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.

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.

Farnesoid X receptor inhibits proliferation of human colorectal cancer cells via the miR­135A1/CCNG2 signaling pathway.

Colorectal cancer (CRC) is among the most common malignancies of the digestive system. Dysregulation of miRNAs and the farnesoid X receptor (FXR) are involved in the progression of CRC. In the present study, the effects of FXR and miR­135A1 in CRC were evaluated. Reverse transcription quantitative­polymerase chain reaction (RT­qPCR) was used to examine the expression of miR­135A1 in patient CRC tissues and adjacent non­tumor tissues, as well as cell lines. The association between miR­135A1 and clinical characteristics of patients with CRC was also investigated. RT­qPCR and western blotting were used to evaluate the expression of miR­135A1 targets. Regulation of cyclin G2 (CCNG2) by miR­135A1was confirmed using luciferase assays. The biological effects of miR­135A1 were assessed in transfected and untransfected CRC cell lines using colony formation assays, cell­cycle analysis by flow cytometry, and CCK­8 assays. miR­135A1 was upregulated in CRC specimens and cell lines. miR­135A1 expression was strongly associated with poor cell differentiation, high expression of carbohydrate antigen (CA)125, CA199, carcinoembryonic antigen and survival rate of patients with CRC. Expression of CCNG2 was downregulated in CRC patients and cell lines, and was further demonstrated to be among the downstream targets of miR­135A1. The present study indicated that inhibition of miR­135A1 expression leads to cell cycle arrest and inhibition of proliferation of CRC cells via increasing CCNG2 expression. In the present study, activation of FXR by GW4064 increased CCNG2 expression via suppression of miR­135A1 expression, and the FXR/miR­135A1/CCNG2 axis was demonstrated to be involved in mediating cell proliferation. In conclusion, activation of FXR by GW4064 suppresses cell proliferation and causes cell cycle arrest in CRC, and the miR­135A1/CCNG2 pathway was suggested to be involved in this step.

Silencing Lin28 promotes apoptosis in colorectal cancer cells by upregulating let­7c targeting of antiapoptotic BCL2L1.

Colorectal cancer (CRC) remains a primary contributor to cancer­associated mortality. The Lin28/let­7 axis has previously been verified to participate in numerous pathophysiological processes involved in CRC. However, the potential roles and underlying mechanisms of this axis in apoptosis during CRC remain to be fully elucidated. The present study aimed to evaluate the role and reveal the molecular mechanisms of the Lin28/let­7 axis in the apoptosis of CRC cells. An MTT assay was conducted to assess the cell viability of HCT116 and HT29 CRC cells, and caspase­3 activity was analyzed to measure the apoptosis of CRC cells. Western blotting and reverse transcription­quantitative polymerase chain reaction were performed to examine the expression of Lin28, B­cell lymphoma 2 (Bcl­2), Bcl­2­associated X protein, Bcl­2­like 1 (BCL2L1) and let­7c. The present study demonstrated that Lin28 was upregulated whereas let­7c was downregulated in CRC tissues and cell lines compared with normal tissues and NCM460 normal colon epithelial cells, respectively. Forced overexpression of let­7c promoted apoptosis in CRC cells, which was at least partially mediated via the targeting of BCL2L1. Furthermore, knockdown of Lin28 decreased viability and promoted apoptosis in CRC cells, whereas this effect was attenuated by let­7c inhibition. The findings of the present study suggest the involvement of the Lin28/let­7c axis in apoptosis during CRC, and indicate the potential role of this pathway as a novel therapeutic target in CRC.

Catalpol protects mice against Lipopolysaccharide/D-galactosamine-induced acute liver injury through inhibiting inflammatory and oxidative response.

The purpose of this study was to investigate the protective effect of catalpol on Lipopolysaccharide (LPS)/D-galactosamine (D-gal)-induced acute liver injury in mice. The mouse model was established by injection of LPS and D-gal. Catalpol (2.5, 5, 10 mg/kg) were pretreated intraperitoneally 1 h before LPS and D-gal. The survival rate, AST, ALT, MDA, MPO activity, hepatic tissue histology, TNF-α level, and NF-κB activation were assayed. The results revealed that catalpol dose-dependently elevated the survival rate. Furthermore, catalpol reduced the activities of AST, ALT, MDA, and MPO. The production of TNF-α was also inhibited by treatment of catalpol. In addition, catalpol inhibited LPS/D-gal-induced NF-κB activation. The expression of Nrf2 and HO-1 were up-regulated by treatment of catalpol. These results indicated that pretreatment with catalpol could attenuate LPS/D-gal-induced acute liver injury in mice and the underlying mechanism may due to the inhibition of NF-κB signaling pathway and the activation of Nrf2 signaling pathway.

LDB2 inhibits proliferation and migration in liver cancer cells by abrogating HEY1 expression.

Hepatocellular carcinoma (HCC) was one of the most common cancers around the world, has very low 5-year survival rate. However, the mechanism of HCC occurrence and development is largely unknown. LDB2 belongs to the LIM-domain binding family and functions as an adaptor for transcriptional regulation. Here we found that LDB2 is downregulated in HCC samples. LDB2 has the ability to inhibit proliferation and migration of hepatocarcinoma cells. We found that the proliferation and migration abilities in HCC sample cells were impaired after LDB2 overexpression and vice versa. In mechanism, we found that LDB2 can recruit BRD7 to HEY1 promoter and then block its expression. HEY1 whose expression is upregulated in HCC acts as an oncogene. In brief, our research reveals a new regulatory mechanism for hepatocarcinoma cell proliferation and migration.

Protective effects of Schisandrin B on cigarette smoke-induced airway injury in mice through Nrf2 pathway.

Schisandrin B (SchB), a dibenzocyclooctadiene derivative isolated from Schisandra chinensis, has been reported to have anti-inflammatory effects. However, the protective effects of SchB on cigarette smoke (CS)-induced lung inflammation remain unclear. This study was to investigate the effects of SchB on CS-induced lung inflammation in mice. The mice were exposed to CS to develop lung inflammation. SchB was given 1h before CS exposure daily for five consecutive days. The levels of inflammatory mediators TNF-α, IL-1ß, and IL-6 in bronchoalveolar lavage fluid (BALF) were measured in this study. SOD, GSH, MPO and MDA contents were also detected. Furthermore, the expression of Nrf-2 and NF-κB were detected by western blot analysis. Histopathological analyses showed that SchB had protective effects against CS-induced lung inflammation. The levels of inflammatory mediators TNF-α, IL-1ß, and IL-6 in BALF were also inhibited by SchB. CS-induced MPO activity and MDA content were inhibited by SchB. The levels of SOD and GSH were up-regulated by SchB. SchB significantly inhibited CS-induced NF-κB activation and up-regulated the expression of Nrf2 and HO-1. In conclusion, these data suggest that SchB protects against CS-induced lung inflammation by activating Nrf2 and inhibiting NF-κB signaling pathway.

Protective effects of tenuigenin on lipopolysaccharide and d-galactosamine-induced acute liver injury.

Tenuigenin (TEN), a major active component of polygala tenuifolia root, has been reported to have a number of biological properties, such as anti-oxidative and anti-inflammatory activities. However, the protective effect of TEN on acute liver injury has not yet been reported. This research aims to detect the protective effect of TEN on lipopolysaccharide (LPS) and d-galactosamine (D-GalN)-induced acute liver injury in mice and to investigate the molecular mechanisms. TEN was administered intraperitoneally 1 h before LPS/D-GalN treatment. The levels of TNF-α, IL-1ß, ALT, and AST were measured. The expression of NF-κB, ASK1, MAPKs, Nrf2, and HO-1 were detected by western blot analysis. The results showed that TEN significantly inhibited LPS/D-GalN-induced serum ALT and AST levels. TEN also inhibited LPS/D-GalN-induced TNF-α and IL-1ß production. Furthermore, LPS/D-GalN-induced hepatic MDA and MPO activities were also inhibited by TEN. In addition, TEN was found to inhibit LPS/D-GalN-induced ASK1 expression, NF-κB and MAPKs activation and up-regulate the expression of Nrf2 and HO-1. In conclusion, TEN protected against LPS/GalN-induced acute liver injury by suppressing inflammatory and oxidative responses.

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.

miR-139-5p inhibits epithelial-mesenchymal transition, migration and invasion of hepatocellular carcinoma cells by targeting ZEB1 and ZEB2.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer deaths worldwide. miRNAs have been suggested to have important roles in HCC development. The purpose of this study was to determine the role of miR-139-5p in regulation of epithelial-mesenchymal transition (EMT) and metastasis of HCC cells. Expression levels of miR-139-5p in 49 HCC specimens with adjacent tissues and five HCC cell lines were assessed by quantitative RT-PCR. We found that miR-139-5p was down-regulated in 89.7% of the HCC tissue samples and all of the HCC cell lines. In addition, luciferase reporter assays validated direct binding of miR-139-5p to the 3' untranslated region of zinc finger E-box binding homeobox 1 (ZEB1) and ZEB2. Ectopic expression of miR-139-5p suppressed and miR-139-in promoted EMT, migration, and invasion in Hep3B and SMMC7721 cells. Furthermore, over-expression of ZEB1 and ZEB2 ablated the inhibitory effects of miR-139-5p on migration and invasion in HCC cells. Our study indicates that miR-139-5p functions as a suppressor of HCC EMT and metastasis by targeting ZEB1 and ZEB2, and it may be a therapeutic target for metastatic HCC.

Piceatannol promotes apoptosis via up-regulation of microRNA-129 expression in colorectal cancer cell lines.

Piceatannol, a naturally occurring analog of resveratrol, has been confirmed as an antitumor agent by inhibiting proliferation, migration, and metastasis in diverse cancer. However, the effect and mechanisms of piceatannol on colorectal cancer (CRC) have not been well understood. This study aimed to test whether piceatannol could inhibit growth of CRC cells and reveal its underlying molecular mechanism. MTT assay was used to detect the cell viability in HCT116 and HT29 cells. Flow cytometry analysis was employed to measure apoptosis of CRC cells. Bcl-2, Bax and caspase-3 levels were analyzed by Western blot and miR-129 levels were determined by real-time RT-PCR. Our study showed that piceatannol inhibited HCT116 and HT29 cells growth in a concentration- and time-dependent manner. Piceatannol induced apoptosis by promoting expression of miR-129, and then inhibiting expression of Bcl-2, an known target for miR-129. Moreover, knock down of miR-129 could reverse the reduction of cell viability induced by piceatannol in HCT116 and HT29 cells. Taken together, our study unraveled the ability of piceatannol to suppress colorectal cancer growth and elucidated the participation of miR-129 in the anti-cancer action of piceatannol. Our findings suggest that piceatannol can be considered to be a promising anticancer agent for CRC.

[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.