Effect of circ MTHFD2 on resistance to pemetrexed in gastric cancer through regulating expression of miR-124.

OBJECTIVE: The aim of this study was to explore the effect of circ MTHFD2 on resistance to pemetrexed (MTA) in gastric cancer by regulating the expression of micro ribonucleic acid (miR)-124. MATERIALS AND METHODS: Human gastric cancer MGC-803 cells were induced by MTA at an increasing concentration. MGC-803/MTA resistant cell model was successfully established after 5 months. The half-maximal inhibitory concentration (IC50) of MTA on the two kinds of cells was detected via cell counting kit-8 (CCK-8) assay. Differentially expressed circular RNAs (circRNAs) were screened using circRNA microarray analysis. Meanwhile, the target miRNAs of circRNAs were predicted using bioinformatics tool and verified via luciferase reporter assay, respectively. In MGC-803 cells, the effects of overexpression and knockdown of circ MTHFD2 on the expression of miR-124 were detected via quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Finally, the effects of circ MTHFD2 on the protein expressions of FDZ5 and multidrug resistance gene-1 (MDR-1) were detected via Western blotting. RESULTS: MGC-803/MTA resistant cell lines were successfully constructed via persistent drug exposure to MTA at an increasing concentration for 5 months. Compared with parental cells, MGC-803/MTA resistant cells showed significantly enhanced drug resistance. Subsequently, differentially expressed circRNAs were screened using circRNA microarray analysis. A total of 673 circRNAs were screened out based on Fold Change >3 and adjusted p-value. The results of qRT-PCR showed that the expression levels of all differentially expressed circRNAs were significantly changed when compared with MGC-803/MTA resistant cells. Compared with MGC-803/MTA resistant cells, the drug resistance of cells increased significantly in circ MTHFD2 overexpression group. However, it markedly decreased in circ MTHFD2 knockdown group. According to the results of bioinformatics and luciferase reporter assay, circ MTHFD2 could target bind to miR-124. In MGC-803/MTA cells, miR-124 could remarkably increase the resistance of MGC-803/MTA cells to MTA. Western blotting results revealed that overexpression of circ MTHFD2 significantly increased the protein expressions of FDZ5 and MDR-1. However, miR-124 mimics reversed the inhibitory effect of circ MTHFD2 on FDZ5 and MDR-1. CONCLUSIONS: Circ MTHFD2 directly binds to miR-124 through the molecular sponge effect. This may induce increased protein expression of MDR-1, ultimately enhancing the drug resistance of MGC-803/MTA cells.

P8 expression is induced in acinar cells during chronic pancreatitis.

The p8 gene is barely expressed in the normal pancreas, but is overexpressed in acute pancreatitis. To elucidate the dynamic expression of p8 mRNA and its significance in the course of chronic pancreatitis, we investigated the p8 expression in spontaneous chronic pancreatitis in the WBN/Kob rat as well as in humans and arginine-treated rat pancreatic acinar AR4-2J cells. p8 mRNA was significantly increased at 12 weeks when chronic pancreatitis first appeared in the WBN/Kob rats. p8 was immunolocalized in the acinar cell nuclei. Acinar cell apoptosis was significantly increased at 12 and 20 weeks in the WBN/Kob rats. In AR4-2J cells, p8 mRNA was significantly induced at 4 hr after arginine addition. Apoptosis of AR4-2J cells was not increased during the strong expression of p8 mRNA. These results suggest that p8 is induced in the acinar cells during chronic pancreatitis as the self-defence mechanism against proapoptotic insults.

Effect of camostat mesilate on the expression of pancreatitis-associated protein (PAP), p8, and cytokines in rat spontaneous chronic pancreatitis.

An oral protease inhibitor, camostat mesilate (CM) has been used clinically for chronic pancreatitis (CP) in Japan, but it lacks enough scientific evidence of its effectiveness. The aim of this study was to analyze the effect of CM on the gene expressions of pancreatitis-associated protein (PAP), p8, and cytokines such as interleukin-6 and transforming growth factor-beta1 in spontaneous CP model (WBN/Kob rats). CM (10 mg/100 g body weight), mixed in MB-3 diet, was administered orally and gene expressions were analyzed by reverse transcription-polymerase chain reaction. In untreated WBN/Kob rats, the gene expressions of all the four factors peaked at 12 weeks, whereas they were significantly suppressed in the CM-treated rats. CM significantly increased the body weight and pancreatic wet weight, and it significantly inhibited inflammatory changes and fibrosis of the pancreas. These results suggest that CM inhibits pancreatic inflammation and fibrosis through the suppression of gene expressions of PAP, p8, and cytokines in CP.

Overexpression of p8 is inversely correlated with apoptosis in pancreatic cancer.

A recently identified gene, p8, has cell growth-promoting activity and is strongly induced in acute pancreatitis. In this study, we detected p8 and single-stranded DNA (ssDNA) for apoptosis by immunohistochemistry in human pancreatic cancer. The p8 was overexpressed (>30% per 1000 cancer cells) in 26 of 44 (59%) pancreatic cancers, and apoptosis (ssDNA-positive cells >10% per 1000 cancer cells) was recognized in 18 of 44 (41%) pancreatic cancers. There was a significant inverse correlation between the p8 overexpression and apoptosis (P < 0.05). Moreover, the expression pattern of high p8 and low ssDNA was seen significantly more often in lower age (<65 years), in moderately or poorly differentiated cancers, and in node-positive cases (P < 0.05). The p8 expression and apoptosis were not significantly correlated with survival. These results suggest that p8 overexpression is involved in antiapoptotic activity and the biological characteristics of pancreatic cancer.

Expression of tumor necrosis factor-alpha, interleukin-6, and interferon-gamma in spontaneous chronic pancreatitis in the WBN/Kob rat.

To clarify the pathophysiological significance of cytokines in chronic pancreatitis (CP), we analyzed tissue expressions of various cytokines in the onset and progression of spontaneous CP in the WBN/Kob rat. Four-week-old male WBN/Kob rats were fed a special pellet diet (MB-3) for 20 weeks, and 6 rats were killed every 4 weeks. Pathologically, CP occurred at 12 weeks and progressed thereafter. The inflammation and fibrosis peaked at 12 and 16 weeks, respectively. By semiquantitative reverse transcription-polymerase chain reaction, the expression of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and interferon (IFN)-gamma mRNAs peaked at 8, 12, and 16 weeks, respectively. Immunohistochemistry showed IL-6 expression in infiltrating inflammatory cells and vascular endothelial cells, whereas TNF-alpha was expressed in both acinar and infiltrating cells. IFN-gamma was localized to acinar, infiltrating and ductal cells, and its expression intensity showed significant correlation with those of fibrosis, type III collagen and alpha-smooth muscle actin. The in situ hybridization results were consistent with the RT-PCR data. These results suggest that tissue expressions of TNF-alpha and IL-6 are involved in the onset of pancreatitis and that IFN-gamma expression is related to the progression of CP.

Expression of clusterin in pancreatic acinar cell injuries in vivo and in vitro.

Clusterin is a secretory glycoprotein that is highly induced in several tissues in response to injury. The pathophysiologic significance of clusterin in the pancreas remains largely unknown. The aim of this work was to examine whether clusterin is expressed in spontaneous chronic pancreatitis in the WBN/Kob rat and to investigate the relationship between clusterin and apoptosis in pancreatic acinar AR4-2J cells. In the in vivo study, 4-week-old male WBN/Kob rats developed chronic pancreatitis at 12 weeks. Clusterin mRNA was expressed after 12 weeks and then decreased. Immunohistochemistry showed clusterin expression in the acinar cells. In the in vitro study, clusterin mRNA and protein were strongly induced in AR4-2J cells treated either with arginine, menadione, tumor necrosis factor-alpha or transforming growth factor-beta1. In the time course study with arginine or menadione, clusterin mRNA was expressed after 4 hours and peaked at 8 and 24 hours, whereas DNA fragmentation peaked at 72 hours. Our results show that clusterin is overexpressed in the pancreas at the onset of chronic pancreatitis in vivo and in cultured acinar cells in response to various stimuli in vitro, suggesting that clusterin is a defense mechanism of the exocrine pancreas.

Expression of p8 in human pancreatic cancer.

The p8 gene is a recently identified gene with mitogenic activity. p8 expression is induced in acute pancreatitis, pancreatic development, and regeneration. However, the expression of p8 in pancreatic cancer is not reported. We investigated p8 expression in 72 human pancreatic tissues, including 38 pancreatic cancers (PCs), by immunohistochemistry. p8 was overexpressed (positive cells >25% in 1,000 cells) in 71% (27 of 38) of PCs, but in only 17% (3 of 18) of chronic pancreatitis cases. There was no overexpression in mucinous cystadenoma or in normal pancreas. The p8 overexpression rate in PC was significantly higher than that in other conditions (P < 0.05). Reverse transcription-PCR analysis confirmed p8 mRNA overexpression (tumor/nontumor ratio >2) in 75% (3 of 4) of PCs. p8 was overexpressed also in human pancreatic cancer cell lines (MIA PaCa-2 and PANC-1). These results suggest that p8 is involved in the development of pancreatic cancer, reflecting its mitogenic activity.

Apoptosis in rat spontaneous chronic pancreatis: role of the Fas and Fas ligand system.

The Fas/Fas ligand (FasL) system is suggested to be correlated to the onset of inflammation and apoptosis in various diseases. However, whether Fas and FasL are expressed in chronic pancreatitis is unknown. The aim of this study was to examine the expression of the Fas/FasL system and to analyze its correlation with apoptosis in a spontaneous chronic pancreatitis model (the WBN/Kob rat). Four-week-old male WBN/Kob rats were fed a special pellet diet (MB-3). Different groups of rats were killed every four weeks, and pancreata were histopathologically examined. Fas and FasL mRNAs in the pancreas were detected with a reverse transcription-polymerase chain reaction method. The cellular localization of Fas and FasL mRNA and protein was determined with in situ hybridization (ISH) and immunohistochemistry (IHC). Apoptosis was detected with a terminal deoxynucleotidyltransferase-mediated method. Fas and FasL mRNA were expressed when the pancreas was still pathologically normal, and showed a biphasic peak at 12 and 20 weeks. ISH and IHC confirmed that Fas and FasL are expressed in the cytoplasm of acinar cells, ductal cells, and lymphocytes. An apoptotic index in acinar cells correlated to the expression of Fas and FasL mRNAs. These results suggest that the expression of the Fas/FasL system is involved in acinar cell apoptosis and the onset and progression of chronic pancreatitis in the WBN/Kob rat.

Antifibrotic effect of the herbal medicine Saiko-keishi-to (TJ-10) on chronic pancreatitis in the WBN/Kob rat.

Chronic pancreatitis is characterized by fibrosis. We reported an anti-inflammatory effect of the herbal medicine Saiko-keishi-to (TJ-10) on chronic pancreatitis. This study aimed to elucidate the antifibrotic effect of TJ-10. Four-week-old male WBN/Kob rats were fed a special pellet diet (MB-3) with or without TJ-10 (80 mg/100 g body weight) for 20 weeks. Pancreata were histopathologically examined at every 4 weeks, and the expression of fibrosis-related factors such as transforming growth factor beta1 (TGF-beta1), fibronectin (FN), alpha-smooth muscle actin (alpha-SMA), and type III collagen was analyzed. In untreated WBN/Kob rats, chronic pancreatitis developed at 12 weeks and progressed with marked fibrosis at 16 weeks, and the expression of TGF-beta1 and FN peaked at 12 weeks. However, in the TJ-10-treated rats, the rate of pancreatic fibrosis and the expression of TGF-beta1, FN, alpha-SMA, and type III collagen at 12 and 16 weeks decreased significantly compared to those in the untreated rats. These results suggest that TJ-10 inhibits the pancreatic fibrosis by the suppression of TGF-beta1 expression.

[Transition of soleus troponin I isoforms and atrophy of testis in tail-suspended rats].

Objective. To observe the transition time of soleus I (TnI) isoforms and to elucidate the relationship between soleus TnI transition and atrophy; and to analyze the time course between testis atrophy and soleus atrophy. Method. Eight groups of male rats were suspended for 3, 4, 5, 7, 14, 21, 28 and 42 d, respectively. Besides, three groups of female rats were suspended for 3, 4 and 5 d respectively. Wet and relative weights (wet weight/body weight) of testis and soleus were measured. The expression of TnI was observed by Western blot. Result. The relative weight of soleus of tail-suspended male rats decreased significantly after 4 d of suspension as compared with control. The degree of rats soleus atrophy in the first 14 d of suspension was greater than that after 14 d. The relative weight of testis showed the same change as that of soleus. There was no significant change in the relative weight of soleus in 4 d of tail-suspended female rats. The significant decrease in the relative weight of tail-suspended female rats began on the 5th day. The Western blot showed that the transition from slow skeletal TnI (ssTnI) to fast skeletal TnI (fsTnI) in the soleus occurred in 14 d of tail-suspension. Conclusion. The overt atrophy of tail-suspended male rats occurs on the 4th day. The soleus TnI transition from ssTnI to fsTnI is on the 14th day. It is suggested that the TnI is not the sensitive protein to gravity. The overt atrophy of female tail-suspended rats occurs at the 5th day. This indicated that the decrease in testosterone level may accelerate the atrophy of the soleus.

Effect of herbal medicine keishi-to (TJ-45) and its components on rat pancreatic acinar cell injuries in vivo and in vitro.

BACKGROUND: In an attempt to clarify the mechanism of the effect of a herbal medicine, Saiko-keishi-to (TJ-10), which is a combination of Keishi-to (TJ-45) and Sho-saiko-to (TJ-9), we investigated the effects of these two herbal medicines and their components on pancreatic acinar cell injury models in vivo and in vitro. METHODS: Four-week-old male WBN/Kob rats were fed an MB-3 pellet diet containing herbal medicine (TJ-9, TJ-10 and TJ-45). Expressions of pancreatitis-associated protein (PAP) and manganese superoxide dismutase (Mn-SOD) were analyzed with a reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. The herbal medicines and two of their components, Keihi (Cinnamomi cortex) and Shakuyaku (Paeoniae radix alba), were tested in vitro using an arginine-treated rat pancreatic acinar AR4-2J cell injury model. The inducible nitric oxide synthase (iNOS) was assayed in in vitro experiments. RESULTS: TJ-45-treated WBN/Kob rats showed no evidence of pancreatitis whereas there were pathological changes of chronic pancreatitis in TJ-9-treated WBN/Kob rats. PAP was not expressed and Mn-SOD expression was increased in the TJ-10-, and TJ-45-treated rats. The herbal medicines and two components suppressed PAP mRNA expression and enhanced Mn-SOD and iNOS mRNA expression in arginine-treated AR4-2J cells. CONCLUSION: These results suggest that the herbal medicine TJ-45 is effective for chronic pancreatitis caused by pancreatic ischemia.

Pancreatitis-associated protein levels in pancreatic juice from patients with pancreatic diseases.

BACKGROUND: Pancreatitis-associated protein (PAP), the acute-phase protein of the pancreas, is overexpressed in acute pancreatitis. Serum PAP levels were reported to be useful as an indicator of the severity, prognosis and healing of acute pancreatitis. Although PAP was originally identified in pancreatic juice, there has been no clinical report on PAP levels in pancreatic juice. This study was conducted to determine levels of PAP in pancreatic juice (PJ-PAP) in various human pancreatic diseases. METHODS: PAP levels in endoscopically aspirated PJ were measured by enzyme-linked immunosorbent assay in 86 patients with pancreatic diseases. RESULTS: 55% of 22 patients with pancreatic cancer (PC) and 25% of 49 patients with chronic pancreatitis (CP) were positive (> 350 ng/ml) for PJ-PAP. PJ-PAP levels were significantly higher in PC than in CP, in which PJ-PAP was also significantly higher than in 15 control subjects. There was no significant correlation between PJ-PAP and serum PAP, and combination assay of serum PAP and/or PJ-PAP detected 80% of PC cases and 44% of CP cases. CONCLUSIONS: We have demonstrated that human PAP could be detected in pancreatic juice from patients with pancreatic diseases. Determination of PAP in pancreatic juice might be helpful for early detection of pancreatic injury.

Effect of herbal medicine Saiko-keishi-to (TJ-10) on rat spontaneous chronic pancreatitis: comparison with other herbal medicines.

BACKGROUND: In an attempt to obtain evidence of the beneficial effects of TJ-10, we investigated the gene expression of PAP, an acute phase protein specific for pancreatitis in rat spontaneous chronic pancreatitis. METHODS: Four-wk-old male WBN/Kob rats were fed with MB-3 pellet diet containing herbal medicine. There were two administration groups for each drug: the prophylactic group administered from 4-12 wk, and the therapeutic group administered from 12-20 wk. Untreated control rats were fed with MB-3 alone. Histopathologic changes and PAP gene expressions were analyzed at 12 and 20 wk. RESULTS: In the prophylactic group, TJ-10-treated WBN/Kob rats showed no evidence of pancreatitis, and there was the amelioration of pancreatitis in the pancreata of the rats treated with other herbal medicines except TJ-24 at 12 wk. PAP mRNA was not expressed in the TJ-10-treated rats, and PAP gene expression was suppressed in rats treated with other drugs except TJ-107. In the therapeutic group, the amelioration of pancreatitis was seen only in TJ-10-treated rats, but PAP gene expression was significantly suppressed in the rats treated with all herbal medicines tested, compared with that in untreated control rats. CONCLUSION: An herbal medicine Saiko-keishi-to (TJ-10) delayed the onset of chronic pancreatitis in the WBN/Kob rat, and suppressed the pancreatitis-associated protein (PAP) gene expression more significantly than other herbal medicines.

Expression of transforming growth factor-beta in spontaneous chronic pancreatitis in the WBN/Kob rat.

Transforming growth factor-beta1 (TGF-beta1) is suggested to be a mediator of fibrosis in chronic pancreatitis, but the serial change of TGF-beta1 expression in the onset and progression of chronic pancreatitis is still unclear. We investigated the TGF-beta1 expression in the spontaneous chronic pancreatitis model. Four-week-old male WBN/Kob rats were fed with special pellet diet (MB-3) for 20 weeks. TGF-beta1 mRNA in the pancreas was detected by reverse transcription-polymerase chain reaction assay from four weeks, and its expression peaked at 12 weeks when the pancreatic fibrosis first appeared. The localizations of TGF-beta1 mRNA and protein were confirmed in the cytoplasm of pancreatic acinar and ductal cells by in situ hybridization and immunohistochemistry, respectively. Although fibronectin expression peaked at 12 weeks and correlated with that of TGF-beta1, its elevated expression tended to be prolonged. Pancreatic fibrosis peaked at 16 weeks after the peak of TGF-beta1 expression. These results suggest that TGF-beta1 expression may be a trigger of the fibrotic process of chronic pancreatitis in the WBN/Kob rat.

Arginine induces apoptosis and gene expression of pancreatitis-associated protein (PAP) in rat pancreatic acinar AR4-2J cells.

Arginine-induced pancreatic acinar cell injury has been reported in vivo, but the mechanism involved is unknown. In this study we investigated the effects of arginine on the cell morphology and pancreatitis-associated protein (PAP) gene expression in rat pancreatic acinar AR4-2J cells in vitro. Arginine inhibited the proliferation of AR4-2J cells in a dose-dependent manner. This decrease in proliferation was due to an increase in apoptosis, as assessed by cell morphology and DNA fragmentation. PAP messenger RNA (mRNA) was expressed at doses of 2.5 and 5.0 mg/ml of arginine, and a time-course study showed that the expression started 2 h after arginine addition and peaked at 6 h. Apoptosis was rarely seen when PAP mRNA was highly expressed, but occurred when PAP mRNA expression was decreased. These results suggest that arginine induces apoptosis and PAP gene expression in pancreatic acinar cells and that PAP might inhibit the induction of apoptosis.

[Advances of capillary electrophoresis and capillary electrochromatography using ionic coating column].

Capillary electrochromatography (CEC) is a promising micro-separation technique, which combines the high selectivity of HPLC and the high efficiency of HPCE. In comparing to CE with fused silica capillary and CEC with ODS stationary phase, capillary electrophoresis and capillary electrochromatography with ionic coating columns can cause EOF more controllable, and provide an alternative separation mechanism. Therefore, the new CEC and CE modes are in favor of optimizing the separation and broadening the range of samples for analysis. The development of capillary electrophoresis and capillary electrochromatography using ionic coating column is reviewed with 48 references.

Expression of pancreatitis-associated protein (PAP) in rat spontaneous chronic pancreatitis: effect of herbal medicine Saiko-keishi-to (TJ-10).

Pancreatitis-associated protein (PAP) is almost absent in the normal pancreas but is overexpressed in acute pancreatitis. However, its expression in chronic pancreatitis (CP) is unknown. An herbal medicine Saiko-keishi-to (TJ-10) has long been used clinically for CP, but there is no experimental evidence of the effect of TJ-10 on CP. The aim of this study was to analyze the expression of PAP and the effect of TJ-10 in a spontaneous chronic pancreatitis model. Four-week-old male WBN/Kob rats were fed with a special pellet diet (MB-3), and TJ-10 (80 mg/100 g body weight/day) was orally administered for 16 weeks. The rats were killed at every 4 weeks, and pancreata were histopathologically examined. PAP messenger RNA (mRNA) in the pancreas was detected with a reverse transcription/polymerase chain reaction (RT-PCR) method. The cellular localization of PAP mRNA and protein was analyzed with in situ hybridization (ISH) and immunohistochemistry (IHC). PAP mRNA was expressed from 8 weeks, when the pancreas was still pathologically normal, and reached its peak at 12 weeks, when the pancreatitis first appeared. Then the expression of PAP mRNA was decreased gradually. TJ-10 suppressed the expression of PAP mRNA completely at 8 and 12 weeks. PAP mRNA was slightly expressed at 16 and 20 weeks. ISH and IHC confirmed the PAP mRNA and protein expression in the cytoplasm of acinar cells. These results suggest that PAP mRNA appears before CP, and its peak coincides with the onset of CP. TJ-10 suppressed the PAP expression and delayed the development of CP in the WBN/Kob rat.

[High frequency electrical knife through fibrobronchoscope in the treatment of tracheal cancer with large airway obstruction].

UNLABELLED: From May 1985 to December 1991, high frequency electrical knife (HFEK) through fibrobronchoscope was used to treat the large airway obstruction by tracheal cancers in 11 patients (cystic adenoid carcinoma: 8 squamous cell carcinoma: 2 and undifferentiated small cell carcinoma: 1). Most of the tracheal cancer patients were inoperable because of extensive lesions or poor lung function. Olympus BF B3R or BF 10 Type Fibrobronchoscope, Olympus UES or Olympus PSD-10 high frequent electricity producer with a home-made electrical knife were applied. After 53 times of HFEK treatment of tracheal cancer, all patients showed remarkable improvement in lung function and exertional dyspnea. CONCLUSION: HFEK is valuable in relieving dyspnea in patients with large airway obstruction with tracheal cancer. After endoscopic cautery to release the obstruction, it is necessary to use radiotherapy and chemotherapy for the patients to get better results.