Mitophagy is the specific autophagic elimination system of mitochondria, which regulates cellular survival via the removal of damaged mitochondria. Recently, we revealed that folate-appended methyl-β-cyclodextrin (FA-M-β-CyD) provides selective antitumor activity in folate receptor-α (FR-α)-expressing cells by the induction of autophagy. In this study, to gain insight into the detailed mechanism of this antitumor activity, we focused on the induction of mitophagy by the treatment of FR-α-expressing tumor cells with FA-M-β-CyD. In contrast to methyl-β-cyclodextrin, FA-M-β-CyD entered KB cells, human epithelial cells from a fatal cervical carcinoma (FR-α (+)) through FR-α-mediated endocytosis. The transmembrane potential of isolated mitochondria after treatment with FA-M-β-CyD was significantly elevated. In addition, FA-M-β-CyD lowered adenosine triphosphate (ATP) production and promoted reactive oxygen species production in KB cells (FR-α (+)). Importantly, FA-M-β-CyD enhanced light chain 3 (LC3) conversion (LC3-I to LC3-II) in KB cells (FR-α (+)) and induced PTEN-induced putative kinase 1 (PINK1) protein expression, which is involved in the induction of mitophagy. Furthermore, FA-M-β-CyD had potent antitumor activity in BALB/c
nu/nu
mice xenografted with KB cells (FR-α (+)) without any significant side effects. Taken together, these findings demonstrate that the autophagic cell death elicited by FA-M-β-CyD could be associated with mitophagy induced by an impaired mitochondrial function.
Mimosa pudica L. rapidly closes its leaves and bends its petioles downward when mechanically stimulated. It has been suggested that the actin cytoskeleton is involved in the bending motion since both cytochalasin B and phalloidin inhibit the motion. In order to clarify the mechanism by which the actin cytoskeleton functions in the motion, we attempted to find actin-modulating proteins in the M. pudica plant by DNase I-affinity column chromatography. The EGTA-eluate from the DNase I column contained proteins with apparent molecular masses of 90- and 42-kDa. The 42-kDa band consisted of two closely migrating components: the slower migrating component was actin while the faster migrating components was a distinct protein. The eluate showed an activity to sever actin filaments and to enhance the rate of polymerization of actin, both in a Ca(2+)-dependent manner. Microsequencing of the faster migrating 42-kDa protein revealed its similarity to proteins in the gelsolin/fragmin family. Our results provide the first biochemical evidence for the presence in a higher plant of a gelsolin/fragmin family actin-modulating protein that severs actin filament in a Ca(2+)-dependent manner.
Objective: Adipocytes secrete a number of molecules such as tumor necrosis factor-alpha, leptin and free fatty acids that can influence the ability of the body to metabolize glucose. Recently, a novel 12.5 kDa cysteine-rich protein, termed resistin, was shown to be secreted by adipocytes. Resistin expression was markedly induced during the conversion of 3T3-L1 cells to mature adipocytes. Expression of resistin has been studied in human, mouse and rat; however, sequence information about an alternative splicing variant (ASV) of resistin mRNA has not been reported. In the present study, we investigated the occurrence of a novel ASV of the resistin gene in human normal tissues. Design and methods: We identified a novel ASV of resistin mRNA in human lung tissue by RT-PCR analysis in human lung tissue. We then investigated a novel ASV of resistin mRNA by real-time PCR analysis in 26 different types of normal human tissues. Results and conclusions: We identified a novel deletion variant of the resistin transcript in the normal human tissues. The deleted transcript of resistin was characterized by an in-frame deletion of 78 bp, corresponding to the complete loss of exon 2 (resistin delta2 ASV). Thus, resistin delta2 ASV causes protein truncation. Our results provide the basis for more detailed studies on the regulation of resistin activity, and should assist in the development of clinical trials with resistin for the central regulation of adipogenesis and adipocyte metabolism.
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