines (A549, A427, MB9812), a breast carcinoma line (ALAB), a normal diploid fibroblast line (HSBP), or normal lymphocytes.     

Improved antitumoral properties of pure antiestrogen RU 58668-loaded liposomes in multiple myeloma

In most of multiple myeloma (MM) cells, the "pure" antiestrogen (AE) RU 58668 (RU) induced either a G1-arrest (LP-1, OPM-2, NCI-H929, U266 cells) or apoptosis (RPMI 8226 cells). In RPMI 8226 cells, RU activates a caspase-dependent cell death pathway leading to the release of cytochrome c, the decrease of the essential MM survival factor Mcl-1, the cleavage of Bid and the activation of caspases-3 and -8. Incorporation of RU in pegylated cholesterol-containing liposomes allowed a controlled RU release, improving its anti-proliferative and apoptotic effects in cells. In RPMI 8226 xenografts, i.v. injected RU-liposomes but not free RU, exhibited antitumor activity. In vivo, RU-liposomes triggered the mitochondrial death pathway, concomitantly with a down-regulation of Mcl-1 and Bid cleavage. The decrease of CD34 immunoreactivity indicated a reduction of angiogenesis. The decrease of VEGF secretion in vitro supported a direct effect of RU on angiogenesis. These pro-apoptotic and antiangiogenic effects were explained by a prolonged exposure to the drug and to the endocytosis capacity of liposomes which might increase RU uptake and bypass a membrane export of free RU. Thus, these combined enhanced activities of RU-liposomes support that such a delivery of an AE may constitute a strategy of benefit for MM treatment.     

Bortezomib Reduces the Tumorigenicity of Multiple Myeloma via Downregulation of Upregulated Targets in Clonogenic Side Population Cells

Side population (SP) cells in cancers, including multiple myeloma, exhibit tumor-initiating characteristics. In the present study, we isolated SP cells from human myeloma cell lines and primary tumors to detect potential therapeutic targets specifically expressed in SP cells. We found that SP cells from myeloma cell lines (RPMI 8226, AMO1, KMS-12-BM, KMS-11) express CD138 and that non-SP cells include a CD138-negative population. Serial transplantation of SP and non-SP cells into NOD/Shi-scid IL-2γnul mice revealed that clonogenic myeloma SP cells are highly tumorigenic and possess a capacity for self-renewal. Gene expression analysis showed that SP cells from five MM cell lines (RPMI 8226, AMO1, KMS-12-BM, KMS-11, JJN3) express genes involved in the cell cycle and mitosis (e.g., CCNB1, CDC25C, CDC2, BIRC5, CENPE, SKA1, AURKB, KIFs, TOP2A, ASPM), polycomb (e.g., EZH2, EPC1) and ubiquitin-proteasome (e.g., UBE2D3, UBE3C, PSMA5) more strongly than do non-SP cells. Moreover, CCNB1, AURKB, EZH2 and PSMA5 were also upregulated in the SPs from eight primary myeloma samples. On that basis, we used an aurora kinase inhibitor (VX-680) and a proteasome inhibitor (bortezomib) with RPMI 8226 and AMO1 cells to determine whether these agents could be used to selectively target the myeloma SP. We found that both these drugs reduced the SP fraction, though bortezomib did so more effectively than VX-680 due to its ability to reduce levels of both phospho-histone H3 (p-hist. H3) and EZH2; VX-680 reduced only p-hist. H3. This is the first report to show that certain oncogenes are specifically expressed in the myeloma SP, and that bortezomib effectively downregulates expression of their products. Our approach may be useful for screening new agents with which to target a cell population possessing strong tumor initiating potential in multiple myeloma.     

硼替佐米增强P16蛋白表达诱导骨髓瘤细胞衰老

目的：研究蛋白酶体抑制剂硼替佐米诱导骨髓瘤RPMI8226、MMH929细胞衰老作用,并进一步探讨其作用机制。方法：硼替佐米0.1-100nmol/L处理骨髓瘤RPMI8226、MMH929细胞48、72h,MTT法检测细胞存活率、药物IC50值。选择药物IC50值1/10剂量处理骨髓瘤RPMI8226、MMH929细胞0、24、48H后检测衰老相关β-半乳糖苷酶染色率。流式细胞术检测细胞周期情况及凋亡率。Western-blot检测相关蛋白表达。结果：硼替佐米处理骨髓瘤细胞RPMI8226、MMH929后48小时IC50值：RPMI8226：19.05 nmol/L,MMH929：18.45nmol/L。以硼替佐米2 nmol/L处理骨髓瘤RPMI8226、MMH929细胞0、24、48H后发现β-半乳糖苷酶染色率、细胞G0/G1期比例明显上升与药物作用时间呈正相关,Western-blot检测细胞周期调控蛋白发现P53、PTEN蛋白无变化,P16蛋白与药物作用时间正相关。结论：硼替佐米通过增强P16蛋白表达诱导骨髓瘤细胞RPMI8226、H929衰老。     

Karyotypic variability of human multiple myeloma cell lines

Cytogenetic analysis of human multiple myeloma (MM) cell lines L363, Karpas 707, RPMI 8226, and U-266 was carried out. During long-term existence in vitro, the number of chromosomes in the cell lines was shown to be preserved at the near-diploid level (L363, Karpas 707, U-266) or to increase up to the hypotriploid level (RPMI 8226). There were complexly rearranged karyotypes with abnormalities of chromosomes of all pairs in all cell lines; however, no identical chromosomal translocations have been revealed. Loci of chromosomes involved in structural rearrangements in these cell lines often coincided with sites of DNA copy number imbalances characteristic for MM in vivo. Distinct types of the karyotypic structure of cell populations differing in the combination of cells with the main and additional structural variants of karyotype and of cells with nonclonal chromosome rearrangements were found in MM cell lines. In general, the karyotypic variability of the MM cell lines corresponds to the dynamics of karyotype of myeloma cells in vivo and, hence, has a tumor-specific character.     

P2X7 receptor activation mediates organic cation uptake into human myeloid leukaemic KG-1 cells

The P2X7 purinergic receptor is an ATP-gated cation channel with an emerging role in neoplasia. In this study we demonstrate that the human KG-1 cell line, a model of acute myelogenous leukaemia, expresses functional P2X7. RT-PCR and immunochemical techniques demonstrated the presence of P2X7 mRNA and protein respectively in KG-l cells, as well as in positive control multiple myeloma RPMI 8226 cells. Flow cytometric measurements demonstrated that ATP induced ethidium⁺ uptake into KG-l cells suspended in sucrose medium (EC₅₀ of ∼3 μM), but not into cells in NaCl medium. In contrast, ATP induced ethidium⁺ uptake into RPMI 8226 cells suspended in either sucrose or NaCl medium (EC₅₀ of ∼3 or ∼99 μM, respectively), as well as into RPMI 8226 cells in KCl medium (EC₅₀ of ∼18 μM). BzATP and to a lesser extent ATPγS and αβ-methylene ATP, but not ADP or UTP, also induced ethidium⁺ uptake into KG-1 cells. ATP-induced ethidium⁺ uptake was completely impaired by the P2X7 antagonists, AZ10606120 and A-438079. ATP-induced ethidium⁺ uptake was also impaired by probenecid but not by carbenoxolone, both pannexin-1 antagonists. ATP induced YO-PRO-1²⁺ and propidium²⁺ uptake into KG-1 cells. Finally, sequencing of full-length P2X7 cDNA identified several single nucleotide polymorphisms (SNPs) in KG-1 cells including H155Y, A348T, T357S and Q460R. RPMI 8226 cells contained A348T, A433V and H521Q SNPs. In conclusion, the KG-1 cell line expresses functional P2X7. This cell line may help elucidate the signalling pathways involved in P2X7-induced survival and invasiveness of myeloid leukaemic cells.     

Design,synthesis, in vitro and in vivo evaluation,and structure-activity relationship (SAR) discussion of novel dipeptidyl boronic acid proteasome inhibitors as orally available anti-cancer agents for the treatment of multiple myeloma and mechanism studies

A series of novel dipeptidyl boronic acid inhibitors of 20S proteasome were designed and synthesized. Aliphatic groups at R¹ position were designed for the first time to fully understand the SAR (structure–activity relationship). Among the screened compounds, novel inhibitor 5c inhibited the CT-L (chymotrypsin-like) activity with IC₅₀ of 8.21?nM and the MM (multiple myeloma) cells RPMI8226, U266B and ARH77 proliferations with the IC₅₀ of 8.99, 6.75 and 9.10?nM, respectively, which showed similar in vitro activities compared with the compound MLN2238 (biologically active form of marketed MLN9708). To investigate the oral availability, compound 5c was esterified to its prodrug 6a with the enzymatic IC₅₀ of 6.74?nM and RPMI8226, U266B and ARH77 cell proliferations IC₅₀ of 2.59, 4.32 and 3.68?nM, respectively. Furthermore, prodrug 6a exhibited good pharmacokinetic properties with oral bioavailability of 24.9%, similar with MLN9708 (27.8%). Moreover, compound 6a showed good microsomal stabilities and displayed stronger in vivo anticancer efficacy than MLN9708 in the human ARH77 xenograft mouse model. Finally, cell cycle results showed that compound 6a had a significant inhibitory effect on CT-L and inhibited cell cycle progression at the G2M stage.     

The novel retinoid AHPN/CD437 induces a rapid but incomplete apoptotic response in human myeloma cells

The synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN/CD437) appears to possess an apoptotic activity superior to classical retinoids in vitro as in vivo. Numerous studies have shown that CD437-induced apoptosis is independent of its nuclear receptor activity, suggesting that CD437 might have a unique mechanism of action. The purpose of this study was to compare CD437- and all-trans retinoic acid (atRA)-induced cell death. CD437 provoked a rapid apoptotic phenotype immediately followed by secondary necrosis in RPMI 8226, U266 and L363 human myeloma cell lines. Nuclear apoptotic features were observed upon both CD437 and atRA treatments. In contrast, membrane blebbing and the subsequent formation of apoptotic bodies, a classical apoptotic event, was only observed upon atRA treatment. In addition, CD437, contrary to atRA, was unable to induce tissue transglutaminase (tTG), an intracellular enzyme involved in the formation of cross-linked protein polymers contributing to apoptotic morphological changes. Taken together, these data suggest that CD437 induces rapid but incomplete apoptotic phenotype in human myeloma cells.     

雷公藤内酯醇对多发性骨髓瘤细胞凋亡及组蛋白H3K4甲基化的影响

目的：探讨雷公藤内酯醇（TPL）对多发性骨髓瘤RPMI8226细胞增殖、凋亡和组蛋白H3K4甲基化的影响。方法：以人多发性骨髓瘤细胞株RPMI8226为研究对象,在不同浓度（10、20、40、80、160 nmol/L） TPL中共培养不同时间（24 h、48 h、72 h）后,采用噻唑蓝（MTT）法检测细胞增殖活性;流式细胞术检测细胞凋亡和细胞周期;Western blot法检测组蛋白H3K4me2、H3K4me3的甲基化状态,实时荧光定量RT-PCR分析组蛋白甲基化酶SMYD3和组蛋白去甲基化酶LSD1的表达水平。结果：TPL对RPMI8226细胞有明显的增殖抑制作用,呈剂量和时间依赖性（P<0.05）;TPL对RPMI8226细胞有明显诱导凋亡的作用,并且随着TPL作用浓度的增加,细胞凋亡比例逐渐增加（P<0.05）;同时TPL还可以诱导RPMI8226细胞周期阻滞于G₂/M期;TPL以浓度依赖性降低组蛋白H3K4me2、H3K4me3的甲基化水平（P<0.05,P<0.01）,并抑制SMYD3和上调LSD1的表达（P<0.05）。结论：TPL可抑制RPMI8226细胞增殖、引起细胞周期阻滞于G₂/M期,并诱导其凋亡;通过抑制组蛋白甲基化酶SMYD3和增强组蛋白去甲基化酶LSD1的表达,降低组蛋白H3K4me3和H3K4me2的甲基化水平,这可能是TPL诱导多发性骨髓瘤细胞凋亡和抗肿瘤作用的机制之一。     

Involvement of apoptosis and autophagy in the death of RPMI 8226 multiple myeloma cells by two enantiomeric sigma receptor ligands

Over-expression of σ receptors by many tumor cell lines makes ligands for these receptors attractive as potential chemotherapeutic drugs. Enantiomeric piperazines (S)-4 and (R)-4 were prepared as potential σ-receptor ligands in a chiral pool synthesis starting from (S)- and (R)-aspartate. Both compounds showed high affinities for the σ₁ and σ₂ receptors. In the human multiple myeloma cell line RPMI 8226, a line expressing high levels of σ receptors, both compounds inhibited cell proliferation with IC₅₀ values in the low μM range. No chiral differentiation between either the σ receptor binding affinity or the cytotoxicity of the two enantiomers was observed. Both compounds induced apoptosis, which was evidenced by nuclear condensation, binding of annexin-V to phosphatidylserine in the outer leaf of the cell membrane, cleavage products of poly(ADP-ribose) polymerase-1 (PARP-1) and caspase-8 as well as the expression of bcl₂ family members bax, bad and bid. However, apoptosis appeared to be caspase independent. Increased levels of the phosphorylated form of the microtubule associated protein light chain 3-II (LC3-II), an autophagosome marker, gave evidence that both compounds induced autophagy. However, further data (e.g., treatment with wortmannin) indicate that autophagy is incomplete and not cytoprotective. Lipid peroxidation (LPO) was observed in RPMI 8226 cells treated with the two compounds, and the lipid antioxidant α-tocopherol attenuated LPO. Interestingly, α-tocopherol reduced significantly both apoptosis and autophagy induced by the compounds. These results provide evidence that, by initiating LPO and changes in mitochondrial membrane potential, both compounds induce apoptosis and autophagy in RPMI 8226 cells.