cytochrome-c-t and aloe-emodin

In this study, aloe-emodin (AE) was less cytotoxic to human noncancerous skin cells (premalignant keratinocytic HaCaT and fibroblast Hs68) than to nonmelanoma cancer cells (epidermoid carcinoma A431 and head and neck squamous cell carcinoma SCC25). Notably, AE induced apoptosis by up-regulating tumor necrosis factor-alpha and Fas ligand and their cognate receptors, downstream adaptor TNF-R1-associated death domain and Fas-associated death domain, and activated caspase-8 in A431 and SCC25 cells. Moreover, AE up-regulated p53, increased intracellular reactive oxygen species levels, depleted intracellular-reduced GSH, up-regulated cytochrome c and Bax, down-regulated Bcl-2, and activated caspase-9 and -3. The combinatory use of AE and 5-fluorouracil (5-Fu) achieved significantly more cell death in A431 and SCC25 cells than only the use of AE or 5-Fu, likely via regulation of caspase-8, -9, and -3 expressions. Incorporating AE into the liposomal formulation accelerated cell death of A431 and SCC25 cells within a short time. Furthermore, skin permeation profiles of drug suggest that the liposomal formulation enhances transdermal delivery of AE. Experimental data demonstrate the feasibility of applying liposome to deliver AE in clinical therapy.

Topics: Administration, Cutaneous; Anthraquinones; Antineoplastic Agents; Apoptosis; Caspase 8; Caspase 9; Cell Line; Cytochromes c; Fas Ligand Protein; Fas-Associated Death Domain Protein; Fluorouracil; Humans; Liposomes; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Skin; Skin Neoplasms; TNF Receptor-Associated Death Domain Protein; Tumor Necrosis Factor-alpha; Tumor Suppressor Protein p53

The purpose of this study was to investigate the anticancer effect of aloe-emodin, an anthraquinone compound present in the leaves of Aloe vera, on two distinct human gastric carcinoma cell lines, AGS and NCI-N87. We demonstrate that aloe-emodin induced cell death in a dose- and time-dependent manner. Noteworthy is that the AGS cells were generally more sensitive than the NCI-N87 cells. Aloe-emodin caused the release of apoptosis-inducing factor and cytochrome c from mitochondria, followed by the activation of caspase-3, leading to nuclear shrinkage and apoptosis. In addition, exposure to aloe-emodin suppressed the casein kinase II activity in a time-dependent manner and was accompanied by a reduced phosphorylation of Bid, a downstream substrate of casein kinase II and a pro-apoptotic molecule. These preclinical studies suggest that aloe-emodin represents a suitable and novel chemotherapeutic drug candidate for the treatment of human gastric carcinoma.

Topics: Anthraquinones; Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Inducing Factor; BH3 Interacting Domain Death Agonist Protein; Carcinoma; Casein Kinase II; Cell Line, Tumor; Cytochromes c; Dose-Response Relationship, Drug; Humans; Phosphorylation; Stomach Neoplasms; Time Factors

The aim of our study was to clarify the apoptosis pathway induced by aloe emodin, an hydroxyanthraquinone present in aloe vera leaves, in rat hepatic stellate cells transformed by simian virus 40 (t-HSC/Cl-6), which retain the features of activated rat stellate cells. Apoptosis was determined by DNA fragmentation, caspase activity assay and western blotting analysis. Treatment of t-HSC/Cl-6 cells with 12.5, 25, or 50 microM aloe emodin inhibited t-HSC/Cl-6 cell viability in a dose- and time-dependent manner. The induction of apoptosis by aloe emodin was confirmed by typical DNA ladder formation and annexin v-propidium iodide flow-cytometric analysis. Aloe emodin treatment of t-HSC/Cl-6 cells caused activation of caspase-3 and caspase-9, detected with a caspase activity assay, although no change was observed in caspase-8 activity. Western blotting showed caspase-3 and caspase-9 active forms and the subsequent proteolytic cleavage of poly(ADP-ribose) polymerase. Aloe emodin induced mitochondrial membrane depolarization. Our data also show that cytochrome c increased in the cytosol but decreased in the mitochondria in a time-dependent manner. Increased Bax and unchanged Bcl-2 levels resulted in an increased Bax/Bcl-2 ratio. Thus, our research provides evidence that aloe emodin-induced apoptosis involves a mitochondria-associated apoptosis pathway.

Topics: Animals; Anthraquinones; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Caspases; Cell Line; Cell Proliferation; Cell Survival; Cytochromes c; DNA Fragmentation; Emodin; Membrane Potentials; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Rats

Previously, we have identified aloe-emodin (AE) as a new type of anticancer agent, with activity that is based on apoptotic cell death promoted by a neuroectodermal tumor-specific drug uptake. We attempt to clarify the intracellular target of AE and the apoptosis-signaling pathway activated by AE in neuroblastoma cell lines. Two-photon excitation microscopy and spectroscopic titrations documented that AE is highly concentrated in susceptible cells and binds to DNA. One of the most important mediators of apoptotic response to genotoxic stimuli, such as anticancer agents, is the p53 tumor suppressor gene. To evaluate the role played by p53 in AE-induced apoptosis a p53 mutant cell line, which lacks transcriptional activity of p53 targeted genes, was tested. AE displayed a reduced growth inhibitory and pro-apoptotic activity in p53 mutant cells (SK-N-BE(2c)) with respect to the p53 wild-type line (SJ-N-KP). This effect was not caused by a reduced drug uptake in the mutant neuroblastoma cell line but was related to a different apoptotic cell phenotype. Whereas SJ-N-KP cells were susceptible to a p53 transcription-dependent pathway of apoptosis, SK-N-BE(2c) cells underwent apoptosis with up-regulation of p53 expression but not of p53-target genes. After AE treatment p53 translocates to the mitochondria inter-membrane space in both neuroblastoma cell lines. Due to its high accumulation in neuroectodermal tumor cells AE could also kill tumor cells harboring p53 mutant genes. This property would further contribute to AE specific anti-tumor activity and might be exploitable in the clinic.

Topics: Anthraquinones; Apoptosis; Benzothiazoles; Blotting, Western; Caspases; Cell Cycle; Child, Preschool; Cytochromes c; DNA Primers; DNA Topoisomerases, Type II; DNA, Neoplasm; Emodin; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Genes, bcl-2; Humans; Male; Microscopy, Immunoelectron; Mitochondria; Mutation; Neuroectodermal Tumors; Poly(ADP-ribose) Polymerases; Protein Transport; Reverse Transcriptase Polymerase Chain Reaction; Thiazoles; Toluene; Tumor Cells, Cultured; Tumor Suppressor Protein p53