Bone morphogenetic protein-4 inhibits proliferation and induces apoptosis of multiple myeloma cells

Bone morphogenetic proteins (BMPs) can be isolated from organic bone matrix and are able to initiate de novo cartilage and bone formation. Here it is shown that BMP-4 inhibited DNA synthesis in a dose-dependent manner in 3 IL-6-dependent multiple myeloma (MM) cell lines (OH-2, IH-1, and ANBL-6). In contrast, no effect on DNA synthesis was observed in 3 IL-6-independent MM cell lines (JJN-3, U266, and RPMI 8226). BMP-4 induced cell cycle growth arrest in the G(0)/G(1) phase in OH-2 and ANBL-6 cells but not in IH-1 cells. BMP-4 induced apoptosis in OH-2 and IH-1 cells, but not significantly in ANBL-6 cells. Furthermore, BMP-4 induced apoptosis in freshly isolated MM cells from 4 of 13 patients. In the OH-2 and ANBL-6 cell lines and in a patient sample, immunoblotting showed that BMP-4 down-regulated IL-6-induced tyrosine phosphorylation of Stat3, suggesting a mechanism for the apparent antagonism between IL-6 and BMP-4. BMP-4 or analogues may be attractive therapeutic agents in MM because of possible beneficial effects on both tumor burden and bone disease.     

LHRH-PE40对胃癌细胞增殖抑制和诱导凋亡的实验研究

目的 观察新型免疫毒素LHRH-PE40对胃癌细胞的抑制作用并探讨其作用机制。方法 将体外培养的胃癌细胞分别加入不同浓度的LHRH-PE40进行干预后，测定肿瘤细胞生长情况的变化，并应用流式细胞仪检测LHRH-PE40诱导肿瘤细胞凋亡情况。结果 免疫毒素LHRH-PE40对人胃癌细胞系BGC-823的增殖有明显的抑制作用，同时促进了细臆的凋亡。结论 免疫毒素LHRH-PE40对胃癌细胞的增殖状态有明显的抑制作用。     

帕瑞昔布对人胰腺癌细胞株BxPC-3、AsPC-1增殖凋亡的影响及其机制

目的:研究帕瑞昔布(parecoxib,PCB)对人胰腺癌细胞株BxPC-3和AsPC-1的增殖、凋亡及其可能的分子机制.方法:BxPC-3、AsPC-1细胞用不同浓度PCB的培养液孵育后,利用MTT法测定细胞活性,计算IC50值,TUNEL法检测处理后细胞的凋亡情况,RT-PCR验证相关蛋白的变化表达.结果:PCB对两种细胞生长呈时间和计量依赖性抑制;PCB处理后BxPC-3、AsPC-1两种细胞IC50值为:400.98μmol/L±10.78μmol/L、256.3μmol/L±2.98μmol/L;TUNEL法检测证明凋亡率增加;RT-PCR显示COX-2表达明显降低.结论:PCB可以抑制胰腺癌细胞增殖,并诱导其凋亡生长,其可能机制是通过抑制COX-2表达来实现的.     

Kininostatin, an angiogenic inhibitor, inhibits proliferation and induces apoptosis of human endothelial cells

We recently reported that domain 5 (D5) of high-molecular-weight kininogen inhibited critical steps required for angiogenesis. Thus, it was named kininostatin. To understand its mechanism of action, we further investigated the effects of D5 on basic fibroblast growth factor (bFGF)-induced endothelial cell proliferation and cell viability. We report here that D5-inhibited cell proliferation of human endothelial cells stimulated by bFGF was associated with a significant reduction of cyclin D1 expression, which is a critical component required for the transition from G(1) to S phase of the cell cycle. However, inhibition of cell proliferation by D5 was not due to an inhibition of extracellular signal-regulated protein kinase activity. Endothelial cells underwent apoptosis when cultured in a serum-free medium, which was prevented by bFGF. D5 reversed the protective effect of bFGF by 80%. Cells treated with D5 in the presence of bFGF showed typical morphological features of apoptosis, which was further confirmed by 2 additional assays: Hoechst 33258 cell staining and DNA fragmentation analysis. We conclude that the inhibition of endothelial cell proliferation and induction of apoptosis together represent a major contribution to the antiangiogenic activity of D5.     

Interleukin-15 blocks apoptosis and induces proliferation of the human myeloma cell line OH-2 and freshly isolated myeloma cells

The growth factor-dependent myeloma cell line OH-2, which has previously been shown to be responsive to interleukin (IL)-6, tumour necrosis factor (TNF)-alpha and lymphotoxin, was examined for response to other growth factors. Enhanced proliferation was found in the presence of IL-10, IL-15, IL-2 and insulin growth factor (IGF)-1. Proliferation was strongest in response to IL-6, intermediate and roughly equipotent in response to IL-15, IL-10 and TNF-alpha, and modest in response to IL-2 and IGF-1. IL-15 was synergistic with TNF-alpha, whereas combinations of IL-15 and the other cytokines were merely additive. IL-15-induced proliferation could not be blocked by neutralizing antibody against gp 130, the common transducer chain of IL-6 and related cytokines. IL-15 and IL-6 prevented apoptosis equally well, both better than TNF-alpha, IL-10, and IGF-1. In four out of six samples of purified primary cells, IL-15 and IL-6 induced proliferation. Furthermore, IL-15 mRNA was detected by RT-PCR in most myeloma cell lines and freshly isolated purified patient samples. IL-15 protein was detectable only in one out of about 20 tested cell supernatants from patients and myeloma cell lines. The OH-2 cell line is multi-responsive to cytokines and is a good system for the study of integration of cytokine signal transduction and growth control in myeloma. IL-15 represents a novel modality of growth regulation in myeloma.     

WWOX induces apoptosis and inhibits proliferation of human hepatoma cell line SMMC-7721

AIM: To investigate the effects of the WWOX gene on the human hepatic carcinoma cell line SMMC-7721.METHODS: Full-length WWOX cDNA was amplified from normal human liver tissues. Full-length cDNA was subcloned into pEGFP-N1, a eukaryotic expression vector. After introduction of the WWOX gene into cancer cells using liposomes, the WWOX protein level in the cells was detected through Western blotting. Cell growth rates were assessed by methyl thiazolyl tetrazolium (MTT) and colony formation assays. Cell cycle progression and cell apoptosis were measured by flow cytometry. The phosphorylated protein kinase B (AKT) and activated fragments of caspase-9 and caspase-3 were examined by Western blotting analysis.RESULTS: WWOX significantly inhibited cell proliferation, as evaluated by the MTT and colony formation assays. Cells transfected with WWOX showed significantly higher apoptosis ratios when compared with cells transfected with a mock plasmid, and overexpression of WWOX delayed cell cycle progression from G1 to S phase, as measured by flow cytometry. An increase in apoptosis was also indicated by a remarkable activation of caspase-9 and caspase-3 and a dephosphorylation of AKT (Thr308 and Ser473) measured with Western blotting analysis.CONCLUSION: Overexpression of WWOX induces apoptosis and inhibits proliferation of the human hepatic carcinoma cell line SMMC-7721.     

Melatonin inhibits cell proliferation and induces caspase activation and apoptosis in human malignant lymphoid cell lines

Abstract: Melatonin exerts strong anti‐tumour activity via several mechanisms, including anti‐proliferative and pro‐apoptotic effects in addition to its potent antioxidant activity. Several studies have investigated the effects of melatonin on haematological malignancies. However, the previous studies investigating lymphoid malignancies have been largely restricted to a single type of malignancy, Burkitt’s lymphoma (BL). Thus, we examined the actions of melatonin on the growth and apoptosis in a small panel of cell lines representing different human lymphoid malignancies including Ramos (Epstein–Barr virus–negative BL), SU‐DHL‐4 (diffuse large B cell lymphoma), DoHH2 (follicular B non‐Hodgkin lymphoma) and JURKAT (acute T cell leukaemia). We showed that melatonin promotes cell cycle arrest and apoptosis in all these cells, although there was marked variations in responses among different cell lines (sensitivity; Ramos/DoHH2 > SU‐DHL‐4 > JURKAT). Melatonin‐induced apoptosis was relatively rapid, with increased caspase 3 and PARP cleavage detected within 0.5–1 h following melatonin addition. Moreover, there was evidence for rapid processing of both caspase 9, as well as a breakdown of the mitochondrial inner transmembrane potential. On the contrary, caspase activation was detected only in SU‐DHL‐4 and Ramos cells following melatonin treatment suggesting that the extrinsic pathway does not make a consistent contribution to melatonin‐induced apoptosis in malignant lymphocytes. Although all cell lines expressed the high‐affinity melatonin receptors, MT1 and MT2, melatonin‐induced caspase activation appeared to be independent these receptors. Our findings confirm that melatonin could be a potential chemotherapeutic/preventive agent for malignant lymphocytes. However, it is necessary to take into account that different lymphoid malignancies may differ in their response to melatonin.     

beta-lapachone,a novel plant product,overcomes drug resistance in human multiple myeloma cells

OBJECTIVE: To evaluate the anti-tumor potential of beta-lapachone in multiple myeloma (MM) cell lines (U266, RPMI8226, and MM.1S); MM cell lines resistant to dexamethasone (MM.1R), melphalan (RPMI8226/LR5), doxorubicin (RPMI8226/DOX40), and mitoxantrone (RPMI8226/ MR20); and MM cells from patients (MM1-MM4). MATERIALS AND METHODS: Cytotoxicity of beta-lapachone was assessed by MTT and [3H]-thymidine uptake assays. Apoptosis was analyzed using propidium iodide staining, DNA fragmentation, TUNEL assay, caspase-9 colorimetric assay, and immunoblotting for caspase-3, poly (ADP-ribose) polymerase (PARP), and caspase-8 cleavage products. Paracrine growth of MM cells was assessed by [3H]-thymidine uptake in cultures of bone marrow stromal cells (BMSCs) and MM cells. Interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) secretion in the culture supernatants was measured by specific enzyme-linked immunosorbent assays (ELISAs). RESULTS: beta-lapachone showed significant cytotoxicity in MM cells (IC(50): 4-8 microM). In contrast, normal peripheral blood mononuclear cells (PBMCs) and BMSCs from MM patients were relatively resistant (IC(50): 8-16 microM). IL-6 did not protect against beta-lapachone-induced apoptosis in MM.1S cells, and dexamethasone showed additive cytotoxicity. beta-lapachone also decreased binding of MM.1S cells to BMSCs; abrogated IL-6 and VEGF secretion triggered by adhesion of BMSCs to MM.1S cells; reduced proliferation of MM.1S cells adherent to BMSCs; and decreased intracellular adhesion molecule-1 (ICAM-1) expression on MM.1S cells. Furthermore, beta-lapachone induced typical PARP cleavage, increased caspase-9 proteolytic activity, and activation of caspase-3, without activation of caspase-8 in U266 cells. CONCLUSION: These studies provide a framework for clinical evaluation of beta-lapachone to improve the outcome for patients with MM.     

The novel inhibitor of histone deacetylase resminostat (RAS2410) inhibits proliferation and induces apoptosis in multiple myeloma (MM) cells

Inhibition of histone deacetylase (HDAC) is a promising mechanism for novel, anti‐myeloma agents. We investigated the effects of the novel HDAC inhibitor resminostat on multiple myeloma (MM) cells in vitro. Resminostat is a potent inhibitor of HDACs 1, 3 and 6 [50% inhibitory concentration (IC₅₀) = 43–72 nmol/l] representing HDAC classes I and II and induces hyperacetylation of histone H4 in MM cells. Low micromolar concentrations of resminostat abrogated cell growth and strongly induced apoptosis (IC₅₀ = 2·5–3 μmol/l in 3 out of 4 MM cell lines) in MM cell lines as well as primary MM cells. At 1 μmol/l, resminostat inhibited proliferation and induced G0/G1 cell cycle arrest in 3 out of 4 MM cell lines accompanied with decreased levels of cyclin D1, cdc25a, Cdk4 and pRb as well as upregulation of p21. Resminostat decreased phosphorylation of 4E‐BP1 and p70S6k indicating an interference with Akt pathway signalling. Treatment with resminostat resulted in increased protein levels of Bim and Bax and decreased levels of Bcl‐xL. Caspases 3, 8 and 9 were activated by resminostat. Furthermore, synergistic effects were observed for combinations of resminostat with melphalan and the proteasome inhibitors bortezomib and S‐2209. In conclusion, we have identified potent anti‐myeloma activity for this novel HDAC inhibitor.     

Anacardic acid (6-nonadecyl salicylic acid), an inhibitor of histone acetyltransferase, suppresses expression of nuclear factor-kappaB-regulated gene products involved in cell survival, proliferation, invasion, and inflammation through inhibition of the inhibitory subunit of nuclear factor-kappaBalpha kinase, leading to potentiation of apoptosis

Anacardic acid (6-pentadecylsalicylic acid) is derived from traditional medicinal plants, such as cashew nuts, and has been linked to anticancer, anti-inflammatory, and radiosensitization activities through a mechanism that is not yet fully understood. Because of the role of nuclear factor-kappaB (NF-kappaB) activation in these cellular responses, we postulated that anacardic acid might interfere with this pathway. We found that this salicylic acid potentiated the apoptosis induced by cytokine and chemotherapeutic agents, which correlated with the down-regulation of various gene products that mediate proliferation (cyclin D1 and cyclooxygenase-2), survival (Bcl-2, Bcl-xL, cFLIP, cIAP-1, and survivin), invasion (matrix metalloproteinase-9 and intercellular adhesion molecule-1), and angiogenesis (vascular endothelial growth factor), all known to be regulated by the NF-kappaB. We found that anacardic acid inhibited both inducible and constitutive NF-kappaB activation; suppressed the activation of IkappaBalpha kinase that led to abrogation of phosphorylation and degradation of IkappaBalpha; inhibited acetylation and nuclear translocation of p65; and suppressed NF-kappaB-dependent reporter gene expression. Down-regulation of the p300 histone acetyltransferase gene by RNA interference abrogated the effect of anacardic acid on NF-kappaB suppression, suggesting the critical role of this enzyme. Overall, our results demonstrate a novel role for anacardic acid in potentially preventing or treating cancer through modulation of NF-kappaB signaling pathway.     

Honokiol overcomes conventional drug resistance in human multiple myeloma by induction of caspase-dependent and -independent apoptosis

Honokiol (HNK) is an active component purified from magnolia, a plant used in traditional Chinese and Japanese medicine. Here we show that HNK significantly induces cytotoxicity in human multiple myeloma (MM) cell lines and tumor cells from patients with relapsed refractory MM. Neither coculture with bone marrow stromal cells nor cytokines (interleukin-6 and insulin-like growth factor-1) protect against HNK-induced cytotoxicity. Although activation of caspases 3, 7, 8, and 9 is triggered by HNK, the pan-caspase inhibitor z-VAD-fmk does not abrogate HNK-induced apoptosis. Importantly, release of an executioner of caspase-independent apoptosis, apoptosis-inducing factor (AIF), from mitochondria is induced by HNK treatment. HNK induces apoptosis in the SU-DHL4 cell line, which has low levels of caspase 3 and 8 associated with resistance to both conventional and novel drugs. These results suggest that HNK induces apoptosis via both caspase-dependent and -independent pathways. Furthermore, HNK enhances MM cell cytotoxicity and apoptosis induced by bortezomib. In addition to its direct cytotoxicity to MM cells, HNK also represses tube formation by endothelial cells, suggesting that HNK inhibits neovascurization in the bone marrow microenvironment. Taken together, our results provide the preclinical rationale for clinical protocols of HNK to improve patient outcome in MM.