Cannabinoid receptor agonists are mitochondrial inhibitors: a unified hypothesis of how cannabinoids modulate mitochondrial function and induce cell death

Time-lapse microscopy of human lung cancer (H460) cells showed that the endogenous cannabinoid anandamide (AEA), the phyto-cannabinoid Δ-9-tetrahydrocannabinol (THC) and a synthetic cannabinoid HU 210 all caused morphological changes characteristic of apoptosis. Janus green assays of H460 cell viability showed that AEA and THC caused significant increases in OD 595 nm at lower concentrations (10-50 μM) and significant decreases at 100 μM, whilst HU 210 caused significant decreases at all concentrations. In rat heart mitochondria, all three ligands caused significant decreases in oxygen consumption and mitochondrial membrane potential. THC and HU 210 caused significant increases in mitochondrial hydrogen peroxide production, whereas AEA was without significant effect. All three ligands induced biphasic changes in either mitochondrial complex I activity and/or mitochondrial complex II-III activity. These data demonstrate that AEA, THC, and HU 210 are all able to cause changes in integrated mitochondrial function, directly, in the absence of cannabinoid receptors.     

Combination of all-<Emphasis Type="Italic">trans</Emphasis> retinoic acid and taxol regressed glioblastoma T98G xenografts in nude mice

Glioblastoma is the most prevalent and highly malignant brain tumor that continues to defy current treatment strategies. This investigation used all-trans retinoic acid (ATRA) and taxol (TXL) as a combination therapy for controlling the growth of human glioblastoma T98G xenografted in athymic nude mice. Histopathological examination revealed that ATRA induced differentiation and combination of ATRA and TXL caused more apoptosis than either treatment alone. Combination therapy decreased expression of telomerase, nuclear factor kappa B (NFκВ), and inhibitor-of-apoptosis proteins (IAPs) indicating suppression of survival factors while upregulated Smac/Diablo. Combination therapy also changed expression of Bax and Bcl-2 proteins leading to increased Bax:Bcl-2 ratio, mitochondrial release of cytochrome c and apoptosis-inducing factor (AIF), and activation of caspase-9. Increased activities of calpain and caspase-3 degraded 270 kD α-spectrin at the specific sites to generate 145 kD spectrin breakdown product (SBDP) and 120 kD SBDP, respectively. Further, increased activity of caspase-3 cleaved inhibitor-of-caspase-activated DNase (ICAD). In situ double immunofluorescent labelings showed overexpression of calpain, caspase-12, caspase-3, and AIF during apoptosis, suggesting involvement of both caspase-dependent and caspase-independent pathways for apoptosis. Our investigation revealed that treatment of glioblastoma T98G xenografts with the combination of ATRA and TXL induced differentiation and multiple molecular mechanisms for apoptosis.     

Signaling events involved in anti-CD20-induced apoptosis of malignant human B cells

Anti-CD20 monoclonal antibodies have been successfully employed in the clinical treatment of non-Hodgkin's lymphomas in both unmodified and radiolabeled forms. Previous publications have demonstrated that the antitumor effects of unmodified anti-CD20 mAb are mediated by several mechanisms including antibody-dependent cellular cytotoxicity, complement-mediated cell lysis, and induction of apoptosis by CD20 cross-linking. In this report, we demonstrate induction of apoptosis by three anti-CD20 monoclonal antibodies [1F5, anti-B1, and C2B8 (Rituximab)]. The magnitude of apoptosis induction was greater with the chimeric Rituximab antibody than with the murine 1F5 and anti-B1 antibodies. Apoptosis could be enhanced with any of the antibodies by cross-linking with secondary antibodies (or Fc-receptor-bearing accessory cells). The signaling events involved in anti-CD20-induced apoptosis were investigated, including activation of protein tyrosine kinases, increases in intracellular Ca²⁺ concentrations, caspase activation, and cleavage of caspase substrates. Our results indicate that anti-CD20-induced apoptosis can be attenuated by PP1, an inhibitor of protein tyrosine kinases Lck and Fyn, chelators of extracellular or intracellular Ca²⁺, and inhibitors of caspases, suggesting that anti-CD20-induced apoptosis may involve modulation of these signaling molecules. We also demonstrated that varying the expression of Bcl-2 did not affect the magnitude of anti-B1-induced apoptosis, possibly because of the sequestering effects of other Bcl-2 family members, such as Bad. These studies identify several of the signal-transduction events involved in the apoptosis of malignant B cells that transpire following ligation of CD20 by anti-CD20 antibodies in the presence of Fc-receptor-expressing cells or secondary goat anti-(mouse Ig) antibodies and which may contribute to the tumor regressions observed in mouse models and clinical trials. Received: 27 May 1999 / Accepted: 1 October 1999     

索拉菲尼联合阿霉素对人肝癌细胞株HepG2的抑制作用

目的：探讨索拉非尼（Sorafenib）和阿霉素（adriamycin）联合用药对肝癌细胞株nepG2的作用及可能的机制。方法：以不同浓度索拉非尼和不同浓度阿霉素分别组成单药组和索拉非尼＋阿霉素联合用药组作用于HepG2细胞,MTT法检测增殖抑制率、流式细胞仪分析细胞周期和凋亡率。结果：索拉非尼、阿霉素单药与联用均能抑制HepG2细胞增殖,呈剂量依赖效应,两药联用有协同效应（P〈0.01）。索拉非尼、阿霉素单药与联用均能诱导HepG2细胞凋亡,并以联合组更为明显,与对照组比较有显著的统计学意义（P〈0.01）。索拉非尼及阿霉素单药作用均可使细胞周期阻滞于G0-G1期,联合用药组G0/Gl期细胞比率低于索拉非尼及阿霉素单药组,S期细胞比率高于单药组;阿霉素能抑制HepG2细胞Survivin mRNA表达诱导细胞的凋亡。结论：索拉非尼与阿霉素联合作用于人肝癌HepG2细胞具有协同作用,其机制可能是通过多途径共同抑制HepG2细胞增殖及诱导细胞凋亡。     

Methylglyoxal induces cellular damage by increasing argpyrimidine accumulation and oxidative DNA damage in human lens epithelial cells

Methylglyoxal (MGO) is a cytotoxic metabolite and modifies tissue proteins through the Maillard reaction, resulting in advanced glycation end products (AGEs), which can alter protein structure and functions. Several MGO-derived AGEs have been described, including argpyrimidine, a fluorescent product of the MGO reaction with arginine residues. Herein, we evaluated the cytotoxic role of MGO in human lens epithelial cell line (HLE-B3). HLE-B3 cells were exposed to 400 μM MGO in the present or absence of pyridoxamine for 24 h. We then examined the formation of argpyrimidine, apoptosis and oxidative stress in HLE-B3 cells. In MGO-treated HLE-B3 cells, the accumulation of argpyrimidine was markedly increased, and caspase-3 and 8-hydroxydeoxyguanosine (8-OHdG) were highly expressed, which paralleled apoptotic cell death. However, pyridoxamine (AGEs inhibitor) prevented the argpyrimidine formation and apoptosis of MGO-treated HLE-B3 cells. These results suggested that the accumulation of argpyrimidine and oxidative DNA damage caused by MGO are involved in apoptosis of HLE-B3 cells.     

Differential apoptotic pathways activated in response to Cu-induced or HOCl-induced LDL oxidation in U937 monocytic cell line

We compared the apoptotic mechanism involved in U937 human monocytic cell line in presence of oxidized low-density lipoproteins (oxLDL) obtained after treatment with hypochlorous acid (HOCl) or copper (Cu).Both types of oxLDL induced U937 apoptotic cell death via the mitochondrial pathway. In contrast to HOCl-oxLDL, Cu-oxLDL induced apoptosis via a caspase-independent mechanism, with no activation of pro-caspase-3, but via the release of apoptosis inducing factor (AIF) from mitochondria.The apoptotic program of the monocyte differs depending on the mode of LDL oxidation, based on differences in the oxidatively modified components of the two oxLDL types.