The proton pump inhibitor lansoprazole (LPZ) inhibits the growth of several cancer cell lines, including A549 and CAL 27. We previously reported that macrolide antibiotics such as azithromycin (AZM) and clarithromycin (CAM) potently inhibit autophagic flux and that combining AZM or CAM with the epidermal growth factor receptor inhibitors enhanced their antitumor effect against various cancer cells. In the present study, we conducted the combination treatment with LPZ and macrolide antibiotics against A549 and CAL 27 cells and evaluated cytotoxicity and morphological changes using cell proliferation and viability assays, flow cytometric analysis, immunoblotting, and morphological assessment. Combination therapy with LPZ and AZM greatly enhanced LPZ‑induced cell death, whereas treatment with AZM alone exhibited negligible cytotoxicity. The observed cytotoxic effect was not mediated through apoptosis or necroptosis. Transmission electron microscopy of A549 cells treated with the LPZ + AZM combination revealed morphological changes associated with necrosis and accumulated autolysosomes with undigested contents. Furthermore, the A549 cell line with ATG5 knockout exhibited complete inhibition of autophagosome formation, which did not affect LPZ + AZM treatment‑induced cytotoxicity, thus excluding the involvement of autophagy‑dependent cell death in LPZ + AZM treatment‑induced cell death. A549 cells treated with LPZ + AZM combination therapy retained the endosomal Alexa‑dextran for extended duration as compared to untreated control cells, thus indicating impairment of lysosomal digestion. Notably, lysosomal galectin‑3 puncta expression induced due to lysosomal membrane permeabilization was increased in cells treated with LPZ + AZM combination as compared to the treatment by either agent alone. Collectively, the present results revealed AZM‑induced autolysosome accumulation, potentiated LPZ‑mediated necrosis, and lysosomal membrane permeabilization, thus suggesting the potential clinical application of LPZ + AZM combination therapy for cancer treatment.

In this paper, a method using cavitation jet is proposed to remove marine creatures adhered to the body of a ship. The flow with the cavitation jet is produced using a high-pressure pump and cavitation nozzle with an orifice plate. Rebound shock waves are expected to be continuously generated in the cavitation jet flow. In order to observe the behaviors of the rebound shock waves, experiments are carried out using the Schlieren method in a water tank. From the results of the visualization, it is found the generation frequency of the rebound shock wave reaches its peak at a close position to the nozzle exit. The behaviors of the rebound shock waves are also investigated under the influence of a wall boundary beside a cavitation jet. The results show that the position where the maximum generation frequency is obtained is at several ten times of an orifice diameter from the nozzle exit, and the generation frequency decreases due to the effect of the wall boundary.

A theoretical model for estimating inactivation effects on marine Vibrio sp. is developed from the viewpoint of the chemical action of the OH radicals induced by interaction of bubbles with shock waves. It consists of a biological probability model for cell viability and a bubble dynamic model for its collapsing motion due to the shock pressures. The biological probability model is built by defining a sterilized space of the OH radicals. To determine the radius of the sterilized space, the Herring equation is solved in the bubble dynamic model in consideration of the effect of the heat conductivity and mass transportation. Furthermore, the pressure waveform of incident shock wave used in the model is obtained with the pressure measurement. On the other hand, a bio-experiment of marine Vibrio sp. is carried out using a high-voltage power supply in a cylindrical water chamber. Finally, the viability ratio of marine bacteria estimated by the theoretical model is examined under the experimental conditions of this study. In addition, we also discuss the influence of bubble initial size for predicting the inactivation effects.

Autophagy, a self-digestive system for cytoplasmic components, is required to maintain the amino acid pool for cellular homeostasis. We previously reported that the macrolide antibiotics azithromycin (AZM) and clarithromycin (CAM) have an inhibitory effect on autophagy flux, and they potently enhance the cytocidal effect of various anticancer reagents in vitro. This suggests that macrolide antibiotics can be used as an adjuvant for cancer chemotherapy. Since cancer cells require a larger metabolic demand than normal cells because of their exuberant growth, upregulated autophagy in tumor cells has now become the target for cancer therapy. In the present study, we examined whether macrolides exhibit cytotoxic effect under an amino acid-starving condition in head and neck squamous cancer cell lines such as CAL 27 and Detroit 562 as models of solid tumors with an upregulated autophagy in the central region owing to hypovascularity. AZM and CAM induced cell death under the amino acid-depleted (AAD) culture condition in these cell lines along with CHOP upregulation, although they showed no cytotoxicity under the complete culture medium. CHOP knockdown by siRNA in the CAL 27 cells significantly suppressed macrolide-induced cell death under the AAD culture condition. CHOP-/- murine embryonic fibroblast (MEF) cell lines also attenuated AZM-induced cell death compared with CHOP+/+ MEF cell lines. Using a tet-off atg5 MEF cell line, knockout of atg5, an essential gene for autophagy, also induced cell death and CHOP in the AAD culture medium but not in the complete culture medium. This suggest that macrolide-induced cell death via CHOP induction is dependent on autophagy inhibition. The cytotoxicity of macrolide with CHOP induction was completely cancelled by the addition of amino acids in the culture medium, indicating that the cytotoxicity is due to the insufficient amino acid pool. These data suggest the possibility of using macrolides for "tumor-starving therapy".

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 metastatic breast cancer therapy.

Underwater explosion through complex medias is one of the research topics related to strong impulsive force in structure(oil plat form, offshore platform and ship) for disaster prevention from industrial accident explosions

This paper reports the observation and analysis of the microbubble motion induced by an underwater shock wave. In the analysis, Herring’s bubble motion equation was numerically solved using an experimental shock wave pressure profile. The pressure attenuation of the rebound shock wave of a microbubble was also estimated by numerical simulation. The motion behaviors of the microbubbles during their interaction with an electric discharge shock wave, such as their rebound, shock wave generation, and microjet formation, were observed by magnified visualization. To improve the observation accuracy, spatial positioning control of the microbubbles was employed. The experimentally determined time variation of the diameter of the microbubbles when they collapsed spherically was in agreement with the results of the numerical analyses, and the latter also revealed a very high pressure of the rebound shock wave. There were, however, discrepancies between the experimental and analytical results for non-spherical collapse. It is thought that spherical collapse produces stronger rebound shock waves and that the probability of such collapse increases with decreasing diameter of the bubble. In addition, it was demonstrated that single and multiple microbubbles moved vigorously after interaction with a shock wave and the latter coalesced into a single bubble within several hundred microseconds.

In 1988 we produced lead azide pellets in house and studied propagation of microunderwater shock wave and its interaction with air bubble. Later we replaced lead azide pellets with silver azide pellets supplied by Chugiku Kayaku Co. Ltd, weighing a few mg to 30 mg [1] and devoted to safely study underwater shock waves and its application to medicine.

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.

The specific 26S proteasome inhibitor bortezomib (BZ) potently induces autophagy, endoplasmic reticulum (ER) stress and apoptosis in multiple myeloma (MM) cell lines (U266, IM-9 and RPMI8226). The macrolide antibiotics including concanamycin A, erythromycin (EM), clarithromycin (CAM) and azithromycin (AZM) all blocked autophagy flux, as assessed by intracellular accumulation of LC3B-II and p62. Combined treatment of BZ and CAM or AZM enhanced cytotoxicity in MM cell lines, although treatment with either CAM or AZM alone exhibited almost no cytotoxicity. This combination also substantially enhanced aggresome formation, intracellular ubiquitinated proteins and induced the proapoptotic transcription factor CHOP (CADD153). Expression levels of the proapoptotic genes transcriptionally regulated by CHOP (BIM, BAX, DR5 and TRB3) were all enhanced by combined treatment with BZ plus CAM, compared with treatment with each reagent alone. Like the MM cell lines, the CHOP+/+ murine embryonic fibroblast (MEF) cell line exhibited enhanced cytotoxicity and upregulation of CHOP and its transcriptional targets with a combination of BZ and one of the macrolides. In contrast, CHOP-/- MEF cells exhibited resistance against BZ and almost completely canceled enhanced cytotoxicity with a combination of BZ and a macrolide. These data suggest that ER stress-mediated CHOP induction is involved in pronounced cytotoxicity. Simultaneously targeting two major intracellular protein degradation systems such as the ubiquitin-proteasome system by BZ and the autophagy-lysosome system by a macrolide antibiotic enhances ER stress-mediated apoptosis in MM cells. This result suggests the therapeutic possibility of using a macrolide antibiotic with a proteasome inhibitor for MM therapy.

2-Aminophenoxazine-3-one (Phx-3) induces apoptosis in several types of cancer cell lines. However, the mechanism of apoptosis induction by Phx-3 has not been fully elucidated. In this study, we investigated the anticancer effects of Phx-3 in the glioblastoma cell line LN229 and analyzed its molecular mechanism. The results indicated that 6- and 20-h treatment with Phx-3 significantly induced apoptosis in LN229 cells, with downregulation of survivin and XIAP. Both ERK and JNK, which are the members of the MAPK family, were activated after treatment with Phx-3. Inhibition of ERK using the specific inhibitor U0126 blocked the Phx-3-induced apoptosis only in part. However, inhibition of JNK using the specific inhibitor SP600125 completely prevented Phx-3-induced apoptosis and restored the phosphorylation states of ERK to the control levels. Enhanced generation of reactive oxygen species (ROS) was detected after 3-h treatment with Phx-3. In addition, the ROS scavenger melatonin almost completely blocked Phx-3-induced JNK activation and apoptosis. This suggests that JNK activation was mediated by Phx-3-induced ROS generation. Although SP600125 and melatonin completely blocked the reduction of mitochondrial membrane potential after a 3-h treatment with Phx-3, extension of Phx-3 exposure time to 20 h resulted in no cancelation of mitochondrial depolarization by these reagents. These reagents also had little effect on the decreased expression of survivin and XIAP during a 3-20-h exposure to Phx-3. These results indicate that the production of ROS following JNK activation is the main axis of Phx-3-induced apoptosis in LN229 cells for short-term exposure to Phx-3, whereas alternative mechanism(s) appear to be involved in apoptosis induction during long-term exposure to Phx-3.

The environmental destruction of marine ecosystem caused by micro-organism included in ship ballast water has been a global problem. The International Maritime Organization (IMO) adopted strict standard rule for control and management of ship ballast water in 2004 [1], after that, many ballast water treatment systems have been proposed and developed all over the world. However, a lot of practical problems have been remaining yet for development of energy-saving and space-saving systems. In general systems, shipping companies have to charge and manage chemicals on board to kill marine bacteria in the ballast water. Therefore, if troubles should happen in the ballast water treatment process, leaking chemicals from the system might contaminate the sea. In order to realize more secure and environmental friendly treatment method, the authors have proposed a new sterilization technique of ship ballast water using underwater shock waves. In the previous research, the result showed that a marine Vibrio sp. was completely inactivated when the excess pressure in cell solution contained in a small aluminum container was over 200 MPa in impact experiments by a gas gun [2]. In general, electric discharge, explosive or high-speed collision of a projectile is used as a power source to produce underwater shock waves and has been applied to engineering and medical fields. However, those power sources are unsuitable for practical use on board from a point of view on energy cost and safety. From the above-mentioned, the authors thought of killing marine bacteria in a large amount of ballast water by exposing to strong pressure pulses and free radicals created from collapse of microbubbles [3-6]. In this idea, the excess pressure of underwater shock waves plays only a role of leading to collapse of microbubbles, so that it does not necessarily need to release an extreme high-energy in water. Therefore, the method of hitting water surface with shock waves produced in gas would be an effective one of underwater shock wave generation on board. In addition, it needs to develop the shock wave generator that can produce shock waves periodically by low driving cost for practical use.

Abstract Abstract 4992 Macroautophagy (hereafter, “autophagy”) is a highly conserved cellular process of self-degradation in eukaryotes. Intracellular proteins and organelles including the endoplasmic reticulum (ER) are engulfed in a double-membrane vesicle called an autophagosome and are delivered to lysosomes for degradation by lysosomal hydrolases. Autophagy has been regarded as a bulk non-selective degradation system for long-lived proteins and organelles, in contrast to the specific degradation of polyubiquitinated short-lived proteins by proteasome. However, recent reports revealed the selective degradation pathway of ubiquitinated protein through autophagy via docking proteins such as p62 and the related protein NBR1, having both a microtubule-associated protein 1 light chain 3 (LC3)-interacting region and a ubiquitin-associated domain. LC3 is essential for autophagy and is associated with autophagosome membranes after processing. By binding ubiquitin via their C-terminal ubiquitin-associated domains, p62-mediated degradation of ubiquitinated cargo occurs by selective autophagy. Thus the two major intracellular degradation systems are directly linked. We have reported on the inhibition of autophagy using the autophagy inhibitor bafilomycin A1enhanced bortezomib (BZ)-induced apoptosis by burdening ER stress in multiple myeloma (MM) cell lines. It was also reported that clarithromycin (CAM) attenuated or blocked autophagy flux, probably mediated through inhibiting the lysosomal function. We therefore investigated whether simultaneous inhibition of protein degradation systems such as the ubiquitin-proteasome system by BZ and the autophagy-lysosome system by a macrolide antibiotic enhances the loading of ER-stress and ER–stress-mediated CHOP (CADD153) induction, followed by transcriptional activation for proapoptotic genes. BZ potently induces autophagy, ER–stress, and apoptosis in MM cell lines (e. g. U266, IM-9, and RPMI8226). The macrolide antibiotics including CAM, concanamycin A, erythromycin (EM), and azithromycin (AZM) all blocked autophagy flux, as assessed by intracellular accumulation of LC3B-II and p62. Combined treatment of BZ and CAM or AZM enhanced cytotoxicity in MM cell lines, although treatment with either CAM or AZM alone exhibited almost no cytotoxicity. This combination also substantially enhanced aggresome formation, intracellular ubiquitinated proteins, and induced the proapoptotic transcription factor CHOP. Expression levels of the proapoptotic genes transcriptionally regulated by CHOP (e. g. BIM, BAX, DR5, and TRB3) were all enhanced by combined treatment with BZ plus CAM, compared with treatment with each reagent alone. Like the MM cell lines, the CHOP+/+ murine embryonic fibroblast (MEF) cell line exhibited enhanced cytotoxicity and up-regulation of CHOP and its transcriptional targets with a combination of BZ and one of the macrolides. In contrast, CHOP−/− MEF cells exhibited resistance against BZ and almost completely canceled enhanced cytotoxicity with a combination of BZ and a macrolide. These data suggest that ER-stress mediated CHOP induction is involved in pronounced cytotoxicity. Simultaneously targeting two major intracellular protein degradation systems such as the ubiquitin-proteasome system by BZ and the autophagy-lysosome system by a macrolide antibiotic enhances ER-stress-mediated apoptosis in MM cells. This result suggests the therapeutic possibility of using a macrolide antibiotic with a proteasome inhibitor for MM therapy. Disclosures: No relevant conflicts of interest to declare.

In A549 cells, the addition of D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (DL-PDMP) led to marked autophagy with massive microtubule-associated protein 1 light chain 3B (LC3B)-II protein expression as an indication of autophagy and a steep decrease of p62 protein as a co-indication of autophagy. The addition of DL-PDMP caused massive autophagy with an increase of CAAT/enhancer binding protein homologous protein (CHOP) expression as the marker of endoplasmic reticulum (ER) stress, lactate dehydrogenase (LDH) release without caspase 3 activation and many autophagic vacuoles/devoid of a cell membrane on morphology. On the other hand, the addition of DL-PDMP caused an increase in cellular or subcellular ceramides (Cers), especially palmitoyl-Cer, based on de novo synthesis of Cer, and led to caspase-independent apoptosis. Marked increases of Cer levels in the nuclear envelope were observed 17 h after the addition. The elevations of Cer synthase activity and longevity-assurance homologue (LASS)5 protein expression were observed in subcellular fractions from 30 min until 2 h after the addition. However, the elevations of Cer synthase activity were independent of reactive oxygen species generation or cytochrome P450 4F2 activity. Since an increase in LASS5 protein expression in subcellular fraction occur in preference to the variation of LC3B-II protein expression via CHOP expression after the addition and Cer accumulation induced by the addition contributes to ER stress, it is thought that an elevation of Cer synthase activity via LASS5 protein expression associate to autophagy via CHOP expression (ER stress) with the addition.

β-Carboline alkaloids are naturally occurring plant substances that have a wide spectrum of neuropharmacological, psychopharmacological, and antitumor effects. Recently, we have demonstrated that harmol, a β-carboline alkaloid, induces apoptosis by caspase-8 activation independently from Fas/Fas ligand interaction in human non-small cell lung cancer (NSCLC) H596 cells. Here, we found that harmol induces autophagy and cell death in human NSCLC A549 cells. Although harmol induced cell death in A549 cells in a significant dose- and time-dependent manner, it did not induce caspase-3, caspase-8, or caspase-9 activity. Furthermore, cleavage of poly-(ADP-ribose)-polymerase was not induced in A549 cells by harmol treatment. Autophagy, but not apoptosis, was detected by electron microscopy in A549 cells treated with 70 µM harmol. Pretreatment of A549 cells with 3-methyladenine, an autophagy inhibitor, as well as small interfering RNA (siRNA)-mediated knockdown of LC3, both suppressed harmol-induced cell death. These suggest that the induction of autophagy by harmol precedes cell death. The cytotoxicity of some anticancer agents is reportedly linked to autophagy induction. The 2 major autophagy regulatory pathways are the Akt/mammalian target of rapamycin (mTOR) pathway and the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. Although harmol treatment showed no effect on the Akt/mTOR pathway, it transiently activated the ERK1/2 pathway. However, inhibition of the ERK1/2 pathway using the mitogen-activated protein kinase (MEK)/ERK inhibitor U0126 partially suppressed autophagy. Therefore, although activation of the ERK1/2 pathway might be related to harmol-induced autophagy, another major pathway may also be involved in A549 cells.

The meat quality of farmed yellowtail Seriola quinqueradiata fed on extruded pellets (EP) containing 0.5% (v/v) red pepper (experimental group) was compared with yellowtail of the same age fed on EP (control group). In 1-year-old yellowtail, the crude lipid content of the dorsal muscle of the experimental group tended to be lower than that of the control group. In contrast, there was no difference in the lipid content of the dorsal muscle between the control group and the experimental group in 2-year-old yellowtail. The muscle texture of the experimental group was significantly firmer than that of the control group, with the effect of red pepper unrelated to fish age and lipid content. Color change of red muscle of the experimental group was significantly lower than that of the control group, and the content of thiobarbituric acid-reactive substances in the red muscle was significantly lower in the experimental group than in the control group. These results are the first to demonstrate that the inclusion of red pepper in the diet is able to reduce the loss of muscle texture firmness and to slow down color change in red muscle of yellowtail.