Ataxia-Telangiectasia Mutated Is Involved in Autolysosome Formation

Ataxia-telangiectasia mutated (ATM), a master kinase of the DNA damage response (DDR), phosphorylates a multitude of substrates to activate signaling pathways after DNA double-strand breaks (DSBs). ATM inhibitors have been evaluated as anticancer drugs to potentiate the cytotoxicity of DNA damage-based cancer therapy. ATM is also involved in autophagy, a conserved cellular process that maintains homeostasis by degrading unnecessary proteins and dysfunctional organelles. In this study, we report that ATM inhibitors (KU-55933 and KU-60019) provoked accumulation of autophagosomes and p62 and restrained autolysosome formation. Under autophagy-inducing conditions, the ATM inhibitors caused excessive autophagosome accumulation and cell death. This new function of ATM in autophagy was also observed in numerous cell lines. Repression of ATM expression using an siRNA inhibited autophagic flux at the autolysosome formation step and induced cell death under autophagy-inducing conditions.Taken together, our results suggest that ATM is involved in autolysosome formation and that the use of ATM inhibitors in cancer therapy may be expanded.

Sensitivity to tumor development by TALEN-mediated Trp53 mutant genes in the susceptible FVB/N mice and the resistance C57BL/6 mice / 한국실험동물학회지

Background@#This study was undertaken to compare the sensitivities of mice strains during tumor induction by transcription activator-like effector nucleases (TALEN)-mediated Trp53 mutant gene. Alterations of their tumorigenic phenotypes including survival rate, tumor formation and tumor spectrum, were assessed in FVB/N-Trp53 em2Hwl /Korl and C57BL/6-Trp53 em1Hwl /Korl knockout (KO) mice over 16 weeks. @*Results@#Most of the physiological phenotypes factors were observed to be higher in FVB/N-Trp53 em2Hwl /Korl KO mice than C57BL/6-Trp53 em1Hwl /Korl KO mice, although there were significant differences in the body weight, immune organ weight, number of red blood cells, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), total bilirubin (Bil-T) and glucose (Glu) levels in the KO mice relative to the wild type (WT) mice. Furthermore, numerous solid tumors were also observed in various regions of the surface skin of FVB/N-Trp53 em2Hwl /Korl KO mice, but were not detected in C57BL/6-Trp53 em1Hwl /Korl KO mice. The most frequently observed tumor in both the Trp53 KO mice was malignant lymphoma, while soft tissue teratomas and hemangiosarcomas were only detected in the FVB/N-Trp53 em2Hwl /Korl KO mice. @*Conclusions@#Our results indicate that the spectrum and incidence of tumors induced by the TALEN-mediated Trp53 mutant gene is greater in FVB/N-Trp53 em2Hwl /Korl KO mice than C57BL/6-Trp53 em1Hwl /Korl KO mice over 16 weeks.

Dose dependence and durability of the therapeutic effects of Asparagus cochinchinensis fermented extract in an ovalbumin-challenged asthma model / 한국실험동물학회지

The butanol extract of Asparagus cochinchinensis roots fermented with Weissella cibaria (BAfW) significantly suppressed the inflammatory response induced by lipopolysaccharide (LPS) treatment in RAW264.7 cells. To investigate the dose dependence and durability of BAfW on the anti-asthma effects, alterations in key parameters were measured in ovalbumin (OVA)-challenged Balb/c mice treated with the different doses of BAfW at three different time points. The number of immune cells, OVA-specific IgE level, thickness of respiratory epithelium and mucus score decreased significantly in a dose-dependent manner in response to treatment with 125 to 500 mg/kg BAfW (P < 0.05), although the highest level was detected in the 500 mg/kg treated group. Moreover, the decrease in these parameters was maintained from 24 to 48 h in the 500 mg/kg of BAfW treated group. At 72 h, the effects of BAfW on the number of immune cells, OVA-specific IgE level and thickness of respiratory epithelium partially disappeared. Overall, this study provides the first evidence that the anti-asthma effect of BAfW may reach the maximum level in OVA-challenged Balb/c mice treated with 500 mg/kg and that these effects can last for 48 h.

Successful development of squamous cell carcinoma and hyperplasia in RGEN-mediated p27 KO mice after the treatment of DMBA and TPA / 한국실험동물학회지

To evaluate the carcinogenicity of p27 knockout (KO) mice with RNA-guided endonuclease (RGENs)-mediated p27 mutant exon I gene (IΔ), alterations in the carcinogenic phenotypes including tumor spectrum, tumor suppressor proteins, apoptotic proteins and cell cycle regulators were observed in p27 (IΔ) KO mice after treatment with 7,12-Dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA)(DT) for 5 months. The target region (544~571 nt) in exon I of the p27 gene was successfully disrupted in p27 (IΔ) KO mice using the RGEN-induced non-homologous end joining (NHEJ) technique. After DT exposure for 5 months, a few solid tumors (identified as squamous cell carcinoma) developed on the surface of back skin of DT-treated p27 (IΔ) KO mice. Also, squamous cell hyperplasia with chronic inflammation was detected in the skin dermis of DT-treated p27 (IΔ) KO mice, while the Vehicle+p27 (IΔ) KO mice and WT mice maintained their normal histological skin structure. A significant increase was observed in the expression levels of tumor suppressor protein (p53), apoptotic proteins (Bax, Bcl-2 and Caspase-3) and cell-cycle regulator proteins (Cyclin D1, CDK2 and CDK4) in the skin of DT-treated p27 (IΔ) KO mice, although their enhancement ratio was varied. Taken together, the results of the present study suggest that squamous cell carcinoma and hyperplasia of skin tissue can be successfully developed in new p27 (IΔ) KO mice produced by RGEN-induced NHEJ technique following DT exposure for 5 months.

Inhibition of endoplasmic reticulum stress in high-fat-diet-induced obese C57BL/6 mice: Efficacy of a novel extract from mulberry (Morus alba) leaves fermented with Cordyceps militaris / 한국실험동물학회지

A few clues about correlation between endoplasmic reticulum (ER) stress and mulberry (Morus alba) leaves were investigated in only the experimental autoimmune myocarditis and streptozotocin-induced diabetes. To investigate whether a novel extract of mulberry leaves fermented with Cordyceps militaris (EMfC) could suppress ER in fatty liver, alterations in the key parameters for ER stress response were measured in high fat diet (HFD)-induced obese C57L/6 mice treated with EMfC for 12 weeks. The area of adipocytes in the liver section were significantly decreased in the HFD+EMfC treated group as compared to the HFD+Vehicle treated group, while their level was higher in HFD+Vehicle treated group than No treated group. The level of the eukaryotic initiation factor 2 alpha (eIF2α) and inositol-requiring enzyme 1 beta (IRE1α) phosphorylation and CCAAT-enhancer-binding protein homologous protein (CHOP) expression were remarkably enhanced in the HFD+Vehicle treated group. However, their levels were restored in the HFD+EMfC treated group, although some differences were detected in the decrease rate. Similar recovery was observed on the ER stress-induced apoptosis. The level of Caspase-3, Bcl-2 and Bax were decreased in the HFD+EMfC and HFD+orlistat (OT) treated group compared to the HFD+Vehicle treated group. The results of the present study therefore provide first evidence that EMfC with the anti-obesity effects can be suppressed ER stress and ER stress-induced apoptosis in the hepatic steatosis of HFD-induced obesity model.

Regulation of gastrointestinal hormones during laxative activity of gallotannin-enriched extract isolated from Galla Rhois in loperamide-induced constipation of SD rats / 한국실험동물학회지

Regulation of gastrointestinal hormones have been reported in animal models for constipation undergoing laxative therapy when administered herbal products. We undertook to investigate whether the laxative activity of gallotannin-enriched extracts isolated from Galla Rhois (GEGR) affects the regulation of gastrointestinal hormones, by examining the concentration of four hormones and the activation of their receptors in the loperamide (Lop)-induced constipation model. Stool parameters, including number, weight and water content, were significantly recovered in the Lop+GEGR treated group, relative to the Lop+vehicle treated group; however, food intake and water consumption were maintained at a constant level. Also, a similar recovery was detected for thickness of mucosa, muscle and flat luminal surface in the Lop+GEGR treated group. Furthermore, concentration of the four gastrointestinal hormones evaluated, namely, cholecystokinin (CCK), gastrin (GAS), somatostatin (SS) and motilin (MTL), were lower in the Lop+vehicle treated group than the No treated group, but were remarkably enhanced in the Lop+GEGR treated group. Moreover, the downstream signaling pathway of MTL and SS receptors were recovered after GEGR administration. Results of the present study therefore indicate that the laxative effects of GEGR treatment may be tightly related with the regulation of gastrointestinal hormones in the Lop-induced constipation model.

Correlation between laxative effects of uridine and suppression of ER stress in loperamide induced constipated SD rats / 한국실험동물학회지

A correlation between endoplasmic reticulum (ER) stress and laxative effects was first reported in a constipation model treated with an aqueous extract of Liriope platyphylla (AEtLP) roots. To investigate the correlation between the laxative effect of uridine (Urd) and ER stress response, alterations in the key parameters for ER stress were measured in loperamide (Lop) induced constipation Sprague Dawley (SD) rats treated with Urd. The efficacy of the laxative effect of Urd was notable on the symptoms of chronic constipation, including alteration of stool parameters and structure of the transverse colon, in Lop induced constipated SD rats. In the PERK/eIF2-ATF4 pathway of ER stress response, the levels of eukaryotic initiation factor 2 alpha (eIF2α) phosphorylation and DNA damage-inducible protein (GADD34) transcripts were significantly enhanced in the Lop+Vehicle treated group. However, the levels were restored in the Lop+Urd treated group, although few differences were detected in the decrease rate. Similar changes were observed for levels of inositol-requiring enzyme 1 beta (IRE1β) phosphorylation and X-box binding protein 1 (XBP-1) transcript in the IRE1α/XBP pathway. Furthermore, the number of ER stress-induced apoptotic cells and Bax and Bcl-2 expression were recovered in the Lop+Urd treated group compared to the Lop+Vehicle treated group. The results of the present study therefore provide first evidence that the laxative effects of Urd may be tightly correlated with the recovery of ER stress response in constipation models.

Hepatotoxicity and nephrotoxicity of saponin-enriched extract of Asparagus cochinchinensis in ICR mice / 한국실험동물학회지

The inhibitory effects of Asparagus cochinchinensis against inflammatory response induced by lipopolysaccharide (LPS), substance P and phthalic anhydride (PA) treatment were recently reported for some cell lines and animal models. To evaluate the hepatotoxicity and nephrotoxicity of A. cochinchinensis toward the livers and kidneys of ICR mice, alterations in related markers including body weight, organ weight, urine composition, liver pathology and kidney pathology were analyzed in male and female ICR mice after oral administration of 150, 300 and 600 mg/kg body weight/day saponin-enriched extract of A. cochinchinensis (SEAC) for 14 days. The saponin, total flavonoid and total phenol levels were found to be 57.2, 88.5 and 102.1 mg/g in SEAC, respectively, and the scavenging activity of SEAC gradually increased in a dose-dependent manner. Moreover, body and organ weight, clinical phenotypes, urine parameters and mice mortality did not differ between the vehicle and SEAC treated group. Furthermore, no significant alterations were measured in alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), blood urea nitrogen (BUN) and the serum creatinine (Cr) in the SEAC treated group relative to the vehicle treated group. Moreover, the specific pathological features induced by most toxic compounds were not observed upon liver and kidney histological analysis. Overall, the results of the present study suggest that SEAC does not induce any specific toxicity in the livers and kidneys of male and female ICR mice at doses of 600 mg/kg body weight/day.

Erratum: Comparative study of fertilization rates of C57BL/6NKorl and C57BL/6N mice obtained from two other sources / 한국실험동물학회지

One of the authors' names was misprinted.

Lipolytic effect of novel extracts from mulberry (Morus alba) leaves fermented with Cordyceps militaris in the primary adipocytes derived from SD rats / 한국실험동물학회지

Mulberry (Morus alba) leaves are known to have therapeutic effects on lipid metabolism including lipogenesis, lipolysis and hyperlipidemia. However, novel compounds with strong lipolytic ability among 27 extracts of the mulberry leaves fermented with Cordyceps militaris (EMfCs) have not yet been identified. Therefore, the cAMP concentration and cell viability were measured in the primary adipocytes of SD (Sprague Dawley) rats and 3T3-L1 cells after treatment of 27 EMfCs. Briefly, mulberry leaves powders amended with three different concentrations (0, 25 and 50%) of silkworm pupae (SWP) powder were fermented with 10% C. militaris (v/w) during three different periods (3, 4 and 6 weeks). A total of 27 extracts were obtained from the fermented mulberry leaves powders using three different solvents (dH2O, 50% EtOH and 95% EtOH). Among the 27 EMfCs treated groups, a significant increase in the concentration of cAMP was detected in primary adipocytes treated with 10 extracts when compared with the Vehicle treated group. However, their cAMP concentration did not agree completely with the non-toxicity, although most extracts showed non-toxicity. Furthermore, the concentration of cAMP and level of free glycerol gradually increased in a dose dependent manner (100, 200 and 400 µg/mL) of 4M3-95 contained cordycepin without any significant toxicity. Overall, the results of this study provide strong evidence that 4M3-95 extract derived from EMfCs can stimulate the lipolysis of primary adipocytes at an appropriate concentration and therefore have the potential for use as lipolytic agents to treat obesity.

Comparison of therapeutic responses to an anticancer drug in three stocks of ICR mice derived from three different sources / 한국실험동물학회지

Korl:ICR mice, established by the Korean National Institute of Food and Drug Safety Evaluation (NIFDS), are characterized based on their genetic variation, response to gastric injury, and response to constipation inducers. To compare the inhibitory responses of ICR stocks obtained from three different sources to the anticancer drug cisplatin (Cis), alterations in tumor volume, histopathological structure, and toxicity were examined in Sarcoma 180 tumor-bearing Korl:ICR, A:ICR (USA source), and B:ICR (Japan source) mice treated with low and high concentrations of Cis (L-Cis and H-Cis, respectively). Tumor size and volume were lower in H-Cis-treated mice than in L-Cis-treated mice in all three ICR stocks with no significant differences among stocks. There was a significant enhancement of the necrotizing areas in the histological structures in the L-Cis- and H-Cis-treated groups relative to that in the untreated group. The necrotizing area changes were similar in the Sarcoma 180 tumor-bearing Korl:ICR, A:ICR, and B:ICR mice. However, there were stock-bases differences in the serum biomarkers for liver and kidney toxic effects. In particular, the levels of AST, ALT and BUN increased differently in the three H-Cis-treated ICR stocks, whereas the levels of ALP and CRE were constant. Taken together, the results of the present study indicate that Korl:ICR, A:ICR, and B:ICR mice have similar overall inhibitory responses following Cis treatment of Sarcoma 180-derived solid tumors, although there were some differences in the magnitude of the toxic effects in the three ICR stocks.

Comparative study of fertilization rates of C57BL/6NKorl and C57BL/6N mice obtained from two other sources / 한국실험동물학회지

C57BL/6N is the most widely used inbred mouse strain applied in a wide variety of research areas including cancer, cardiovascular biology, developmental biology, diabetes and obesity, genetics, immunology, neurobiology, and sensorineural research. To compare the fertilization rates of C57BL/6NKorl mice with two commercial C57BL/6N stocks, differences in reproductive organ structures, sperm and egg numbers, fertilization rates, and embryo development rates among C57BL/6NKorl (Korea FDA source), C57BL/6NA (USA source), and C57BL/6NB (Japan source) mice were determined. Among the stocks, no significant differences were detected in organ weight and histological structure of male and female reproductive organs, although body weight was higher in C57BL/6NKorl mice than that in the other groups. The concentration and morphology of sperm and eggs in C57BL/6NKorl mice were similar to those of C57BL/6NA and C57BL/6NB mice. Furthermore, the three stocks had similar in vitro fertilization and embryo development rates, although these rates tended to be higher in C57BL/6NB mice. Pup body weight was higher in C57BL/6NKorl and C57BL/6NB mice than that in C57BL/6NA mice. The results of the present study suggest that C57BL/6NKorl, C57BL/6NA, and C57BL/6NB mice obtained from three different sources have similar fertilization and embryo development rates, although there were slight differences in the magnitude of their responses rates.