[Screening for active components of Sophorae Flos on inhibiting AGEs formation based on non-enzymatic glycation reaction].

Sugar-poison caused blood-heat is the pathological basis of many complications of diabetes. Advanced glycation end products( AGEs) are considered as the potential glycotoxic factor that can cause blood-heat. Sophorae Flos hold the effect of removing pathogenic heat from blood. In this study,chromatographic non-enzymatic glycation reaction system of bovine serum albumin( BSA)/methylglyoxal( MGO) and Sophorae Flos was established to identify active components in Sophorae Flos inhibiting AGEs formation. The HPLC was used to analyze chromatograms before and after the incubation of Sophorae Flos and methylglyoxal. Changes of chromatographic peaks of eight compounds was found. It is speculated that this change may be due to new substance produced by the reaction of active components in Sophorae Flos and methylglyoxal,and these active components may be flavonoid component rutin. Further investigation for the effects of rutin and MGO reaction( 1 ∶ 1,1 ∶ 3,3 ∶ 1) for 6 days on the formation of AGEs was performed. The results showed that the inhibition activity of rutin on AGEs production was most obvious when the reaction ratio was 1 ∶3,and the most inhibition was in 24 h and stabilized after 3 d. The product of the reaction of rutin with MGO was identified by LC-ESI-MS/MS,which indicated that the newly formed seven substances were the mono-and di-MGO adducts of rutin. This study showed that rutin is the active component on Sophorae Flos for removing pathogenic heat from blood by forming new compounds to inhibit the formation of sugar poison products,which provides reference for rational application of Sophorae Flos.

[Discussion on influence factors, mechanism and traditional Chinese medicine pathogenesis of idiosyncratic drug-induced liver injury].

Idiosyncratic drug-induced liver injury (IDILI) is a kind of unique adverse drug reaction with relative high morbidity compared with other idiosyncratic diseases. Its occurrence, however, has nothing to do with pharmacological effects and clinical dosage of drugs administered, and only a small number of susceptible individuals will suffer from it. Especially to deserve to be mentioned, the proportion of TCM-induced IDILI showed an ascending trend year by year. So in this article, the author has reviewed some facts related with TCM-induced IDILI, including the predisposing causes and occurrence mechanism, and tries to provide reference for the prevention, diagnosis and treatment of TCM-induced IDILI through the analysis of characteristics and research status of TCM-induced IDILI and exploration of the internal relationship between Chinese medicine constitution type and IDILI.

Pim-2 Modulates Aerobic Glycolysis and Energy Production during the Development of Colorectal Tumors.

Tumor cells have higher rates of glucose uptake and aerobic glycolysis to meet energy demands for proliferation and metastasis. The characteristics of increased glucose uptake, accompanied with aerobic glycolysis, has been exploited for the diagnosis of cancers. Although much progress has been made, the mechanisms regulating tumor aerobic glycolysis and energy production are still not fully understood. Here, we demonstrate that Pim-2 is required for glycolysis and energy production in colorectal tumor cells. Our results show that Pim-2 is highly expressed in colorectal tumor cells, and may be induced by nutrient stimulation. Activation of Pim-2 in colorectal cells led to increase glucose utilization and aerobic glycolysis, as well as energy production. While knockdown of Pim-2 decreased energy production in colorectal tumor cells and increased their susceptibility to apoptosis. Moreover, the effects of Pim-2 kinase on aerobic glycolysis seem to be partly dependent on mTORC1 signaling, because inhibition of mTORC1 activity reversed the aerobic glycolysis mediated by Pim-2. Our findings suggest that Pim-2-mediated aerobic glycolysis is critical for monitoring Warburg effect in colorectal tumor cells, highlighting Pim-2 as a potential metabolic target for colorectal tumor therapy.

Application of tumoroids derived from advanced colorectal cancer patients to predict individual response to chemotherapy.

Therapeutic approaches of advanced colorectal cancer are more complex, here we present a living biobank of patient-derived tumoroids from advanced colorectal cancer patients and show examples of how these tumoroids can be used to to simulate cancer behavior ex vivo and provide more evidence for tumoroids could be utilized as a predictive platform during chemotherapy treatment to identify the chemotherapy response. Morphological, histological and genomic characterization analysis of colorectal cancer tumoroids was conducted. Further, we treated colorectal cancer tumoroids with different drugs to detect cellular activities to evaluate drug sensitivity using CellTiter-Glo 3 D cell viability assay. Then the drug sensitivity of tumoroids was compared with clinical outcomes. Our results implied that tumoroids recapitulated the histological features of the original tumours and genotypic profiling of tumoroids showed a high-level of similarity to the matched primary tumours. Dose-response curves, area under the curve and tumour inhibitory rate of each therapeutic profiling calculations in tumoroids demonstrated a great diversity and we gained 88.24% match ratio between the sensitivity data of tumoroids with their paired patients' clinical outcomes. tumour inhibitory rate of each treatment parameters in tumoroids performed positive correlation with progression-free survival while area under the curve of each treatment parameters performed negative correlation with progression-free survival of the corresponding patients. In summary, We presented a living biobank of tumoroids from advanced colorectal cancer patients and show tumoroids got great potential for predicting clinical responses to chemotherapy treatment of advanced colorectal cancer.

In vitro inhibition of Huanglian [Rhizoma coptidis (L.)] and its six active alkaloids on six cytochrome P450 isoforms in human liver microsomes.

Huanglian (Rhizoma Coptidis) as a popular herb has been used for the treatment of various diseases such as diarrhea, eye inflammation and women's abdominal ailments. Alkaloids are considered to be responsible for its pharmacological effects. In this investigation, Huanglian and its six alkaloids (coptisine, epiberberine, berberine, jateorrhizine, palmatine and magnoflorine) were systematically evaluated for their inhibition of six cytochrome P450 isoforms (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) in human liver microsomes by the LC-MS/MS method. Huanglian showed the strongest inhibition of CYP2D6, followed by CYP1A2 and CYP3A4_T. The IC50 values were 5.8 µg/mL, 36.8 µg/mL and 59.2 µg/mL, respectively. Of the constituents tested, coptisine and epiberberine showed strong inhibition of CYP2D6 with IC50 values of 4.4 µM and 7.7 µM; berberine, jateorrhizine and palmatine showed weak inhibition of CYP2D6 with IC50 values of 45.5 µM, 49.4 µM and 92.6 µM, respectively; jateorrhizine showed moderate inhibition of CYP3A4_T with an IC50 value of 13.3 µM; coptisine showed weak inhibition of CYP1A2 with an IC50 value of 37.3 µM. In addition, activation was observed in coptisine/CYP2C9 and palmatine/CYP2C9/CYP2C19. Other CYP450 isoforms were not affected markedly by the six alkaloids. In conclusion, Huanglian showed in vitro inhibition of CYP2D6, the inhibition might be contributed mostly by protoberberine alkaloids, especially coptisine and epiberberine. Herb-drug interactions may occur through the CYP2D6 inhibition.

lncRNA GAS5 inhibits malignant progression by regulating macroautophagy and forms a negative feedback regulatory loop with the miR­34a/mTOR/SIRT1 pathway in colorectal cancer.

Long non­coding RNA growth arrest specific 5 (GAS5) exerts inhibitory effects through the modulation of several target microRNAs (miRs) in cancer. However, its potential roles and underlying relationship during colorectal cancer (CRC) progression are unclear. Therefore, we explored the role of the negative feedback loop formed by the GAS5/miR­34a axis and mammalian target of rapamycin/sirtuin 1 (mTOR/SIRT1) pathway on macroautophagy and apoptosis in CRC. Expression of GAS5, miR­34a, SIRT1 and mTOR in CRC patients and cell lines was detected by quantitative reverse transcription polymerase chain reaction. Online bioinformatic analysis was used to predict the downstream miRs of GAS5. Luciferase assay and western blotting were performed to demonstrate miR­34a as a downstream target gene of GAS5 in CRC cells. The effects of the GAS5/miR­34a axis on apoptosis, macroautophagy, and the mTOR/SIRT1 pathway were assessed by flow cytometry, transmission electron microscopy and western blotting, respectively. Our results suggested that GAS5 was downregulated and acted as a molecular sponge of miR­34a during CRC progression. miR­34a participated in regulating GAS5­suppressed CRC cell macroautophagy and induced apoptosis through the mTOR/SIRT1 pathway. GAS5­mediated macroautophagy was maintained in an equilibrium state that might have a protective effect on CRC cell apoptosis. The mTOR signaling pathway suppressed GAS5 expression and formed a negative regulation feedback loop with miR­34a in CRC cells. Our results suggested that the GAS5/miR­34a/SIRT1/mTOR negative regulatory feedback loop mediated CRC cell macroautophagy, and maintained the cells in an autonomous equilibrium state, but not excessive activation state, which functions as a strong antiapoptotic phenotype during human CRC progression.

[Surgical treatment and prognosis of cancer of hepatic flexure of colon invading the duodenum in 65 patients].

OBJECTIVE: To discuss surgical treatment of right colon carcinoma of hepatic flexure invading the duodenum. METHODS: Sixty-five patients with right colon carcinoma of hepatic flexure invading the duodenum, treated in our department from 1987 to 2007, were included in this study. Their clinicopathological data were retrospectively reviewed and analyzed. All the cases were divided into three types (local invasion, regional invasion, and cancer with internal fistula) according to duodenal defect, including local invasion (< 2.0 cm), wide invasion (> 2.0 cm) and the presence of internal fistula. RESULTS: 25 patients with local invasion underwent en bloc resection of the duodenal wall. Pedicled ileal flap was used to cover the large duodenal defect measuring 2.0 - 3.0 cm in 5 patients. Dudenojejunostomy was used to reconstruct the large defect measuring more than 5 cm in 3 patients. Conservative resection of right-sided colon was performed in 18 patients with wide invasion. 4 patients underwent pancreaticoduodenectomy combined with right hemicolectomy for colon cancer involving the pancreatic head. 10 underwent duodenal diverticularization. One patient with anastomotic leakage healed within 3 weeks. Other patients were cured without postoperative complications. The total 3-year and 5-year survival rates after surgery were 53.8% and 9.2%, respectively. CONCLUSION: The surgical procedure to be performed is usually decided according to the cancer location, extent, and duodenal defect and invasion, which are important for prolonging life time, improving of quality of life and prognosis in these patients.

[Diagnosis and surgical treatment of carcinoid tumors of the appendix in 64 patients].

OBJECTIVE: To summarize the experience in the diagnosis and surgical treatment of carcinoid tumors of the appendix. METHODS: From 1972 to 2006, 64 patients with carcinoid tumors of the appendix received surgical treatment in our hospitals. The clinical data of those patients were retrospectively analyzed. RESULTS: Of the 64 cases, only 6 cases (9.4%) were correctly diagnosed preoperatively, while 58 (90.6%) not confirmed, with a misdiagnosis rate of 90.6%. All patients underwent surgical treatment, including appendectomy in 54, ileocecectomy in 4, right hemicolectomy in 2 and right hemicolectomy with regional lymph node dissection in 4 cases. The operation modes were determined according to the doctor's judgments based on the age of the patients, the nature, size, location, infiltration depth and lymph node metastasis of the tumors. Of the 64 patients, 58 were followed up with a longest follow-up period of 13 years, while 6 lost follow-up. Fifty-seven of those were still surviving, only one died of liver metastasis at 13 years after operation. CONCLUSION: Carcinoid tumor of the appendix is rare with a high rate of misdiagnosis before operation. Surgical resection is the only effective treatment for this disease and proper operation mode is the key to achieve good survival.

miR-34a mediates oxaliplatin resistance of colorectal cancer cells by inhibiting macroautophagy via transforming growth factor-ß/Smad4 pathway.

AIM: To investigate whether microRNA (miR)-34a mediates oxaliplatin (OXA) resistance of colorectal cancer (CRC) cells by inhibiting macroautophagy via the transforming growth factor (TGF)-ß/Smad4 pathway. METHODS: miR-34a expression levels were detected in CRC tissues and CRC cell lines by quantitative real-time polymerase chain reaction. Computational search, functional luciferase assay and western blotting were used to demonstrate the downstream target of miR-34a in CRC cells. Cell viability was measured with Cell Counting Kit-8. Apoptosis and macroautophagy of CRC cells were analyzed by flow cytometry and transmission electron microscopy, and expression of beclin I and LC3-II was detected by western blotting. RESULTS: Expression of miR-34a was significantly reduced while expression of TGF-ß and Smad4 was increased in CRC patients treated with OXA-based chemotherapy. OXA treatment also resulted in decreased miR-34a levels and increased TGF-ß and Smad4 levels in both parental cells and the OXA-resistant CRC cells. Activation of macroautophagy contributed to OXA resistance in CRC cells. Expression levels of Smad4 and miR-34a in CRC patients had a significant inverse correlation and overexpressing miR-34a inhibited macroautophagy activation by directly targeting Smad4 through the TGF-ß/Smad4 pathway. OXA-induced downregulation of miR-34a and increased drug resistance by activating macroautophagy in CRC cells. CONCLUSION: miR-34a mediates OXA resistance of CRC by inhibiting macroautophagy via the TGF-ß/Smad4 pathway.

Inhibitory effects of limonin on six human cytochrome P450 enzymes and P-glycoprotein in vitro.

Among the various possible causes for drug interactions, pharmacokinetic factors such as inhibition of drug-metabolizing enzymes and transporters, especially cytochrome P450 (CYP) isoenzymes and P-glycoprotein (P-gp), are regarded as the most frequent and clinically important. Limonin is a widely used dietary supplement and one of the most prevalent citrus limonoids, which are known to have inhibitory effects on CYPs and P-gp. In this study, the in vitro inhibitory effects of limonin on the major human CYP isoenzymes (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) activities in human liver microsomes were examined using liquid chromatography-tandem mass spectrometry. The inhibitory effects of limonin on P-gp activity in a human metastatic malignant melanoma cell line WM-266-4 were examined using a calcein-AM fluorometry screening assay. It demonstrates that limonin has negligible inhibitory effects on human CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and P-gp. However, potent inhibition of CYP3A4 by limonin is observed with IC50 values of 6.20 µM (CYP3A4/testosterone) and 19.10 µM (CYP3A4/midazolam). This finding has important implications with regard to food-drug interactions between limonin and several narrow therapeutic index drugs that are metabolized by CYP3A4.

[Expressions and clinical implications of kruppel-like factor 6 and APC in human colorectal carcinoma].

OBJECTIVE: To investigate the expressions and clinical implications of kruppel-like factor 6(KLF-6) and APC in human colorectal carcinoma. METHODS: The expressions of KLF-6 and APC in tumor and normal tissues from 32 patients with colorectal carcinoma were investigated by RT-PCR and immunohistochemical technique. RESULTS: The expression rates of KLF-6 and APC mRNA were 37.5% and 34.3% in tumor tissue, 96.9% and 93.8% respectively in normal tissues (both P< 0.05). The expression rates of KLF-6 and APC protein were 28.1% and 25.0% in colorectal carcinomas, 81.3% and 84.43% respectively in normal tissues (both P< 0.05). There was a significant correlation between the expressions of KLF-6 and APC in colorectal carcinomas (P < 0.05). The expressions of KLF-6 and APC were significantly correlated with tumor differentiation, depth of infiltration, lymph node metastasis and clinical stage (P< 0.05). CONCLUSION: Down-regulations of KLF-6 and APC might play an important role in the carcinogenesis, development, metastasis of human colorectal carcinoma.