Hiroko Kokuba

The E3 ligase synoviolin controls body weight and mitochondrial biogenesis through negative regulation of PGC‐1β

Obesity is a major global public health problem, and understanding its pathogenesis is critical for identifying a cure. In this study, a gene knockout strategy was used in post‐neonatal mice to delete synoviolin (Syvn)1/Hrd1/Der3, an ER‐resident E3 ubiquitin ligase with known roles in homeostasis maintenance. Syvn1 deficiency resulted in weight loss and lower accumulation of white adipose tissue in otherwise wild‐type animals as well as in genetically obese (ob/ob and db/db) and adipose tissue‐specific knockout mice as compared to control animals. SYVN1 interacted with and ubiquitinated the thermogenic coactivator peroxisome proliferator‐activated receptor coactivator (PGC)‐1β, and Syvn1 mutants showed upregulation of PGC‐1β target genes and increase in mitochondrion number, respiration, and basal energy expenditure in adipose tissue relative to control animals. Moreover, the selective SYVN1 inhibitor LS‐102 abolished the negative regulation of PGC‐1β by SYVN1 and prevented weight gain in mice. Thus, SYVN1 is a novel post‐translational regulator of PGC‐1β and a potential therapeutic target in obesity treatment.

Targeting the integrated networks of aggresome formation, proteasome, and autophagy potentiates ER stress-mediated cell death in multiple myeloma cells

The inhibitory effects of macrolide antibiotics including clarithromycin (CAM) on autophagy flux have been reported. Although a macrolide antibiotic exhibits no cytotoxicity, its combination with bortezomib (BZ), a proteasome inhibitor, for the simultaneous blocking of the ubiquitin (Ub)-proteasome and autophagy-lysosome pathways leads to enhanced multiple myeloma (MM) cell apoptosis induction via stress overloading of the endoplasmic reticulum (ER). As misfolded protein cargo is recruited by histone deacetylase 6 (HDAC6) to dynein motors for aggresome transport, serving to sequester misfolded proteins, we further investigated the cellular effects of targeting proteolytic pathways and aggresome formation concomitantly in MM cells. Pronounced apoptosis was induced by the combination of vorinostat [suberoylanilide hydroxamic acid (SAHA); potently inhibits HDAC6] with CAM and BZ compared with each reagent or a 2-reagent combination. CAM/BZ treatment induced vimentin positive-aggresome formation along with the accumulation of autolysosomes in the perinuclear region, whereas they were inhibited in the presence of SAHA. The SAHA/CAM/BZ combination treatment maximally upregulated genes related to ER stress including C/EBP homologous protein (CHOP). Similarly to MM cell lines, enhanced cytotoxicity with CHOP upregulation following SAHA/CAM/BZ treatment was shown by a wild-type murine embryonic fibroblast (MEF) cell line; however, a CHOP-deficient MEF cell line almost completely canceled this pronounced cytotoxicity. Knockdown of HDAC6 with siRNA exhibited further enhanced CAM/BZ-induced cytotoxicity and CHOP induction along with the cancellation of aggresome formation. Targeting the integrated networks of aggresome, proteasome, and autophagy is suggested to induce efficient ER stress-mediated apoptosis in MM cells.

Macrolides sensitize EGFR-TKI-induced non-apoptotic cell death via blocking autophagy flux in pancreatic cancer cell lines.

Pancreatic cancer is one of the most difficult types of cancer to treat because of its high mortality rate due to chemotherapy resistance. We previously reported that combined treatment with gefitinib (GEF) and clarithromycin (CAM) results in enhanced cytotoxicity of GEF along with endoplasmic reticulum (ER) stress loading in non-small cell lung cancer cell lines. An epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) such as GEF induces autophagy in a pro-survival role, whereas CAM inhibits autophagy flux in various cell lines. Pronounced GEF-induced cytotoxicity therefore appears to depend on the efficacy of autophagy inhibition. In the present study, we compared the effect on autophagy inhibition among such macrolides as CAM, azithromycin (AZM), and EM900, a novel 12-membered non-antibiotic macrolide. We then assessed the enhanced GEF-induced cytotoxic effect on pancreatic cancer cell lines BxPC-3 and PANC-1. Autophagy flux analysis indicated that AZM is the most effective autophagy inhibitor of the three macrolides. CAM exhibits an inhibitory effect but less than AZM and EM900. Notably, the enhancing effect of GEF-induced cytotoxicity by combining macrolides correlated well with their efficient autophagy inhibition. However, this pronounced cytotoxicity was not due to upregulation of apoptosis induction, but was at least partially mediated through necroptosis. Our data suggest the possibility of using macrolides as ‘chemosensitizers’ for EGFR-TKI therapy in pancreatic cancer patients to enhance non-apoptotic tumor cell death induction.

Harmol induces autophagy and subsequent apoptosis in U251MG human glioma cells through the downregulation of survivin.

The β-carboline alkaloids are plant substances that exhibit a wide spectrum of neuropharmacological, psychopharma-cological and antitumor effects. In the present study, we found that harmol, a β-carboline alkaloid, induced autophagy and suppression of survivin expression, and subsequently induced apoptotic cell death in U251MG human glioma cells. Autophagy was induced within 12 h by treatment with harmol. When treated for over 36 h, however, apoptotic cell death was induced. Harmol treatment also reduced survivin protein expression. Small interfering RNA (siRNA)-mediated knockdown of survivin enhanced the harmol-induced apoptosis. Knockdown of survivin by siRNA also induced autophagy. Therefore, harmol-induced apoptosis is a result of the reduction in survivin protein expression. Treatment with 3-methyladenine (3-MA) in the presence of harmol did not affect the expression of survivin and diminished harmol-induced cell death. Treatment with chloroquine in the presence of harmol did not suppress the reduction of survivin expression and increased harmol-induced cell death. From these results, harmol-induced reduction of survivin expression was closely related to autophagy. It is assumed that when isolation membrane formation is inhibited by treatment with 3-MA, reduction of survivin protein expression and apoptotic cell death were not induced. However, when isolation membrane formation is started and an autophagosome is formed, survivin expression is suppressed and apoptosis is executed. Harmol treatment reduced phosphorylation of Akt, mammalian target of rapamycin (mTOR) and its downstream targets p70-ribosomal protein S6 kinase and 4E-binding protein 1, resulting in induction of autophagy. Conversely, activation of the Akt/mTOR pathway inhibited harmol-induced autophagy and cell death. These findings indicate that harmol-induced autophagy involves the Akt/mTOR pathway. Taken together, autophagy induced by harmol represented a pro-apoptotic mechanism, and harmol suppressed the expression of survivin and subsequently induced apoptosis.

Targeting bortezomib-induced aggresome formation using vinorelbine enhances the cytotoxic effect along with ER stress loading in breast cancer cell lines

The ubiquitin-proteasome and autophagy-lysosome pathways are two major self-digestive systems for cellular proteins. Ubiquitinated misfolded proteins are degraded mostly by proteasome. However, when ubiquitinated proteins accumulate beyond the capacity of proteasome clearance, they are transported to the microtubule-organizing center (MTOC) along the microtubules to form aggresomes, and subsequently some of them are degraded by the autophagy-lysosome system. We previously reported that macrolide antibiotics such as azithromycin and clarithromycin block autophagy flux, and that concomitant treatment with the proteasome inhibitor bortezomib (BZ) and macrolide enhances endoplasmic reticulum (ER) stress-mediated apoptosis in breast cancer cells. As ubiquitinated proteins are concentrated at the aggresome upon proteasome failure, we focused on the microtubule as the scaffold of this transport pathway for aggresome formation. Treatment of metastatic breast cancer cell lines (e.g., MDA-MB-231 cells) with BZ resulted in induction of aggresomes, which immunocytochemistry detected as a distinctive eyeball-shaped vimentin-positive inclusion body that formed in a perinuclear lesion, and that electron microscopy detected as a sphere of fibrous structure with some dense amorphous deposit. Vinorelbine (VNR), which inhibits microtubule polymerization, more effectively suppressed BZ-induced aggresome formation than paclitaxel (PTX), which stabilizes microtubules. Combined treatment using BZ and VNR, but not PTX, enhanced the cytotoxic effect and apoptosis induction along with pronounced ER stress loading such as upregulation of GRP78 and CHOP/GADD153. The addition of azithromycin to block autophagy flux in the BZ plus VNR-containing cell culture further enhanced the cytotoxicity. These data suggest that suppression of BZ-induced aggresome formation using an inhibitory drug such as VNR for microtubule polymerization is a novel strategy for meta-static breast cancer therapy.

Review: Exploration of placentation from human beings to ocean-living species

This review covers four topics. 1) Placental pathology in Himalayan mountain people. To determine morphological changes of the placenta at high altitude, pathological examination was made of 1000 Himalayan placentas obtained in Nepal and Tibet and the results compared with Japanese placentas delivered at sea level. Characteristic findings in the placental villi of the Himalayan group included high incidences of villous chorangiosis and chorangioma. These processes were clarified by ultrastructural observation. 2) Placentation in Sirenians. The giant Takikawa sea cow, which lived 5 million years ago, was discovered on Hokkaido, Japan. It was an ancestor of the dugong as well as the manatees. Sirenia, the sea cow group, shares a common ancestor with Proboscidea, the elephants, even though they now inhabit quite different environments. A comparison was made of their zonary endothelial type of placentation. 3) Placentation in sharks and rays. The remarkable placentation of hammerhead sharks and manta rays is described. 4) Placentation in the Antarctic minke whale. Placental tissue samples of this whale were obtained from the Japan Institute of Cetacean Research. In an ultrastructural study of the utero-placental junction, microfilamental processes of the allantochorionic zone and crypt formation were visualized.

Amino acid starvation culture condition sensitizes EGFR-expressing cancer cell lines to gefitinib-mediated cytotoxicity by inducing atypical necroptosis

The maintenance of the intracellular level of amino acids is crucial for cellular homeostasis. This is carried out via the regulation of both the influx from the extracellular environment and the recycling of intracellular resources. Since epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors, including gefitinib (GEF) have been reported to induce the apoptosis of several cancer cell lines, in the present study, we examined whether the cytotoxic effects of GEF are further enhanced under amino acid starvation (AAS) culture conditions. Under AAS culture conditions, the cell killing effect of GEF was synergistically pronounced in the EGFR-expressing cell lines, namely, CAL 27, Detroit 562, A549 and PANC-1 cells compared with those treated with either GEF or AAS alone. The addition of essential amino acids, but not non-essential amino acids to the cell culture medium resulted in the cancellation of this pronounced cytotoxicity. The knockdown of L-type amino acid transporter 1 (LAT-1) by siRNA also enhanced GEF-induced cytotoxicity. Therefore, the shortage of the intracellular amino acid pool appears to determine the sensitivity to GEF. Notably, this enhanced cytotoxicity is not mediated by the induction of apoptosis, but is accompanied by the pronounced induction of autophagy. The presence of necrostatin-1, an inhibitor of receptor-interacting serine/threonine-protein kinase 1 (RIPK-1), but not that of Z-VAD-fmk, attenuated the cytotoxic effects of GEF under AAS culture conditions. Electron microscopy demonstrated that the CAL 27 cells treated with GEF under AAS culture conditions exhibited swelling of the cytosol and organelles with an increased number of autophagosomes and autolysosomes, but without chromatin condensation and nuclear fragmentation. Autophagic cell death was excluded as the inhibition of autophagy did not attenuate the cytotoxicity. These results strongly suggest the induction of necroptosis in response to GEF under AAS culture conditions. However, we could not detect any phosphorylation of RIPK-1 and mixed lineage kinase domain like pseudokinase (MLKL), as well as any necrosome formation. Therefore, the enhanced cytotoxic effect of GEF under AAS culture conditions is thought to be mediated by atypical necroptosis.

Visualization of ceramide channels in lysosomes following endogenous palmitoyl-ceramide accumulation as an initial step in the induction of necrosis

In this study, we showed that the dual addition of glucosyl ceramide synthase and ceramidase inhibitors to A549 cell culture led to the possibility of ceramide channel formation via endogenous palmitoyl-ceramide accumulation with an increase in cholesterol contents in the lysosome membrane as an initial step prior to initiation of necrotic cell death. In addition, the dual addition led to black circular structures of 10–20 nm, interpreted as stain-filled cylindrical channels on transmission electron microscopy. The formation of palmitoyl-ceramide channels in the lysosome membrane causes the liberation of cathepsin B from lysosomes for necrotic cell death. On the other hand, necrotic cell death in the dual addition was not caused by oxidative stress or cathepsin B activity, and the cell death was free from the contribution of the translation of Bax protein to the lysosome membrane.