Involvement of MAPK, Bcl-2 family, cytochrome c, and caspases in induction of apoptosis by 1,6-O,O-diacetylbritannilactone in human leukemia cells

1,6-O,O-diacetylbritannilactone (OODBL) isolated from Inula britannica, exhibits potent antitumor activity against several human cancer cell lines. However, the molecular mechanism of OODBL in the induction of anticancer activity is still unclear. In the present study, we demonstrated that OODBL induced the occurrence of apoptosis in human leukemic (HL-60) cells and cell arrest at the S phase. On the other hand, activation of caspase-8, -9, and -3, phosphorylation of Bcl-2 and Bid, and increased release of cytochrome c from mitochondria into cytosolic fraction were detected in OODBL-treated HL-60 cells. We further demonstrated that production of reactive oxygen species (ROS), activation of mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) signaling pathways may play an important role in OODBL-induced apoptosis. The results from the present study highlight the molecular mechanisms underlying OODBL-induced anticancer activity.     

Xanthohumol induces apoptosis in cultured 40-16 human colon cancer cells by activation of the death receptor- and mitochondrial pathway

Xanthohumol (XN) is one of the major prenylflavonoids found in hop cones (Humulus lupulus L.). In this study, we investigated the cell growth inhibitory potential of XN on cultured human colon cancer cells. Cell proliferation was measured by sulforhodamine B staining. Poly(ADP-ribose)polymerase (PARP) cleavage, activation of caspases-3, -7, -8, and -9, and Bcl-2 family protein expression were detected by Western blot analyses. XN significantly reduced proliferation of the HCT 116-derived colon cancer cell line 40--16. Half-maximal inhibitory concentrations decreased from 4.1 microM after 24 h treatment to 3.6 and 2.6 microM after 48 and 72 h incubation, respectively. Treatment with 15 microM XN for 48 h and with 5 microM for 72 h led to the detection of the cleaved 89 kDa fragment of 116 kDa PARP as an indication of apoptosis induction. Concomitantly, we observed activation and cleavage of the effector caspases-3 and -7, induced by activation of the initiator caspases -8 and -9. Expression of anti-apoptotic Bcl-2 was down regulated when the cells were treated with XN for 48--72 h. We conclude that induction of apoptosis by downregulation of Bcl-2 and activation of the caspase cascade may contribute to the chemopreventive or therapeutic potential of XN.     

Osteocytes: Their Lacunocanalicular Structure and Mechanoresponses

Osteocytes connect with neighboring osteocytes and osteoblasts through their processes and form an osteocyte network. Shear stress on osteocytes, which is induced by fluid flow in the lacunae and canaliculi, has been proposed as an important mechanism for mechanoresponses. The lacunocanalicular structure is differentially developed in the compression and tension sides of femoral cortical bone and the compression side is more organized and has denser and thinner canaliculi. Mice with an impaired lacunocanalicular structure may be useful for evaluation of the relationship between lacunocanalicular structure and mechanoresponses, although their bone component cells are not normal. We show three examples of mice with an impaired lacunocanalicular structure. Ablation of osteocytes by diphtheria toxin caused massive osteocyte apoptosis, necrosis or secondary necrosis that occurred after apoptosis. Osteoblast-specific Bcl2 transgenic mice were found to have a reduced number of osteocyte processes and canaliculi, which caused massive osteocyte apoptosis and a completely interrupted lacunocanalicular network. Osteoblast-specific Sp7 transgenic mice were also revealed to have a reduced number of osteocyte processes and canaliculi, as well as an impaired, but functionally connected, lacunocanalicular network. Here, we show the phenotypes of these mice in physiological and unloaded conditions and deduce the relationship between lacunocanalicular structure and mechanoresponses.     

P53基因、Bcl-XL基因表达对淋巴性肿瘤细胞株辐射敏感性的影响

本文运用PCR－SSCP银染法检测人淋巴细胞恶性肿瘤株8226、U266、Raji、Jurkat、Daudi p53基因状态,RT－PCR半定量检测了Bcx-xL的mRNA的相对表达,DNA片段释放法检测细胞受照后24h的细胞DNA链断裂量,探讨了p53基因功能状态及Bcl-xL基因表达对细胞株8226、U266、Raji、Jurkat、Dsaudi辐射敏感性的影响。结果表明,淋巴细胞恶性肿瘤株8226、U266、Raji、Jurkat、Daudi均存在p53基因突变,高表达Bcl-xL基因的细胞株较低表达细胞株有较强的辐射耐性。     

p53和Bcl—2／Bax在辐射诱导胸腺细胞凋亡中的作用

采用流式细胞术对昆明小鼠接受７５ｍＧｙ和２ＧｙＸ射线全身照射后，胸腺细胞内细胞凋亡相关基因ｐ５３，Ｂｃｌ－２和Ｂａｘ蛋白的表达进行了比较研究。结果表明，７５ｍＧｙＸ射线全身照射后２４ｈ，ｐ５３基因蛋白产物的表达显著降低，而２ＧｙＸ射线全身照射后２４ｈ，ｐ５３基因蛋白表达则显著增高。     

Anionic long‐circulating liposomes for delivery of radioiodinated antisense oligonucleotides

Long‐circulating liposomes have been widely used to enhance efficacy of gene therapy. Antisense therapy might increase the efficacy of radiation or chemotherapy; therefore, we undertook to optimize the composition of the liposomal delivery vehicles. The radiolabeling efficiency, radiochemistry purity, and specific radioactivity of radioiodinated antisense oligonucleotides (ASON) were 71.66 ± 7.73, 98.33 ± 0.39%, 4.09 ± 0.11 MBq/nmol. Radioiodinated ASON remained stable in 0.01 M HEPES buffer and human serum even after incubation for 4 hours. Mean diameter of the anionic long‐circulating liposomes (ALCL) was 504 ± 31.76 nm with a polydispersity index (PDI) of 0.107 ± 0.008 before extrusion, 115 ± 8.5 nm with a PDI of 0.103 ± 0.002 after extrusion. The zeta potential of ALCL was ?29.23 ± 0.45 mV. ALCL prepared for this study provided 70.28 ± 1.84% encapsulation efficiency. Compared with other liposome formulations, the ALCL mediated enhanced cellular uptake (32.51 ± 1.44%) by MCF‐7 breast cancer cells (p<0.05). Practical applications: Drug delivery systems can in principle provide enhanced efficacy and/or reduced toxicity for anticancer agents. LCL have become a commonly used carrier for gene therapy recently. ALCL are promising for the mediated radiation and antisense therapy for breast cancer which is the leading cause for women.     

Synthetic Inhibitors of Extended Helix–Protein Interactions Based on a Biphenyl 4,4′‐Dicarboxamide Scaffold

Turn Bak : We present rationally designed scaffolds that mimic the spatial projection of the i, i+4, i+7, and i+11 residues of an α‐helix. A library of biphenyl derivatives was shown by competition fluorescence polarization and ITC to mimic Bak and disrupt the Bak/Bcl‐x_L protein–protein interaction. ¹⁵N HSQC experiments confirmed that the surface of Bcl‐x_L normally occupied by Bak was the target area of our new synthetic inhibitors.

     

Svf1 inhibits reactive oxygen species generation and promotes survival under conditions of oxidative stress in Saccharomyces cerevisiae

Aberrant regulation of apoptosis, or programmed cell death, contributes to the aetiology of several diseases, including cancers, immunodeficiencies and neurodegenerative illnesses. We hypothesized that key features of mammalian cell death regulation may be conserved in single celled organisms such as the budding yeast Saccharomyces cerevisiae. We previously identified the yeast gene SVF1 in a screen for mutations that could be functionally complemented by exogenous expression of the human anti-apoptotic gene Bcl-x(L). Anti-apoptotic Bcl-2 family members have been shown to promote redox stability through upregulation of antioxidant pathways in mammalian cells. Here we demonstrate that the Svf1 protein is required for yeast survival under conditions of oxidative stress, including cold stress. Cells lacking SVF1 are hypersensitive to conditions associated with increased reactive oxygen species (ROS) generation and to direct chemical precursors of ROS, and demonstrate increased levels of ROS under these conditions. Hypersensitivity to oxidative stress can be reversed by treatment with the antioxidant N-acetylcysteine or expression of exogenous SVF1, although exogenous expression of Bcl-x(L) did not protect cells from cold stress. Exogenous SVF1 expression in mammalian cells confers resistance to H(2)O(2) exposure. Our data are consistent with previous observations suggesting a key role of oxidative stress response in mammalian apoptotic regulation and validate the use of S. cerevisiae as a model for studying programmed cell death.     

Novel Bcl-2 inhibitors: Discovery and mechanism study of small organic apoptosis-inducing agents

Apoptosis as a novel target for cancer chemotherapy has generated an intense demand for new apoptosis-inducing agents. The newly revealed role of protein families involved in the apoptosis pathway, and resistance to cytotoxic therapies have opened new avenues for the development of novel anticancer strategies. We have established a novel strategy to rapidly obtain protein-targeted, instead of conventional DNA-targeted, apoptosis inducers as antitumor leads. First, a novel organic non-DNA intercalative compound S1 (8-oxo-3-thiomorpholin-4-yl-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile, M(W) = 331) was found with an IC50 of 10(-7)-10(-8) microM against diverse cancer cell lines. Further biological evaluation demonstrated that it was an apoptosis-inducer both in vivo and in vitro. The treatment of hydroperitoneum hepatoma cells (H22 cell line) with S1 at various concentrations (from 0.01 to 10 microM) for 24 h triggered these cells to enter the apoptosis process. The antitumor efficiency was also tested in the H22 xenotransplant models in mice. At a dosage of 0.3 mg kg(-1), S1 exhibited significant antitumor activity with a much longer survival time, a decrease in tumor size, and increased apoptosis cells in tumor tissue. More importantly, studies of the molecular mechanism of apoptosis induction by S1 revealed that S1 inactivated the Bcl-2 protein by binding to it, depolarizing the mitochondrial membrane, and then activating caspase 9, followed by caspase 3. Finally, structure-based virtual modification was performed by computer modeling. As a result, a derivative, S2 (8-oxo-3-[(thienylmethyl)amino]-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile, M(W) = 341) was identified that possessed a lower binding energy to Bcl-2, and demonstrated better antitumor potency, even on the Bcl-2-overexpressing human acute myeloid leukemia (HL-60) cells (IC50 = 1.3 microM) in vitro. S1 and S2 are the well-defined Bcl-2 inhibitors that give us a promising platform for the development of new therapeutic agents.