Multiple myeloma regression mediated by bruceantin.

PURPOSE: Bruceantin has been shown to induce cell differentiation in a number of leukemia and lymphoma cell lines. It also down-regulated c-MYC, suggesting a correlation of down-regulation with induction of cell differentiation or cell death. In the present study, we focused on multiple myeloma, using the RPMI 8226 cell line as a model. EXPERIMENTAL DESIGN: The effects of bruceantin on c-MYC levels and apoptosis were examined by immunoblotting, 4',6-diamidino-2-phenylindole staining, evaluation of caspase-like activity, and 3,3'-dihexyloxacarbocyanine iodide staining. The potential of bruceantin to inhibit primary tumor growth was assessed with RPMI 8226 xenografts in SCID mice, and apoptosis in the tumors was evaluated by the terminal deoxynucleotidyl transferase-mediated nick end labeling assay. RESULTS: c-MYC was strongly down-regulated in cultured RPMI 8226 cells by treatment with bruceantin for 24 h. With U266 and H929 cells, bruceantin did not regulate c-MYC in this manner. Apoptosis was induced in the three cell lines. In RPMI 8226 cells, apoptosis occurred through proteolytic processing of procaspases and degradation of poly(ADP-ribose) polymerase. The mitochondrial pathway was also involved. Because RPMI 8226 cells were the most sensitive, they were used in a xenograft model. Bruceantin treatment (2.5-5 mg/kg) resulted in a significant regression of tumors without overt toxicity. Apoptosis was significantly elevated in tumors derived from animals treated with bruceantin (37%) as compared with the control tumors (14%). CONCLUSIONS: Bruceantin interferes with the growth of RPMI 8226 cells in cell culture and xenograft models. These results suggest that bruceantin should be reinvestigated for clinical efficacy against multiple myeloma and other hematological malignancies.     

FANCF基因沉默增强多药耐药多发性骨髓瘤细胞株RPMI8226/R对马法兰的敏感性

目的:使用小片段干扰RNA（siRNA）沉默FANCF基因表达,观察其对FA/BRCA途径的影响及对多药耐药多发性骨髓瘤细胞株RPMI8226/R对交联剂马法兰的敏感性变化。方法:FANCF siRNA瞬时转染多药耐药细胞株RPMI8226/R,采用CCK-8法检测转染前后细胞对马法兰的敏感性变化,RT-PCR,western-blot法检测FA/BRCA途径中FANCF、FANCD2、BRCA2基因表达量的改变,彗星实验检测DNA损伤,流式细胞仪检测细胞凋亡率。结果:FANCF siRNA转染耐药骨髓瘤细胞后,对马法兰的IC50值由17.29μmol/L降至4.88μmol/L;western-blot检测出瞬时转染FANCF siRNA后FA/BRCA途径中FANCF、FANCD2、BRCA2 蛋白表达量明显较转染前减低;彗星实验检测出转染后耐药细胞的DNA损伤增多;转染后马法兰诱导细胞凋亡率由（77.67±0.62）%升高至（88.37±0.25）%,提示FANCF基因沉默可使耐药RPMI8226/R细胞对化疗药物马法兰重新恢复敏感性。结论:FANCF基因沉默可抑制FA/BRCA途径中FANCD2及下游BRCA2基因表达,从而阻断FA/BRCA途径介导的DNA修复,增强马法兰对多药耐药骨髓瘤细胞株RPMI8226/R的细胞毒性,增加细胞凋亡率,为解决临床耐药问题提供一个新的思路。     

Selective cyclooxygenase 2 inhibitor NS-398 induces apoptosis in myeloma cells via a Bcl-2 independent pathway

NS-398, a selective inhibitor of cyclooxygenase 2 (COX-2), has been reported to inhibit growth and induce apoptosis in several cancer cell lines that overexpress COX-2. However it has not been extensively studied in multiple myeloma (MM). Here, we studied the effects of COX-2 inhibitors on MM cell lines and primary myeloma patient cells. We investigated the effects of NS-398 on proliferation and apoptosis in three myeloma cell lines (PCM6, U266 and RPMI8226) and isolated CD138-positive cells from MM patients. Furthermore, the combined effects of NS-398 plus dexamethasone (Dex) or thalidomide (Thal) were investigated. All myeloma cell lines express COX-2. NS-398 inhibited growth and induced apoptosis in PCM6, RPMI8226 and CD138-positive MM cells in a time- and dose-dependent manner. At low concentrations (10 microM), NS-398 primarily induced growth arrest without affecting cell viability, but at higher concentrations (over 25 microM), apoptosis was induced. During the process of apoptosis, the number of Fas-positive cells increased. Downstream signals of Fas, such as caspase 8, 3 and 9, were also activated. On the other hand, protein levels of the Bcl-2 family did not change, although mitochondrial transmembrane potential ((Delta)(psi)m) was decreased. Combined incubation with Dex or Thal enhanced NS-398-induced growth inhibition and apoptosis in RPMI8226 cells. The combined effect of Dex was more potent than that of Thal. Our findings suggests that COX-2 plays an important role in regulation of apoptosis in myeloma cells, and COX-2 inhibitors might serve as an effective tool for future chemoprevention and/or treatment of myeloma.     

Targeting SIRT1 to inhibit the proliferation of multiple myeloma cells

Multiple myeloma (MM) is the second most common hematopoietic malignancy and remains an incurable disease. Thus, novel drugs and therapeutic methods are required for patients with MM. The present study aimed to investigate the effect of sirtuin 1 (SIRT1) inhibitor cambinol on the proliferation and apoptosis of myeloma cell lines, RPMI8226 and U266. Moreover, the present study evaluated the underlying molecular mechanisms of proliferation inhibition and apoptosis induced by cambinol. A Cell Counting Kit-8 assay was used to measure the viability of RPMI8226 and U266 cells treated with cambinol. Apoptosis and the cell cycle were analyzed via flow cytometry. The expression levels of caspase-3, poly(ADP-ribose) polymerase 1 (PARP), p53, acetylated p53 (Ac-p53), Bcl-2, cyclin D1 and p21 were detected in cells treated with cambinol using western blot analysis. The results demonstrated that cambinol inhibited the proliferation of RPMI8226 and U266 cells in a time- and dose-dependent manner. Increased apoptosis and G₁ cell cycle arrest, together with enhanced procaspase-3 degradation and PARP cleavage were identified in cambinol-treated cells compared with controls. Western blotting results also revealed the upregulation of p53 acetylation and p21, as well as the downregulation of Bcl-2 and cyclin D1 in cells treated with cambinol. In conclusion, the present results suggest that cambinol inhibits the proliferation and induces apoptosis in RPMI8226 and U266 cells by regulating acetylation of p53 via the targeting of SIRT1.     

Resveratrol inhibits myeloma cell growth, prevents osteoclast formation, and promotes osteoblast differentiation

Multiple myeloma is characterized by the accumulation of clonal malignant plasma cells in the bone marrow, which stimulates bone destruction by osteoclasts and reduces bone formation by osteoblasts. In turn, the changed bone microenvironment sustains survival of myeloma cells. Therefore, a challenge for treating multiple myeloma is discovering drugs targeting not only myeloma cells but also osteoclasts and osteoblasts. Because resveratrol (trans-3,4',5-trihydroxystilbene) is reported to display antitumor activities on a variety of human cancer cells, we investigated the effects of this natural compound on myeloma and bone cells. We found that resveratrol reduces dose-dependently the growth of myeloma cell lines (RPMI 8226 and OPM-2) by a mechanism involving cell apoptosis. In cultures of human primary monocytes, resveratrol inhibits dose-dependently receptor activator of nuclear factor-kappaB (NF-kappaB) ligand-induced formation of tartrate-resistant acid phosphatase (TRACP)-positive multinucleated cells, TRACP activity in the medium, up-regulation of cathepsin K gene expression, and bone resorption. These inhibitions are associated with a down-regulation of RANK expression at both mRNA and cell surface protein levels and a decrease of NFATc1 stimulation and NF-kappaB nuclear translocation, whereas the gene expression of c-fms, CD14, and CD11a is up-regulated. Finally, resveratrol promotes dose-dependently the expression of osteoblast markers like osteocalcin and osteopontin in human bone marrow mesenchymal stem cells (hMSC-TERT) and stimulates their response to 1,25(OH)2 vitamin D3 [1,25(OH)2D3]. Moreover, resveratrol up-regulates dose-dependently the expression of 1,25(OH)2D3 nuclear receptor. Taken together, these results suggest that resveratrol or its derivatives deserve attention as potential drugs for treating multiple myeloma.     

茶多酚诱导人多发性骨髓瘤RPMI8226细胞的凋亡及其作用机制

目的:研究茶多酚对人多发性骨髓瘤RPMI 8226细胞生长及凋亡的影响,并探讨其可能的作用机制.方法:通过MTT法、瑞氏染色法和FCM法分别检测不同浓度的茶多酚对RPMI 8226细胞增殖和凋亡的影响;FCM法检测对细胞线粒体膜电位的影响;蛋白质印迹法检测△caspase-3片段和凋亡相关蛋白bc1-2的表达;ELISA法检测对RPMI 8226细胞分泌细胞因子白细胞介素-6(interleukin-6,IL-6)水平的影响.结果:与对照组相比,茶多酚浓度129.6和259.1 μmol/L处理RPMI 8226细胞1～3 d后均能明显抑制细胞的增殖,且呈时间-剂量相关性;统计显示,24 h时的半数细胞抑制浓度(half inhibitory concentration,IC50)值为165.7μmol/L.茶多酚(129.6μmol/L)作用24 h后,RPMI 8226细胞出现典型的凋亡形态学改变;茶多酚(129.6和259.1 μmol/L)作用24 h后,RPMI 8226细胞凋亡率分别为(24.6±2.3)％和(43.5±3.9)％,与对照组比较差异均有统计学意义;细胞的线粒体膜电位降低,活性△caspase-3片段产生,bc1-2蛋白表达水平下降,IL-6分泌水平降低.结论:茶多酚能抑制RPMI 8226细胞增殖并诱导其凋亡,其作用机制可能与产生活性△caspase-3片段,抑制bc1-2蛋白表达,降低细胞分泌IL-6水平相关.     

Habb-e-Asgandh Suppresses Cell Proliferation and Induces Apoptosis through Mitochondria Dysfunction in Multiple Myeloma Cells (RPMI8226)

Objective: This study was conducted to assess the anti-neoplastic properties of Habb-e-Asgandh in multiple myeloma cells (RPMI8226). Methods: Multiple myeloma cells (RPMI8226) were cultured according to the ATCC’s instruction. The anti-proliferative effect of HeA was assessed by MTT assay and proliferating cellnuclear antigen (PCNA) activity. Cell cycle analysis, cellular apoptosis, and mitochondria membrane potential analysis was done by flow cytometry. Total antioxidants, migratory potential, angiogenesis and inflammatory biomarkers were also estimated after treatment of RPMI8226 with HeA. Results: LD30 and LD50 dose of HeA was 0.3mg/ml and 0.5mg/ml respectively determined by MTT assay and also confirmed by a reduced PCNA activity. Cell cycle analysis of RPMI8226 cells revealed that sub-G0/G1 phase increases upon treatment with HeA alone or in combination with lenalidomide. Annexin V-FITC/PI is used to detect early apoptosis, late apoptosis and necrotic cells and results showed that percentage of apoptotic cells increased in RPMI8226 cells after treatment with HeA. Also, HeA induces loss of mitochondria membrane potential (MMP) in MM cells in-vitro as measured by cationic JC1 dye staining. Upon treatment, the abnormal overexpression of oncogenic protein, AKT serine/threonine kinase has also been reduced. Furthermore, anti-oxidants level also increased while migratory potential, angiogenesis and inflammation decreased in multiple myeloma cell line upon treatment with HeA. Conclusion: Collectively, our results demonstrated that integrative therapy of habb-e-asgandh efficiently eliminates the need to use higher dose of lenalidomide for multiple myeloma treatment.     

Apomine, an inhibitor of HMG-CoA-reductase, promotes apoptosis of myeloma cells in vitro and is associated with a modulation of myeloma in vivo

Apomine, a novel 1,1 bisphosphonate ester, increases the rate of degradation of HMG-CoA reductase, inhibiting the mevalonate pathway and thereby blocking cholesterol biosynthesis. We have investigated whether Apomine can induce myeloma cell apoptosis in vitro and modulate myeloma disease in vivo. Apomine induced a dose-dependent increase in apoptosis in NCI H929, RPMI 8226 and JJN-3 human myeloma cells. Apomine, unlike the bisphosphonate, alendronate, had no measurable effect on osteoclastic bone resorption in vitro. To investigate the effect of Apomine in vivo, 5T2MM murine myeloma cells were injected into C57BL/KaLwRij mice. After 8 weeks all animals had a serum paraprotein and were treated with Apomine (200 mg/kg), or vehicle, for 4 weeks. Animals injected with 5T2MM cells and treated with vehicle developed osteolytic bone lesions, reduced cancellous bone area, decreased bone mineral density (BMD) and increased osteoclast number. Apomine caused a decrease in serum paraprotein and a decrease in tumor burden. Apomine inhibited the development of osteolytic lesions and prevented the tumor-induced decreases in BMD. Apomine had no effect on osteoclast number in contrast to what had been seen previously with the bisphosphonate, zoledronic acid, suggesting that these are direct effects of Apomine on myeloma cells. This demonstrates that Apomine is able to promote myeloma cell apoptosis in vitro and inhibit the development of multiple myeloma and lytic bone disease in vivo. The use of bisphosphonate esters such as Apomine represents a novel therapeutic approach in the treatment of myeloma and, indirectly, the associated bone disease.     

An osteoprotegerin-like peptidomimetic inhibits osteoclastic bone resorption and osteolytic bone disease in myeloma

Multiple myeloma is a B-cell malignancy characterized by the uncontrolled growth of plasma cells in the bone marrow and the development of osteolytic bone disease. Myeloma cells express the receptor activator of nuclear factor kappaB ligand (RANKL), induce RANKL expression in the bone marrow, and down-regulate expression of the decoy receptor osteoprotegerin, thereby promoting bone resorption. Targeting this system in myeloma has clear therapeutic potential. However, osteoprotegerin also binds tumor necrosis factor-related apoptosis inducing ligand (TRAIL) and prevents TRAIL-induced apoptosis of myeloma cells. Whether or not osteoprotegerin can bind TRAIL and prevent apoptosis in vivo and the relative importance of osteoprotegerin binding to TRAIL and RANKL are unclear. In the present study, we have investigated the ability of an osteoprotegerin-like peptidomimetic (OP3-4), designed to block the RANKL/RANK interaction, to inhibit osteoclastic bone resorption and TRAIL-induced apoptosis in vitro and myeloma bone disease in vivo. OP3-4 inhibited osteoclast formation (P < 0.01) and bone resorption (P < 0.01) in vitro. However, OP3-4 had no effect on TRAIL-induced apoptosis of RPMI 8226 myeloma cells. Treatment of 5T2MM myeloma-bearing mice with OP3-4 decreased osteoclast number and the proportion of bone surface covered by osteoclasts (P < 0.05). Treatment also prevented the tumor-induced decrease in cancellous bone area and the development of osteolytic lesions (P < 0.05). OP3-4 also reduced tumor burden when compared with the control (P < 0.05). These data suggest that OP3-4 and the selective inhibition of RANKL, but not TRAIL activity, are effective in preventing myeloma bone disease and offer a novel therapeutic approach to treating this aspect of myeloma. [Cancer Res 2007;67(1):202-8].     

Glucocorticoid effects on myeloma cells in culture: correlation of growth inhibition with induction of glucocorticoid receptor messenger RNA

Glucocorticoids are widely used for the treatment of multiple myeloma. To investigate the direct actions of glucocorticoids on myeloma cells, we have used three cell lines of human multiple myeloma, OPM-1, OPM-2, and RPMI 8226. When growth curves of these cells were examined, OPM-1 cells were resistant, while OPM-2 were sensitive to dexamethasone (DEX). In cultures of OPM-2 cells, addition of DEX led to virtual cessation of growth, with only 16% of the residual cells viable after 4 days. RPMI 8226 appeared to be slightly sensitive, showing some slowing of growth for several days in DEX, with later recovery. Viabilities of OPM-1 and RPMI 8226 cells were not affected. Secretion of immunoglobulin (Ig-lambda) was also partially suppressed, by 30% in OPM-2 and 14% in OPM-1. No significant suppression was observed in RPMI 8226. To explore the mechanism of these differential responses to the steroid, glucocorticoid receptor (GR) was examined. Binding assays showed high affinity binding sites in all three cell lines: 64 +/- 11 fmol/10(6) cells in OPM-1, 78 +/- 14 in OPM-2, and 62 +/- 16 in RPMI 8226. Nuclear transfer of GR and DNA-cellulose binding after heat activation appeared similar in all three cell lines. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cytosol proteins labeled with [3H]dexamethasone mesylate showed a GR of Mr 95,000 in all three. When GR mRNA was studied in these cells, all of them had GR mRNA of approximately 7 kilobases, but OPM-2 and RPMI 8226 had 3 times more GR mRNA than OPM-1. OPM-2 GR mRNA was induced 2-fold by DEX treatment at 5 x 10(-9) M or greater. OPM-1 GR mRNA was much less sensitive, with no response at less than 10(-6) M DEX and only 1.5-fold induction at that concentration. These results demonstrate that some myeloma cells can be killed by a direct action of glucocorticoids. The quantity and affinity of GR in the cells were not predictive of this response. Therefore, we propose that the resistance of OPM-1 and the relative resistance of RPMI 8226 to glucocorticoid inhibition of cell growth is by post-receptor mechanisms. The high sensitivity of induction of GR mRNA in OPM-2 may correlate with glucocorticoid-evoked cell kill.     

依托泊苷对人骨髓瘤细胞 RPMI8226增殖与凋亡的影响

目的：探讨依托泊苷对人骨髓瘤细胞 RPMI8226的增殖抑制作用及其分子机制。方法：将依托泊苷作用于骨髓瘤细胞,用 MTT 法检测细胞增殖抑制率,光学显微镜和电子显微镜观察细胞形态及超微结构变化,流式细胞术检测细胞凋亡率和细胞周期分布,半定量 RT －PCR 检测 Bax 及 Caspase －3 mRNA 表达量的变化,Western blot 检测 Bax 及 Caspase －3蛋白表达量变化。结果：依托泊苷可抑制 RPMI8226细胞增殖,抑制率呈时间（r ＝0．926）和浓度（r ＝0．938）依赖性增强。24h 后在光学显微镜下可见依托泊苷组细胞数量减少,排列紊乱,细胞形态变得不规则,可见凋亡细胞及坏死细胞。48h 电子显微镜下可见细胞典型凋亡改变,凋亡小体形成。流式细胞术检测结果显示,依托泊苷组 RPMI8226细胞凋亡率明显增高（P ＜0．05）;依托泊苷作用48h 后将 RPMI8226细胞阻滞于 S 期（P ＜0．05）;依托泊苷作用48h,Bax、Caspase －3 mRNA 及蛋白表达量增加（P ＜0．05）。结论：依托泊苷可诱导 RPMI8226细胞凋亡,可能与细胞周期阻滞,激活细胞内、外源性凋亡通路有关。     

Triptolide inhibits transcription factor NF-kappaB and induces apoptosis of multiple myeloma cells

Triptolide has been reported to be effective in the treatment of auto-immune diseases. This study investigates the cytotoxic function of triptolide on multiple myeloma (MM) cells. We found that triptolide inhibited the proliferation of both RPMI8226 and U266 cells in a dose-dependent manner (10-80 ng/mL). Triptolide induced apoptosis in MM cells through activation of the cystein protease caspase 8, 9 and 3, and subsequent cleavage of the DNA repair enzyme poly (ADP-ribose) polymerase. Apoptosis was confirmed with cell-cycle analysis and annexin V staining. Moreover, triptolide down-regulated nuclear factor (NF)-kappaB activity in MM cell lines. In addition, triptolide also induced chemosensitivity to doxorubicin and suppressed cell proliferation of fresh MM cells. Therefore, triptolide appears to be a potent inducer of apoptosis in myeloma cells, and might have some benefit in the treatment of myeloma patients.     

三氧化二砷诱导骨髓瘤细胞凋亡机制研究

目的 了解三氧化二砷（As2O3)诱导多发性骨髓瘤细胞（MM）凋亡的可能机制及其与维甲酸和干扰素的相互作用。方法 联合应用As2O3和巯基还原剂（DTT）、谷胱甘肽耗褐剂（BSO）、全反式维甲酸（ATRA）或干扰素（ＩＦＮ－α）处理MM细胞系RPM18226和U266细胞;应用台盼蓝拒染法计数细胞活力,经细胞形态学和流式细胞仪等判定细胞凋亡的程度;通过测定细胞内荧光染料Rhodamine123的色强度分析线粒体跨膜电位（△ψm)。结果 BSO可加强As2O3诱导的RPM18226和U266细胞线粒体△ψm下降和凋亡,而DTT则有部分拮抗的作用,ATRA诱导RPMI8226细胞闻过则喜亡,但它和As2O3之间无协同效尖,ATRA并不诱导U266细胞凋亡。此外,INF-α,既不抑制RPMIS226和U266细胞的生长和活力,也不改变As2O3对这些细胞的作用。结论 As2O3诱导多发性骨髓瘤细胞凋亡和巯基有关,但ATRA干扰素和As2O3无协同效应。     

Canonical nuclear factor kappaB pathway inhibition blocks myeloma cell growth and induces apoptosis in strong synergy with TRAIL.

PURPOSE: Intrinsic activation of nuclear factor kappaB (NF-kappaB) characterizes various hematologic malignancies. In this study, we specifically address the role of NF-kappaB blockade in mediated antimyeloma activity using the IkappaB kinase-2 pharmacologic inhibitor, AS602868. EXPERIMENTAL DESIGN: Human myeloma cell lines (n = 16) and primary myeloma cells (n = 10) were tested for their sensitivity to AS602868 in terms of proliferation and apoptosis. Both in vitro and in vivo experiments were conducted. Functional mechanisms regarding the apoptotic pathways triggered by AS602868 were studied. The potential proapoptotic synergy between AS602868 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was also evaluated. RESULTS: Our results show that AS602868 efficiently targeted the canonical NF-kappaB pathway in myeloma cells and potently inhibited their growth in inducing apoptosis through Bax and caspase-3 activation. AS602868 also induced apoptosis in primary myeloma cells even in the presence of bone marrow mononuclear cells. Moreover, the IkappaB kinase-2 inhibitor targeted the paracrine effect on the bone marrow environment. Indeed, it decreased the intrinsic and myeloma-induced secretion of interleukin-6 from bone marrow stromal cells. In addition, AS602868 inhibited myeloma cell growth in the MM.1S xenograft myeloma model. Of particular interest, AS602868 strongly increased myeloma sensitivity to TRAIL in blocking TRAIL-induced NF-kappaB activation and in decreasing the expression of antiapoptotic proteins such as cFLIP and cIAP-1/2. CONCLUSIONS: Taken together, our data point out the interest to inhibit the canonical NF-kappaB pathway in myeloma and clearly encourage clinical evaluation of novel therapies based on targeting NF-kappaB, especially in combination with TRAIL.     

三氧化二砷诱导人骨髓瘤细胞RPMI 8226凋亡的机制探讨

目的:探讨三氧化二砷（As2O3）诱导人骨髓瘤细胞RPMI 8226凋亡的机制。方法:不同浓度As2O3作用RPMI 8226细胞48h后,用MTT法计算细胞增殖抑制率。流式细胞仪检测1.0、2.0、5.0μmol/L As2O3干预RPMI 8226细胞48h后的凋亡率;透射电镜观察As2O3作用前后RPMI 8226细胞超微结构的变化;RT-PCR、Western Blot法检测As2O3作用前后Bcl-2及Caspase-3表达的变化。结果:不同浓度的As2O3对RPMI 8226细胞均有增殖抑制作用(P<0.05）。1.0、2.0、5.0μmol/L As2O3干预RPMI 8226细胞48h后,细胞凋亡率随As2O3浓度的增加而呈上升趋势,与对照组相比,差异具有统计学意义（P<0.05）;As2O3干预RPMI 8226细胞48h后,电镜下可见典型的凋亡小体;RT-PCR、Western Blot结果均显示上述浓度的As2O3干预RPMI 8226细胞48h后,Bcl-2 mRNA及蛋白表达下调（P<0.05）,Caspase-3 mRNA及蛋白表达上调（P<0.05）。结论:As2O3可能通过激活Caspase-3、下调Bcl-2的表达从而诱导RPMI 8226细胞凋亡。     

The novel retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphtalene carboxylic acid can trigger apoptosis through a mitochondrial pathway independent of the nucleus

The novel retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphtalene carboxylic acid (AHPN/CD437), a retinoic acid receptor (RAR)gamma activator, has been found to inhibit the growth and to induce apoptosis of a wide variety of malignant cell types including solid tumors and various leukemias. Interestingly, CD437 is able to induce apoptosis in some all-trans-retinoic acid (ATRA)-resistant models. In a number of experimental systems, the early apoptotic stage that precedes nuclear chromatinolysis consists in mitochondrial alterations, including a disruption of the inner mitochondrial transmembrane potential (delta(psi)m) mediated by the mitochondrial permeability transition (MPT). Similarly CD437 causes RPMI 8226, a human myeloma cell line, to undergo a rapid delta(psi)m disruption that precedes other apoptotic alterations such as the generation of reactive oxygen species and DNA fragmentation. The same sequence of events is observed during the CD437-induced apoptosis in L363, a RARgamma-negative human myeloma cell line, as well as RPMI 8226 cytoplasts (anucleate cells). Indeed, RPMI 8226 cells and cytoplasts manifest a similar degree in delta(psi)m loss, phosphatidylserine exposure, and caspase activation in response to CD437, which indicates that nuclear effects cannot account for the apoptogenic potential of CD437. The mitochondrial release of cytochrome c, the activation of caspases as well as nuclear signs of CD437-induced apoptosis are fully prevented by the MPT inhibitory compound cyclosporin A. Purified mitochondria can be directly induced to undergo MPT with CD437 but not with ATRA. In a cell-free in vitro system consisting of exposing mitochondrial supernatants to isolated nuclei, only supernatants from CD437-treated mitochondria provoke chromatin condensation, whereas supernatants from mitochondria treated with ATRA, or with the combination of CD437 and cyclosporin A, remain inactive. In conclusion, these results suggest that the rapid execution of CD437-induced apoptosis is a nucleus-independent (and probably RARgamma-independent) phenomenon involving mitochondria and MPT.     

黄连解毒汤抑制多发性骨髓瘤RPMI8226细胞增生及促凋亡作用的研究

 目的 研究中药黄连解毒汤（HLJDT）对人多发性骨髓瘤（MM）细胞RPMI8226增生和凋亡的作用。方法 以含10 ％胎牛血清的RPMI1640培养基常规培养RPMI8226细胞,分别以不同浓度的HLJDT作用不同的时间后,应用锥虫蓝拒染法测定细胞活力变化,MTT法检测细胞的增生变化;采用流式细胞术测定细胞周期的变化;应用流式细胞仪检测药物细胞的凋亡现象,荧光显微镜下观察AO／EB染色后的细胞凋亡形态变化;比色法检测半胱氨酸蛋白水解酶3（caspase3）活性变化。结果 与对照组相比,200～800 ng／ml浓度的HLJDT可明显抑制细胞增生影响（P＜0.01）,并呈时间和剂量依赖性;使细胞周期阻滞在G0／G1期细胞增多,S期细胞减少,并呈剂量依赖性改变; MM细胞凋亡百分率显著增高（P＜0.01）,免疫荧光显微镜下可见典型的凋亡细胞形态学变化;使caspase3活性增强,且呈剂量和时间依赖性（P＜0.01）。结论 HLJDT能有效抑制MM细胞RPMI8226增生,促其凋亡,G0／G1期细胞比例增加和S期细胞比例减少可能是原因之一;caspase3活性增强可能参与其凋亡过程,具体机制研究还有待完善。     

Production of growth factors by human myeloma cells

Using in vitro-growing myeloma cell lines, we studied the growth factors involved in human multiple myeloma, and particularly the potential of autocrine secretion and response to B-cell growth factor (BCGF) of RPMI 8226, the best-documented Epstein-Barr virus-negative human myeloma cell line. We found that three myeloma cell lines (RPMI 8226, U266, and IM9) produce an autostimulatory growth factor (AGF) and thus increase their own proliferation by 2- to 3-fold in cells cultured at low density. Optimal AGF production was obtained after 24 h of culture at a cell density ranging from 2.5 to 5 million cells/ml. The three myeloma cell lines produce type II BCGF, able to induce the proliferation of highly purified human peripheral blood B-cells, only after anti-mu activation. The BCGF produced by RPMI 8226 can be absorbed onto RPMI 8226 cells together with the RPMI 8226 AGF, and the two are copurified on gel filtration in a peak with an apparent molecular weight of 70,000. RPMI 8226 can be efficiently activated by human high molecular weight BCGF II (Mr 50,000) and less extensively by BCGF I (Mr 12,000). RPMI 8226 does not produce either detectable IL1 or interferons gamma and alpha and IL1 and gamma-IFN had no stimulating effect on RPMI 8226 proliferation. Our findings support the conclusion that RPMI 8226 produces a BCGF II working as an AGF.     

Green tea component, catechin, induces apoptosis of human malignant B cells via production of reactive oxygen species.

PURPOSE: Green tea polyphenol, (-)-epigallocatechin-3-gallate, has been shown to inhibit cellular proliferation and induce apoptosis of various cancer cells. The aim of this study was to investigate the possibility of (-)-epigallocatechin-3-gallate as a novel therapeutic agent for the patients with B-cell malignancies including multiple myeloma. EXPERIMENTAL DESIGN: We investigated the effects of (-)-epigallocatechin-3-gallate on the induction of apoptosis in HS-sultan as well as myeloma cells in vitro and further examined the molecular mechanisms of (-)-epigallocatechin-3-gallate-induced apoptosis. RESULTS: (-)-Epigallocatechin-3-gallate rapidly induced apoptotic cell death in various malignant B-cell lines in a dose- and time-dependent manner. (-)-Epigallocatechin-3-gallate-induced apoptosis was in association with the loss of mitochondrial transmembrane potentials (Deltapsim); the release of cytochrome c, Smac/DIABLO, and AIF from mitochondria into the cytosol; and the activation of caspase-3 and caspase-9. Elevation of intracellular reactive oxygen species (ROS) production was also shown during (-)-epigallocatechin-3-gallate-induced apoptosis of HS-sultan and RPMI8226 cells as well as fresh myeloma cells. Antioxidant, catalase, and Mn superoxide dismutase significantly reduced ROS production and (-)-epigallocatechin-3-gallate-induced apoptosis, suggesting that ROS plays a key role in (-)-epigallocatechin-3-gallate-induced apoptosis in B cells. Furthermore, a combination with arsenic trioxide (As2O3) and (-)-epigallocatechin-3-gallate significantly enhanced induction of apoptosis compared with As2O3 alone via decreased intracellular reduced glutathione levels and increased production of ROS. CONCLUSIONS: (-)-Epigallocatechin-3-gallate has potential as a novel therapeutic agent for patients with B-cell malignancies including multiple myeloma via induction of apoptosis mediated by modification of the redox system. In addition, (-)-epigallocatechin-3-gallate enhanced As2O3-induced apoptosis in human multiple myeloma cells.