Anti-hyperglycemic Effect of Long-Term Bis(hinokitiolato)zinc Complex ([Zn(hkt)2]) Ingestion on Insulin Resistance and Pancreatic Islet Cells Protection in Type 2 Diabetic KK-Ay Mice.

Zinc (Zn) is a trace element with anti-diabetes mellitus (anti-DM) effects. Zn complexes exhibit stronger insulin-like activity than Zn ions. Bis(hinokitiolato)zinc complex ([Zn(hkt)2]) was recently reported to be a potent anti-DM candidate. We examined the effects of [Zn(hkt)2] on insulin resistance and pancreatic islet cells through in vivo long-term ingestion studies. In an in vivo study, we performed 4-month long-term [Zn(hkt)2] administration experiments in KK-Ay mice as a type 2 DM animal model. Ingestion of [Zn(hkt)2] resulted in lower blood glucose levels compared with the non-treated KK-Ay mice (control group). Additionally, [Zn(hkt)2] treatment decreased plasma insulin concentration compared with that of the non-treated KK-Ay group. [Zn(hkt)2] treatment resulted in a significant suppression of islet cell enlargement and a significantly decreased number of insulin-positive cells compared with the non-treated KK-Ay control group. The [Zn(hkt)2] treatment group showed the increasing tendency in the amount of Zn levels in peripheral organs; liver, muscle, adipose, and pancreas, compared with the non-treated KK-Ay control group. However, the Zn level in the pancreas of the [Zn(hkt)2] treatment group did not show the significant increase compared with the non-treated KK-Ay control group. This accumulation of Zn in pancreas suggested that [Zn(hkt)2] mainly effects on the peripheral tissue, and [Zn(hkt)2] has the less effect on the pancreas directly. Thus, we concluded that [Zn(hkt)2] exerted the main effect on peripheral organs by ameliorating insulin resistance.

Apoptosis, necroptosis and autophagy in colorectal cancer: Associations with tumor aggressiveness and p53 status.

OBJECTIVE: Cleaved caspase-3 (CC3), phosphorylated-mixed-lineage kinase domain-like protein (p-MLKL), and microtubule-associated protein-1 light chain-3B (LC3B) have pivotal functions in apoptosis, necroptosis, and autophagy, respectively. In vitro studies have shown that interaction of these proteins are complex and their roles in cancer can be influenced by many factors. However, these findings are not adequately assessed in human tissues. Here, we determined CC3, p-MLKL, and LC3B expression in colorectal cancers (CRCs), and assessed their associations with clinicopathological parameters, and with KRAS and p53 status. METHODS: We immunohistochemically assessed 113 CRC specimens for levels of CC3, p-MLKL, LC3B, and p53. KRAS gene status was analyzed using the Scorpion- amplification refractory mutation system. RESULTS: High levels of CC3 (CC3High) and LC3B (LC3BHigh) were detected in 38% and 35% of the 113 CRCs, respectively, but no or only a few p-MLKL-positive cells were observed in any of the tumors. CC3High was significantly associated with high pT status (P = 0.03), vascular invasion (P = 0.03) and high pStage (P = 0.04) and was marginally associated with lymph node (P = 0.06) and distant metastases (P = 0.06). LC3BHigh was also significantly associated with high pT status (P = 0.02) and lymphatic invasion (P = 0.002), and was marginally associated with nerve plexus invasion (P = 0.06). In combined analysis, compared with CC3Low/LC3BLow tumors, tumors that were either CC3High, LC3BHigh, or both were significantly associated with high pT status (P = 0.0007), lymphatic invasion (P = 0.03), vascular invasion (P = 0.003), distant metastasis (P = 0.04) and high pStage (P = 0.04). LC3BHigh was significantly associated with a mutant-type expression pattern of p53 (P = 0.003). CONCLUSION: To the best of our knowledge, this is the first study to examine the combination of CC3/LC3B and p-MLKL expression in clinical CRC samples and to correlate these expression data with clinicopathological parameters and EGFR and p53 status. Our results suggest that necroptosis is a rare process in CRC, apoptosis and autophagy are upregulated in aggressive CRCs, and p53 mutation may lead to the upregulation of autophagy.

Autophagy in normal tissues of camel (Camelus dromedarius) with focus on immunoexpression of LC3 and LC3B.

Autophagy is a highly regulated intracellular pathway for degradation and recycling of cytoplasmic protein aggregates and entire organelles. The autophagic pathway is stimulated by nutrient starvation, which prompted us to study the desert camel. Various organs of the camel undergo ecological and physiological stress due to food and water deprivation, dehydration and long exposure to solar radiation. We investigated the immunohistochemical expression of specific biomarkers of autophagy under normal conditions as a baseline for later work on stressed individuals. The autophagy-specific biomarkers, microtubule-associated protein1 light chain 3 (LC3), and its cleaved variant, LC3B, were strongly expressed in the cytosol of all tissues examined. The cytosolic immunoreactivity of LC3 was relatively weak, diffuse and vacuolar, while that of LC3B was stronger, punctate and at lower levels. LC3 appears to be associated with the autophagosomal membranes, either free or lysosome-bounded. LC3B is specific for the autophagosome-lysosome complexes and their degraded, granular contents. Autophagy was strongly expressed in CNS neurons and intestinal neural elements, which suggests a protective function for the nervous system. Autophagic markers also were seen in deformed immune-competent cells with fragmented nuclei in lymph nodes, spleen and gut-associated lymphoid tissue (GALT), which suggests a "suicidal" activity of eliminating unneeded cells. Autophagy, as measured by LC3 and LC3B expression, may participate in a general regulatory mechanism in tissues of the desert camel.

Distribution of inflammatory cells in adventitia changed with advancing atherosclerosis of human coronary artery.

AIM: Since atherosclerosis was recognized as an inflammatory disease in 1990, the infiltration of macrophages and T lymphocytes has been reported to be predominant in human atherosclerotic lesions. Although adventitis accompanying atherosclerosis was also described in many reports, it is still unclear whether T lymphocytes or B lymphocytes are predominant in the adventitis. In this study, the authors immunohistochemically investigated the correlation between the transition of infiltrating inflammatory cells in the adventitia with atherosclerosis and the type of coronary atherosclerosis. METHODS: Sixty-four coronary atherosclerotic lesions from a surgical specimen and 47 autopsy cases were used for immunohistochemical study of CD45RO, CD20, CD68 and others. Atherosclerosis was classified into type I, II, III, IV according to the 1995 AHA classification. RESULTS: T lymphocyte infiltration in the adventitia was predominantly recognized in about 80% (38/48) of cases, but B lymphocyte infiltration was occasionally recognized in about 20% (10/48). Among 10 cases with B lymphocyte infiltration, small lymph follicles formed in 3 cases. This inflammatory response in adventitia subsided in type III and augmented again in type IV. CONCLUSION: This result suggested that other inflammatory stimuli were induced in the adventitia in type IV coronary atherosclerosis.

Expression of chromosomal regional maintenance protein-1 may be associated with subcellular survivin expression in human gastric and colorectal carcinoma.

Survivin, a member of the inhibitor of apoptosis protein family, is a potential prognostic marker and molecular target for anticancer therapies. Chromosomal regional maintenance protein-1 (CRM-1) mediates the nuclear export of proteins such as survivin. The aims of the present study were to compare the expression and subcellular localization of CRM-1 in human gastric and colorectal carcinomas and to assess the association between CRM-1 and survivin expression in these tumor types. The nuclear and cytoplasmic CRM-1 expression rates in gastric carcinoma were 61% (42/69) and 29% (20/69), respectively, while the nuclear and cytoplasmic CRM-1 expression rates in colorectal carcinoma were 55% (43/78) and 37% (29/78), respectively. Nuclear and cytoplasmic CRM-1 expression was found to be significantly correlated with nuclear and cytoplasmic survivin expression in colorectal carcinoma, but not gastric carcinoma. These results indicate that CRM-1 expression patterns differ between gastric and colorectal carcinomas and thus, we hypothesize that CRM-1-mediated nuclear export of survivin may be deregulated in gastric carcinoma. Therefore, CRM-1 may exhibit different functions in gastric and colorectal carcinoma.

Distribution of ghrelin-immunoreactive cells in human gastric mucosa: comparison with that of parietal cells.

BACKGROUND: Ghrelin is a novel 28-amino-acid peptide isolated from rat and human stomach. This peptide is an endogenous ligand for the growth hormone secretagogue receptor and stimulates the release of growth hormones. Although the principal site of ghrelin synthesis has been reported to be in the stomach, there have been no reports of detailed descriptions of the distribution of ghrelin-immunoreactive cells in the stomach. We examined the distribution of ghrelin-immunoreactive cells in human gastric mucosa. METHODS: We studied the tumor-free mucosa along the lesser curvature in 30 patients with gastric cancer. Sections were stained with hematoxylin and eosin and then subjected to ghrelin immunohistochemistry. The distribution of ghrelin-immunoreactive and parietal cells was studied by continuous cell counting in restricted fields along the lamina muscularis mucosa. RESULTS: Ghrelin-immunoreactive cells were found in all regions, but were distributed mainly in the fundic region. The distribution of ghrelin-immunoreactive cells was positively correlated with that of parietal cells. CONCLUSIONS: The results of this study suggested that the distribution of ghrelin-immunoreactive cells follows that of parietal cells.

Organic cation transporter 2 for predicting cisplatin-based neoadjuvant chemotherapy response in gastric cancer.

Some studies have shown the usability of neoadjuvant chemotherapy (NAC) in gastric cancer (GC). Nevertheless there are a few predictive markers of the effectiveness of NAC in GC. The aim of this study is to assess the predictive impact of organic cation transporter 2 (OCT2) expression on response to neoadjuvant chemotherapy (NAC) in gastric cancer. We retrospectively assessed 66 patients with advanced gastric cancer received NAC with S-1/cisplatin or paclitaxel/cisplatin. Expression levels of OCT2 were assessed by immunohistochemistry in pre-chemotherapy biopsies and correlated with clinicopathologic parameters including pathologic response. High expression level of OCT2 (OCT2(high)) was significantly associated with intestinal type according to Laurén classification (P = 0.03) and low histologic grade (P = 0.03). In univariate analysis of the entire cohort, no variables showed any significant association with a response, although intestinal type (P = 0.09), low histologic grade (P = 0.09), and OCT2(high) (P = 0.07) tended to be more frequent in responders compared with non-responders. When the two treatment groups were separately assessed in the univariate analysis, a significantly higher rate of OCT2(high) was observed in responders compared with non-responders in the S-1/cisplatin group (P = 0.001). In addition, multivariate analysis identified OCT2(high) as the sole independent predictor of response (P = 0.04). However, in the paclitaxel/cisplatin group, no variables were associated with response. Taken together, our results suggest that OCT2(high) may represent a potential predictor of response to NAC with S-1/cisplatin in gastric cancer.

Phosphorylation status of Akt and caspase-9 in gastric and colorectal carcinomas.

The serine/threonine protein kinase B/Akt plays a central role in the coordination of multiple signal transduction processes involved in transcriptional regulation, cell survival, and apoptosis. Activation of Akt kinase is a prognostic factor in several types of cancers; however, its role in gastrointestinal cancers is not fully understood. Caspase-9 is an Akt substrate that belongs to the caspase family of proteases, which function as initiators of the mitochondrial apoptotic pathway. Little is known about the role of caspase-9 phosphorylation, which downregulates the apoptotic activity of the enzyme. In this study, we investigated the expression of phosphorylated (p)-Akt and phosphorylated (p)-caspase-9 in gastric and colorectal carcinoma and the relationship between p-Akt and p-caspase-9 expression and clinicopathological parameters of gastric and colorectal cancer patients. In total, 75 samples of advanced gastric adenocarcinoma (37 well-to-moderately differentiated and 38 poorly differentiated) and 76 samples of advanced colorectal adenocarcinoma (69 well-to-moderately differentiated and 7 poorly differentiated) were analyzed for p-Akt and p-caspase-9 expression by immunohistochemistry. Our results reveal a correlation between p-Akt and p-caspase-9 expression in gastric and colorectal cancers. Levels of p-caspase-9 were significantly higher in colorectal cancer than in gastric cancer, indicating tumor-specific regulation. Although the biological role of p-Akt/p-caspase-9 signaling remains unclear, we suggest that phosphorylation of caspase-9 may be a useful tool to assess the state of gastrointestinal cancer and the effects of anti-cancer therapy.

Smac/DIABLO expression in human gastrointestinal carcinoma: Association with clinicopathological parameters and survivin expression.

Lack of apoptosis is a key factor in carcinogenesis and tumor progression. Survivin is a member of the inhibitor of apoptosis protein (IAP) family. Second mitochondria-derived activator of caspases/direct inhibitor of apoptosis-binding protein with low pI (Smac/DIABLO) is an antagonist of IAPs. Recently, Smac/DIABLO was identified as a potent therapeutic target. However, the clinical significance of Smac/DIABLO in gastrointestinal carcinomas remains unclear. In the present study, Smac/DIABLO expression was analyzed by immunohistochemistry in 72 gastric adenocarcinomas and 78 colorectal adenocarcinomas. The expression of Smac/DIABLO was significantly higher in colorectal carcinoma than in gastric carcinoma. Additionally, a correlation was found between the expression of Smac/DIABLO and nuclear survivin in well- to moderately-differentiated colorectal adenocarcinomas (r=0.245; P<0.01). Based on these results, it was hypothesized that gastric and colorectal carcinomas differ in the level of Smac/DIABLO expression. Our previous studies revealed that the expression of cleaved caspase-9 was significantly lower in colorectal carcinoma than in gastric carcinoma (P<0.0001). Conversely, the expression levels of microtubule-associated protein 1 light chain 3 (LC3), an autophagy marker, and survivin were significantly higher in colon cancer than in gastric cancer (P<0.0001 and P<0.01, respectively). Taken together, these results indicate that not only LC3 and survivin expression, but also Smac/DIABLO expression, are significantly higher in colorectal carcinoma than in gastric carcinoma. We hypothesize that the analysis of Smac/DIABLO, survivin and LC3 expression in colorectal carcinoma is likely to aid cancer therapy due to the involvement of these markers in apoptosis and/or autophagy.

Immunohistochemical expression of nuclear and cytoplasmic survivin in gastrointestinal carcinoma.

Survivin is a protein that is highly expressed in many embryonic tissues, as well as most human tumors. Prior studies have reported both positive and negative correlations between survivin expression and cancer prognosis, but these associations remain controversial. In the present study, we assessed the expression of nuclear and cytoplasmic survivin in gastrointestinal carcinomas. Using these data, we determined the correlation between nuclear and cytoplasmic survivin and, further, investigated correlations between survivin expression and clinicopathological parameters. Seventy-two advanced gastric adenocarcinomas and 78 colorectal adenocarcinomas were analyzed for survivin expression by immunohistochemistry. Expression of both nuclear and cytoplasmic survivin was significantly higher in colorectal carcinomas than in gastric carcinomas (P < 0.01). There was a positive correlation between nuclear and cytoplasmic expression of survivin (r = 0.42, P < 0.001). In gastric carcinomas, the level of survivin protein expression was associated with tumor differentiation, patient age, and lymphatic invasion (P < 0.05, 0.01, and 0.01, respectively). In colorectal carcinomas, the level of nuclear survivin expression was significantly higher in females than in males (P < 0.05). There were no significant associations between survivin expression and most of the clinicopathological parameters. Nevertheless, there was a trend towards an inverse correlation between nuclear survivin expression and tumor aggressiveness in gastric carcinoma; there was a similar trend for cytoplasmic survivin expression. In summary, our results suggest that levels of nuclear and cytoplasmic survivin expression differ between gastric carcinoma and colorectal carcinoma.

Immunohistochemical analysis of cell death pathways in gastrointestinal adenocarcinoma.

Caspase-8 and caspase-9 play crucial roles in the extrinsic and intrinsic apoptotic pathways, respectively. The nuclear translocation of apoptosis-inducing factor (AIF) is involved in caspase-independent apoptosis. Microtubule-associated protein 1 light chain 3 (LC3) plays a pivotal role in autophagy. In the present study, we analyzed the expression of cleaved caspase-8 (CC8), cleaved caspase-9 (CC9), AIF, and LC3 in 160 gastrointestinal adenocarcinomas. The nuclear expression of AIF was rare. The expression of CC8 in gastric and colorectal adenocarcinomas did not differ, whereas the percentage of CC9-positive tumors in gastric adenocarcinomas was significantly higher than in colorectal adenocarcinomas. In contrast, the percentage of LC3-positive tumors in gastric adenocarcinomas was significantly lower than in colorectal adenocarcinomas. CC8 and CC9 occasionally co-existed in the same tumor cells in gastric adenocarcinoma. However, LC3-positive tumor cells in colorectal adenocarcinomas were constantly negative for CC8. No correlation was identified between the expression of any markers and clinicopathological parameters. These results suggest that different cell death pathways are activated in a manner that depends upon the primary site and cell type. The extrinsic and intrinsic apoptotic pathways may be mutually regulated in gastric adenocarcinomas. Also, autophagy may function as a cellular guardian to avoid apoptosis in colorectal adenocarcinomas.

Immunohistochemical characterization of pyrimidine synthetic enzymes, thymidine kinase-1 and thymidylate synthase, in various types of cancer.

Thymidine kinase-1 (TK-1) and thymidylate synthase (TS) are key enzymes for salvage and de novo pyrimidine synthesis, respectively. Numerous studies have suggested that increased TS levels are associated closely with resistance to fluoropyrimidine-based chemotherapy. TAS-102 is a novel drug containing trifluorothymidine, which is phosphorylated by TK-1 to its active monophosphated form, that in turn can inhibit TS. TAS-102 has been shown to exhibit antitumor activity in fluoropyrimidine-resistant human cancer cells. TAS-102 is currently undergoing clinical trials for use in gastrointestinal cancers. In the present study, we used immunohistochemistry to investigate the expression of TK-1 and TS in various types of cancer. TK-1 and TS expression was markedly different between cancer types. High TK-1 expression was detected prominently in gastrointestinal adenocarcinomas and esophageal and uterine squamous cell carcinomas. Gastrointestinal adenocarcinomas and squamous cell uterine carcinomas were often accompanied by high TS expression, indicating activation of pyrimidine synthesis through both the salvage and de novo pathways. These results led us to consider that TAS-102 may also be effective for esophageal and uterine squamous cell carcinomas, as well as for gastrointestinal adenocarcinomas, even in fluoropyrimidine-resistant cases with high TS expression. In contrast, thyroid papillary carcinomas, lung adenocarcinomas, hepatocellular carcinomas, pancreatic ductal carcinomas, and renal cell carcinomas, which exhibit low TK-1 expression, may be resistant to TAS-102. In non-small cell lung cancers, high TK-1 expression was demonstrated in squamous cell carcinomas, but not in adenocarcinomas. This result suggests that TAS-102 efficacy and the pyrimidine synthetic pathway may differ depending on histological type. Our results indicate that administration of TAS-102 could be selected on the basis of the immunohistochemical evaluation of TK-1 and TS.

Immunohistochemical study of enterochromaffin-like cell in human gastric mucosa.

Enterochromaffin-like (ECL) cell has been identified as the histamine-containing argyrophil cell in rat gastric mucosa and vigorously studied. However, there are few reports of the distribution of ECL cell in human stomach. The aim of the present study was to determine the precise distribution of ECL cell by immunohistochemical staining of histidine decarboxylase (HDC) and gastrin-cholecystokinin B receptor (CCK-BR) in human stomach, and the correlation between their distribution and that of parietal cells. Thirty specimens of surgically resected stomach were used. Parietal cell, Grimelius-silver-positive cell, gastrin, HDC- and CCK-BR-immunoreactive cell were studied on continuous cell counting in the restricted field along the lamina muscularis from the oral to the anal ends. The percentage of HDC-immunoreactive cells of endocrine cells was smaller (15%) than that of a previous report (35%) in the fundic region. HDC- and CCK-BR-immunoreactive cells were found not only in the fundic region, but also in the intermediate and pyloric regions. In the pyloric region, HDC- and CCK-BR-immunoreactive cells were found mainly in the mucosa with intestinal metaplasia. Double-positive cells were also found, but only in small numbers. This suggests that ECL cell, or a cell sharing its function, is present in the pyloric region.