Autophagy is a crucial process for cells to maintain homeostasis and survival through degradation of cellular proteins and organelles, including mitochondria and endoplasmic reticula (ER). We previously demonstrated that temozolomide (TMZ), an alkylating agent for brain tumor chemotherapy, induced reactive oxygen species (ROS)/extracellular signal-regulated kinase (ERK)-mediated autophagy to protect glioma cells from apoptosis. In this study, we investigated the role of mitochondrial damage and ER stress in TMZ-induced cytotoxicity. Mitochondrial depolarization and mitochondrial permeability transition pore (MPTP) opening were observed as a prelude to TMZ-induced autophagy, and these were followed by the loss of mitochondrial mass. Electron transport chain (ETC) inhibitors, such as rotenone (a complex I inhibitor), sodium azide (a complex IV inhibitor), and oligomycin (a complex V inhibitor), or the MPTP inhibitor, cyclosporine A, decreased mitochondrial damage-mediated autophagy, and therefore increased TMZ-induced apoptosis. TMZ treatment triggered ER stress with increased expression of GADD153 and GRP78 proteins, and deceased pro-caspase 12 protein. ER stress consequently induced autophagy through c-Jun N-terminal kinases (JNK) and Ca2+ signaling pathways. Combination of TMZ with 4-phenylbutyrate (4-PBA), an ER stress inhibitor, augmented TMZ-induced cytotoxicity by inhibiting autophagy. Taken together, our data indicate that TMZ induced autophagy through mitochondrial damage- and ER stress-dependent mechanisms to protect glioma cells. This study provides evidence that agents targeting mitochondria or ER may be potential anticancer strategies.

The results indicate that triflavin effectively prevents thrombocytopenia, possibly through the following 2 mechanisms: (1) Triflavin markedly inhibits platelet aggregation, resulting in decreased thromboxane A2 formation. (2) It inhibits the adhesion of platelets to subendothelial matrixes, thereby leading to a reversal in the distribution of platelets in blood and liver in LPS-treated rats.

Examination of an ammonium sulfate-enriched fraction (70-1 00% saturation) of heat-shock proteins (HSPs) by nondenaturing polyacrylamide gel electrophoresis revealed the presence of a high molecular m a s complex (280 kD) in soybean (Glycine max) seedlings. This complex cross-reacted with antibodies raised against soybean class I low-molecular-mass (LMW) HSPs. Dissociation of the complex by denaturing polyacrylamide gel electrophoresis showed the complex to contain at least 15 polypeptides of the 15-to 18-kD class I L M W HSPs that could be detected by staining, radiolabeling, and western blotting. A similar LMW-HSP complex was observed in mung bean (Vigna radiafa L.; 295 kD), in pea (Pisum safivum 1.; 270 kD), and in rice (Oryza sativa 1.; 310 kD). l h e complex was stable under high salt conditions (250 mM KCI), and the integrity was not affected by 1 % Nonidet P-40 and 3 & n L RNase treatment. l h e size of the isolated HSP complex in vitro was conserved to 55°C; however, starting at 37.5"C, it changed to higher molecular forms in the presence of soluble proteins. l h e isolated HSP complex was able to protect up to 75% of the soluble proteins from heat denaturation in vitro.The induction of HSP synthesis in response to thermal stress occurs in a11 organisms that have been examined, ranging from bacteria to humans (Schlesinger et al., 1982;Vierling, 1991). Whereas the physiological functions of HSPs have not been clearly established, these proteins are well correlated with the acquisition of thermotolerance in a time-and temperature-dependent manner. Under HS conditions the expression of the LMW HSPs in soybean (Glycine max) is increased, yielding final concentrations of up to 1% of the total cellular proteins (Hsieh et al., 1992). Based on this correlation, it has been hypothesized that accumulation of HSPs is a n essential component of a process that prevents heat damage (Lin et al., 1984;Key et al., 1985;Kimple et al., 1990). Some HSPs become selectively localized in cellular organelles in a temperature-dependent fashion and relocate to the cytoplasm after a 28°C incubation period (Lin et al., 1984). Isolated mitochondria with associated HSPs are functional in oxidative phosphorylation under thermal stress, suggesting that association of HSPs with organelles provide protection from heat damage (Chou et al., 1989 (Jinn et al., 1993). In soybean, depletion of the 15-to 18-kD HSPs in the HSP-enriched fraction resulted in the loss of the thermostabilizing ability, and this ability was again restored when these HSPs were added. The protection provided by these HSP-enriched fractions is effective mainly for membrane-associated proteins (Jinn et al., 1993).The LMW HSPs of plants and a11 other eukaryotes have a conserved carboxyl-terminal region homologous to the a-crystallin of the eye lens at the amino acid sequence leve1 (Ingolia and Craig, 1982; Augusteyn and Koretz, 1987;Lindquist and Craig, 1988). The LMW HSPs in vertebrates (Arrigo and Welch, 1987;Merck et al., 1993), Drosopkila (Arrigo and Pauli, 1988), ye...

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