The human allicin-proteome: S-thioallylation of proteins by the garlic defence substance allicin and its biological effects.

A single clove of edible garlic (Allium sativum L.) of about 10 g produces up to 5 mg of allicin (diallylthiosulfinate), a thiol-reactive sulfur-containing defence substance that gives injured garlic tissue its characteristic smell. Allicin induces apoptosis or necrosis in a dose-dependent manner but biocompatible doses influence cellular metabolism and signalling cascades. Oxidation of protein thiols and depletion of the glutathione pool are thought to be responsible for allicin's physiological effects. Here, we studied the effect of allicin on post-translational thiol-modification in human Jurkat T-cells using shotgun LC-MS/MS analyses. We identified 332 proteins that were modified by S-thioallylation in the Jurkat cell proteome which causes a mass shift of 72 Da on cysteines. Many S-thioallylated proteins are highly abundant proteins, including cytoskeletal proteins tubulin, actin, cofilin, filamin and plastin-2, the heat shock chaperones HSP90 and HSPA4, the glycolytic enzymes GAPDH, ALDOA, PKM as well the protein translation factor EEF2. Allicin disrupted the actin cytoskeleton in murine L929 fibroblasts. Allicin stimulated the immune response by causing Zn2+ release from proteins and increasing the Zn2+-dependent IL-1-triggered production of IL-2 in murine EL-4 T-cells. Furthermore, allicin caused inhibition of enolase activity, an enzyme considered a cancer therapy target. In conclusion, our study revealed the widespread extent of S-thioallylation in the human Jurkat cell proteome and showed effects of allicin exposure on essential cellular functions of selected targets, many of which are targets for cancer therapy.

Nuclear heat shock protein 110 expression is associated with poor prognosis and hyperthermo-chemotherapy resistance in gastric cancer patients with peritoneal metastasis.

AIM: To investigate the significance of heat shock protein 110 (HSP110) in gastric cancer (GC) patients with peritoneal metastasis undergoing hyperthermo-chemotherapy. METHODS: Primary GC patients (n = 14) with peritoneal metastasis or positive peritoneal lavage cytology who underwent distal or total gastrectomy between April 2000 and December 2011 were enrolled in this study. The patients underwent postoperative intraperitoneal hyperthermo-chemotherapy using a Thermotron RF-8 heating device two weeks after surgery. We analyzed nuclear HSP110 expression in surgically resected tumors using immunohistochemistry. Additionally, the effect of HSP110 suppression on hyptherthermo-chemosensitivity was assessed in vitro in the MKN45 GC cell line using the HSP inhibitor KNK437. RESULTS: HSP110 immnohistochemical staining in 14 GC patients showed that five (35.7%) samples belonged to the low expression group, and nine (64.3%) samples belonged to the high expression group. Progression-free survival was significantly shorter in the HSP110 high-expression group than in the low-expression group (P = 0.0313). However, no significant relationships were identified between HSP110 expression and the clinicopathological characteristics of patients. Furthermore, high HSP110 expression was not an independent prognostic factor in GC patients with peritoneal metastasis (P = 0.0625). HSP110 expression in MKN45 cells was suppressed by KNK437 at the hyperthermic temperature of 43 °C in vitro. Comparison of MKN45 cell proliferation in the presence and absence of KNK437 at 43 °C, revealed that proliferation was significantly decreased when HSP110 was inhibited by KNK437. Additionally, HSP110 suppression via HSP inhibitor treatment increased cellular sensitivity to hyperthermo-chemotherapy in vitro. CONCLUSION: The expression of nuclear HSP110 in GC patients might be a new marker of chemosensitivity and a therapeutic target for patients who are tolerant to existing hyperthermo-chemotherapies.

A Systems Biology Approach Using Transcriptomic Data Reveals Genes and Pathways in Porcine Skeletal Muscle Affected by Dietary Lysine.

Nine crossbred finishing barrows (body weight 94.4 ± 6.7 kg) randomly assigned to three dietary treatments were used to investigate the effects of dietary lysine on muscle growth related metabolic and signaling pathways. Muscle samples were collected from the longissimus dorsi of individual pigs after feeding the lysine-deficient (4.30 g/kg), lysine-adequate (7.10 g/kg), or lysine-excess (9.80 g/kg) diet for five weeks, and the total RNA was extracted afterwards. Affymetrix Porcine Gene 1.0 ST Array was used to quantify the expression levels of 19,211 genes. Statistical ANOVA analysis of the microarray data showed that 674 transcripts were differentially expressed (at p ≤ 0.05 level); 60 out of 131 transcripts (at p ≤ 0.01 level) were annotated in the NetAffx database. Ingenuity pathway analysis showed that dietary lysine deficiency may lead to: (1) increased muscle protein degradation via the ubiquitination pathway as indicated by the up-regulated DNAJA1, HSP90AB1 and UBE2B mRNA; (2) reduced muscle protein synthesis via the up-regulated RND3 and ZIC1 mRNA; (3) increased serine and glycine synthesis via the up-regulated PHGDH and PSPH mRNA; and (4) increased lipid accumulation via the up-regulated ME1, SCD, and CIDEC mRNA. Dietary lysine excess may lead to: (1) decreased muscle protein degradation via the down-regulated DNAJA1, HSP90AA1, HSPH1, and UBE2D3 mRNA; and (2) reduced lipid biosynthesis via the down-regulated CFD and ME1 mRNA. Collectively, dietary lysine may function as a signaling molecule to regulate protein turnover and lipid metabolism in the skeletal muscle of finishing pigs.

Acupuncture regulates the unfolded protein response and inhibits apoptosis in a rat model of heroin relapse.

OBJECT: To explore the unfolded protein response (UPR) in the hippocampus of rats undergoing heroin relapse and the mechanisms underlying the acupuncture-mediated inhibition of brain damage caused by heroin relapse. METHODS: 60 Sprague-Dawley rats (30 females and 30 males) were randomly divided into four groups: Control group, Heroin group, Heroin+acupuncture group, and Heroin+methadone group (n=15 each). In the latter three groups, a model of heroin addiction was established by successive increments of intramuscular heroin injections for 8 days, according to the exposure (addiction)→detoxification method. A UPR RT2 Profiler PCR array was used to screen for differentially expressed genes in the hippocampus. Apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining. The protein expression levels of the following three differentially expressed genes were detected by Western blot to validate the results of the PCR array: heat shock protein (HSP)70, HSP105, and valosin-containing protein (Vcp). RESULTS: The UPR RT2 Profiler PCR Array detection results indicated that acupuncture increased the expression levels of the molecular chaperones HSP70, HSP105, and Vcp. The degree of neuronal apoptosis in the hippocampus of rats in the Heroin+acupuncture and Heroin+methadone groups was significantly reduced compared with the untreated Heroin group (p<0.01). Protein expression of HSP70, HSP105, and Vcp in the Heroin+acupuncture and Heroin+methadone groups was significantly higher than the Heroin group (p<0.01). CONCLUSIONS: The positive effects of acupuncture on brain damage caused by heroin may be closely related to up-regulation of HSP70, HSP105, and Vcp, and reduced apoptosis.

Hirsutinolide Series Inhibit Stat3 Activity, Alter GCN1, MAP1B, Hsp105, G6PD, Vimentin, TrxR1, and Importin α-2 Expression, and Induce Antitumor Effects against Human Glioma.

We report that hirsutinolide series, 6, 7, 10, 11, 20, and 22, and the semisynthetic analogues, 30, 31, 33, and 36, inhibit constitutively active signal transducer and activator of transcription (Stat)3 and malignant glioma phenotype. A position 13 lipophilic ester group is required for activity. Molecular modeling and nuclear magnetic resonance structural analyses reveal direct hirsutinolide:Stat3 binding. One-hour treatment of cells with 6 and 22 also upregulated importin subunit α-2 levels and repressed translational activator GCN1, microtubule-associated protein (MAP)1B, thioredoxin reductase (TrxR)1 cytoplasmic isoform 3, glucose-6-phosphate 1-dehydrogenase isoform a, Hsp105, vimentin, and tumor necrosis factor α-induced protein (TNAP)2 expression. Active hirsutinolides inhibited anchorage-dependent and three-dimensional spheroid growth, survival, and migration of human glioma lines and glioma patients' tumor-derived xenograft cells harboring constitutively active Stat3. Oral gavage delivery of 6 or 22 inhibited human glioma tumor growth in subcutaneous mouse xenografts. The inhibition of Stat3 signaling represents part of the hirsutinolide-mediated mechanisms to induce antitumor effects.

Patients with colorectal tumors with microsatellite instability and large deletions in HSP110 T17 have improved response to 5-fluorouracil­based chemotherapy.

BACKGROUND & AIMS: Patients with colorectal tumors with microsatellite instability (MSI) have better prognoses than patients with tumors without MSI, but have a poor response to 5-fluorouracil­based chemotherapy. A dominant-negative form of heat shock protein (HSP)110 (HSP110DE9) expressed by cancer cells with MSI, via exon skipping caused by somatic deletions in the T(17) intron repeat, sensitizes the cells to 5-fluorouracil and oxaliplatin.We investigated whether HSP110 T(17) could be used to identify patients with colorectal cancer who would benefit from adjuvant chemotherapy with 5-fluorouracil and oxaliplatin. METHODS: We characterized the interaction between HSP110 and HSP110DE9 using surface plasmon resonance. By using polymerase chain reaction and fragment analysis, we examined how the size of somatic allelic deletions in HSP110 T(17) affected the HSP110 protein expressed by tumor cells. We screened 329 consecutive patients with stage II­III colorectal tumors with MSI who underwent surgical resection at tertiary medical centers for HSP110 T(17). RESULTS: HSP110 and HSP110DE9 interacted in a1:1 ratio. Tumor cells with large deletions in T(17) had increased ratios of HSP110DE9:HSP110, owing to the loss of expression of full-length HSP110. Deletions in HSP110 T(17) were mostly biallelic in primary tumor samples with MSI. Patients with stage II­III cancer who received chemotherapy and had large HSP110 T(17) deletions (≥5 bp; 18 of 77 patients, 23.4%) had longer times of relapse-free survival than patients with small or no deletions (≤4 bp; 59 of 77 patients, 76.6%) in multivariate analysis (hazard ratio, 0.16; 95% confidence interval, 0.012­0.8; P = .03). We found a significant interaction between chemotherapy and T17 deletion (P =.009). CONCLUSIONS: About 25% of patients with stages II­III colorectal tumors with MSI have an excellent response to chemotherapy, due to large, biallelic deletions in the T(17) intron repeat of HSP110 in tumor DNA.

Expression of a mutant HSP110 sensitizes colorectal cancer cells to chemotherapy and improves disease prognosis.

Heat shock proteins (HSPs) are necessary for cancer cell survival. We identified a mutant of HSP110 (HSP110ΔE9) in colorectal cancer showing microsatellite instability (MSI CRC), generated from an aberrantly spliced mRNA and lacking the HSP110 substrate-binding domain. This mutant was expressed at variable levels in almost all MSI CRC cell lines and primary tumors tested. HSP110ΔE9 impaired both the normal cellular localization of HSP110 and its interaction with other HSPs, thus abrogating the chaperone activity and antiapoptotic function of HSP110 in a dominant-negative manner. HSP110ΔE9 overexpression caused the sensitization of cells to anticancer agents such as oxaliplatin and 5-fluorouracil, which are routinely prescribed in the adjuvant treatment of people with CRC. The survival and response to chemotherapy of subjects with MSI CRCs was associated with the tumor expression level of HSP110ΔE9. HSP110 may thus constitute a major determinant for both prognosis and treatment response in CRC.

Expression of material mRNA in the hypothalamus and frontal cortex in a rat model of fatal hypothermia.

Diagnosis of fatal hypothermia is considered to be difficult in forensic practice because of the lack of any specific pathological findings. The mechanism that induces abnormal behavior such as undressing or hiding during the state of hypothermia has not been clarified. In order to solve these problems, we made a rat model of fatal hypothermia and investigated the expression of some mRNA within the hypothalamus and the frontal cortex. The expression of aldehyde dehydrogenase 6 family, member A1 (ALDH6A1), cocaine- and amphetamine-regulated transcript peptide (CARTPT), desmin (DES), heat shock 70kDa protein 4 (HSPA4), serotonin receptor 2A (HTR2A), opioid receptor, delta 1 (OPRD1) and transthyretin (TTR) supposedly related to fatal hypothermia was determined using quantitative real-time PCR. The expression of OPRD1 in the hypothalamus of fatal hypothermia was significantly increased, while the expression of TTR within the frontal cortex was significantly decreased compared to that in the control. These findings suggest that OPRD1 and TTR may be involved in thermoregulation at a low ambient temperature.

Effect of herbal medicine Juzentaihoto on hepatic and intestinal heat shock gene expression requires intestinal microflora in mouse.

AIM: To evaluate the role of intestinal microflora in the effects of multi-herbal medicine on gene expression in the gut and liver. METHODS: The multi-herbal medicine Juzentaihoto (JTX) was administered to five germ-free mice and regular mice for 2 wk. Among the results of the comprehensive gene chip analysis of the intestine and liver, we featured heat shock proteins (HSPs) 70 and 105 because their gene expression changed only in the presence of microflora. Real-time RT-PCR was performed to confirm the expression levels of these HSP genes. To determine whether JTX acts directly on the HSP genes, sodium arsenite (SA) was used to induce the heat shock proteins directly. To examine the change of the intestinal microflora with administration of JTX, the terminal restriction fragment polymorphism (T-RFLP) method was used. To identify the changed bacteria, DNA sequencing was performed. RESULTS: Heat shock protein gene expression, documented by gene chip and real-time RT-PCR, changed with the administration of JTX in the regular mice but not in the germ-free mice. JTX did not suppress the direct induction of the HSPs by SA. T-RFLP suggested that JTX decreased unculturable bacteria and increased Lactobacillus johnsoni. These data suggested that JTX changed the intestinal microflora which, in turn, changed HSP gene expression. CONCLUSION: Intestinal microflora affects multi-herbal product JTX on the gene expression in the gut and liver.

The heat-shock protein Apg-2 binds to the tight junction protein ZO-1 and regulates transcriptional activity of ZONAB.

The tight junction adaptor protein ZO-1 regulates intracellular signaling and cell proliferation. Its Src homology 3 (SH3) domain is required for the regulation of proliferation and binds to the Y-box transcription factor ZO-1-associated nucleic acid binding protein (ZONAB). Binding of ZO-1 to ZONAB results in cytoplasmic sequestration and hence inhibition of ZONAB's transcriptional activity. Here, we identify a new binding partner of the SH3 domain that modulates ZO-1-ZONAB signaling. Expression screening of a cDNA library with a fusion protein containing the SH3 domain yielded a cDNA coding for Apg-2, a member of the heat-shock protein 110 (Hsp 110) subfamily of Hsp70 heat-shock proteins, which is overexpressed in carcinomas. Regulated depletion of Apg-2 in Madin-Darby canine kidney cells inhibits G(1)/S phase progression. Apg-2 coimmunoprecipitates with ZO-1 and partially localizes to intercellular junctions. Junctional recruitment and coimmunoprecipitation with ZO-1 are stimulated by heat shock. Apg-2 competes with ZONAB for binding to the SH3 domain in vitro and regulates ZONAB's transcriptional activity in reporter gene assays. Our data hence support a model in which Apg-2 regulates ZONAB function by competing for binding to the SH3 domain of ZO-1 and suggest that Apg-2 functions as a regulator of ZO-1-ZONAB signaling in epithelial cells in response to cellular stress.