Effects of ß-carboline alkaloids from Peganum harmala on the FAK/PI3K/AKT/Mtor pathway in human gastric cancer cell line SGC-7901 and tumor-bearing mice.

This study investigates the effects of ß-carboline alkaloids from Peganum harmala on FAK/PI3K/AKT/mTOR pathway in gastric cancer cell line SGC-7901 and tumor-bearing mice. Western blot, immunohistochemistry and RT-PCR were performed to detect protein and mRNA expressions of BCL-2, Bax, FAK, PI3K, AKT and mTOR. Mice model of gastric tumor was established with SGC-7901 cells. TUNEL assay was used to detect apoptosis. HE staining was used to observe morphological changes. In vitro, the protein and mRNA expressions of FAK, PI3K, AKT and mTOR in ß-carboline alkaloids groups were significantly lower than those in control and fluorouracil groups (P<0.05). BCL-2 decreased while Bax increased. In vivo, the tumor weights of ß-carboline alkaloids and fluorouracil groups were significantly lower than those of control group (P<0.05). FAK, PI3K, AKT and mTOR proteins in tumor tissues of ß-carboline alkaloids and fluorouracil groups were significantly lower than control group (P<0.05). Additionally, ß-carboline alkaloids treatment in vivo caused obvious cell necrosis and apoptosis. Conclusively, ß-carboline alkaloids can reduce FAK, PI3K, AKT and mTOR expressions at both protein and mRNA levels in SGC-7901 cells and tumor tissues formed by SGC-7901 cells. They may be targets of ß-carboline in FAK/PI3K/AKT/mTOR pathway.

Ras GTPase-Activating-Like Protein IQGAP1 (IQGAP1) Promotes Breast Cancer Proliferation and Invasion and Correlates with Poor Clinical Outcomes.

BACKGROUND Breast cancer is one of the most common female cancers in the world. As a key integrator of cell signaling pathways, IQGAP1 contributes to the development and progression of several cancers. However, the exact effects and molecular mechanisms of IQGAP1 in breast cancer progression remain poorly understood. MATERIAL AND METHODS In the present study, IQGAP1 expression was measured in 96 paired breast cancer samples and the corresponding adjacent non-cancerous tissues by immunohistochemistry and quantitative polymerase chain reaction. To further explore the biological function of IQGAP1 in breast cancer cells, we knocked down IQGAP1 expression in MCF-7 cells and overexpressed it in SK-BR-3 cells. RESULTS IQGAP1 was specifically upregulated in breast cancer tissues compared with the corresponding adjacent non-cancerous tissues. Moreover, IQGAP1 expression was positively correlated with breast cancer survival rate. IQGAP1 also promoted breast cancer cell proliferation and cell cycle progression and suppressed apoptosis. CONCLUSIONS In conclusion, our results suggest that IQGAP1 plays an important role in the cell proliferation and invasion of human breast cancer cells, thus indicating that IQGAP1 may be a potential therapeutic target for the treatment of human breast cancer.

CircRNA LDLR promotes proliferation and aerobic glycolysis of gastric cancer cells by targeting CHD1 with miR-449b-5p.

Background/aim: Circular RNAs can serve as detection biomarkers and therapeutic targets for tumors. Our study aimed to elucidate the mechanisms associated with circRNA LDLR (circLDLR) in gastric cancer (GC) proliferation and aerobic glycolysis. Materials and methods: Expression signatures of circLDLR, miR-449b-5p, and CHD1 were examined in GC samples using quantitative PCR. Proliferation ability of MKN-45 cells was assessed via CCK-8 and EdU assays, and cell apoptosis was measured by flow cytometry. Glucose uptake, lactate production, ATP/ADP ratios, and NAD+/NADH ratios in cell supernatants were quantified to evaluate aerobic glycolysis. Subcellular isolation assay, quantitative PCR, immunoblot analysis, RNA immunoprecipitation (RIP), and dual luciferase reporter assay were employed to investigate the relationship between genes. Results: Expression of circLDLR and CHD1 was elevated, while miR-449b-5p expression decreased in GC. Functionally, overexpression of circLDLR enhanced proliferation and aerobic glycolysis and hampered apoptosis of MKN-45 cells. However, upregulation of miR-449b-5p or downregulation of CHD1 reversed these effects. CircLDLR acted as an miRNA spongeand regulated the expression of miR-449b-5p, thereby affecting CHD1 and accelerating GC malignant progression. Conclusion: CircLDLR drives the proliferation and aerobic glycolysis of GC cells by targeting CHD1 with miR-449b-5p, which is an ideal potential target for early diagnosis and clinical treatment of GC.

[Effect of Kang'ai injection on hepatic fibrosis in rats].

OBJECTIVE: To investigate the inhibition role of Kang'ai injection (KAI) in rats with hepatic fibrosis. METHOD: Animal model with liver fibrosis were induced by 0.01% concentration of diethylnitrosamine (DEN). 30 female Wistar rats (160-200 g) were randomly divided into 3 groups: the KAI-DEN group, the DEN group and the blank control group. The KAI-DEN group was administered Kang'ai injection (1 mL x k(-1), intraperitoneal injection, once a week) and the DEN group was administered normal saline intraperitoneal injection. HE staining and VG special staining of liver tissue were used to evaluate liver fibrosis. RESULT: Compared with the DEN group, relatively less structural damage and less pseudolobular formation in the KAI-DEN group. Collagen area of the blank control group, the KAI-DEN group and the DEN group were (6.52 +/- 2.64)% , (17.41 +/- 1.112)% and (20.180 +/- 2.519)% , respectively. The difference was statistically significant, P < 0.05. CONCLUSION: Kang'ai injection could inhibit the formation of DEN-induced liver fibrosis.

MiR-30a-5p hampers proliferation of lung squamous cell carcinoma through targeting FBXO45.

OBJECTIVE: Studies have elaborated the inhibition of miR-30a-5p on the proliferation of cancer cells. However, the regulatory mechanism of how miR-30a-5p works in lung squamous cell carcinoma (LUSC) cells is obscure. METHODS: Data of miRNAs/mRNAs in LUSC tissue (The Cancer Genome Atlas (TCGA)) were accessed. A differential upstream miRNA (miR-30a-5p) was obtained by differential analysis. Downstream target mRNAs were predicted and screened by several databases. The function pathways of target protein in cells were determined by gene set enrichment analysis (GSEA). Abnormal expression levels of FBXO45 and miR-30a-5p were evaluated in three LUSC cell lines. The expression levels of FBXO45 mRNA and miR-30a-5p were analyzed by qRT-PCR. Western blot method was employed to assess protein levels of FBXO45, Cyclin E1, Cdk4 and Cyclin D1. How the two researched genes interact was testified by dual-luciferase method. Cell proliferative ability was compared by CCK-8 and colony formation methods. Moreover, cell cycle was tested by flow cytometry. RESULTS: MiR-30a-5p was tested to be noticeably down-regulated in LUSC cell lines. Up-regulated FBXO45 in LUSC was targeted by miR-30a-5p. Overexpressing miR-30a-5p modulated proliferation and cell cycle in LUSC via inhibiting FBXO45. CONCLUSION: MiR-30a-5p hindered FBXO45 expression to repress the proliferation of LUSC. FBXO45/miR-30a-5p may shed light on future molecular treatment of LUSC.

Differentially Expressed mRNAs and Potential Mechanisms of Radiation-Induced TUT4-/- Esophageal Cell Injury.

Radiation-induced esophageal injury remains a limitation for the process of radiotherapy for lung and esophageal cancer patients. Esophageal epithelial cells are extremely sensitive to irradiation, nevertheless, factors involved in the radiosensitivity of esophageal epithelial cells are still unknown. Terminal uridyl transferase 4 (TUT4) could modify the sequence of miRNAs, which affect their regulation on miRNA targets and function. In this study, we used transcriptome sequencing technology to identify mRNAs that were differentially expressed before and after radiotherapy in esophageal epithelial cells. We further explored the mRNA expression profiles between wild-type and TUT4 knockout esophageal epithelial cells. Volcano and heatmap plots unsupervised hierarchical clustering analysis were performed to classify the samples. Enrichment analysis on Gene Ontology functional annotations and Kyoto Encyclopedia of Genes and Genomes pathways was performed. We annotated differential genes from metabolism, genetic information processing, environmental information processing, cellular processes, and organismal systems human diseases. The aberrantly expressed genes are significantly enriched in irradiation-related biological processes, such as DNA replication, ferroptosis, and cell cycle. Moreover, we explored the distribution of transcription factor family and its target genes in differential genes. These mRNAs might serve as therapeutic targets in TUT4-related radiation-induced esophageal injury.

Establish immune-related gene prognostic index for esophageal cancer.

Background: Esophageal cancer is a tumor type with high invasiveness and low prognosis. As immunotherapy has been shown to improve the prognosis of esophageal cancer patients, we were interested in the establishment of an immune-associated gene prognostic index to effectively predict the prognosis of patients. Methods: To establish the immune-related gene prognostic index of esophageal cancer (EC), we screened 363 upregulated and 83 downregulated immune-related genes that were differentially expressed in EC compared to normal tissues. By multivariate Cox regression and weighted gene coexpression network analysis (WGCNA), we built a prognostic model based on eight immune-related genes (IRGs). We confirmed the prognostic model in both TCGA and GEO cohorts and found that the low-risk group had better overall survival than the high-risk group. Results: In this study, we identified 363 upregulated IRGs and 83 downregulated IRGs. Next, we found a prognostic model that was constructed with eight IRGs (OSM, CEACAM8, HSPA6, HSP90AB1, PCSK2, PLXNA1, TRIB2, and HMGB3) by multivariate Cox regression analysis and WGCNA. According to the Kaplan-Meier survival analysis results, the model we constructed can predict the prognosis of patients with esophageal cancer. This result can be verified by the Gene Expression Omnibus (GEO). Patients were divided into two groups with different outcomes. IRGPI-low patients had better overall survival than IRGPI-high patients. Conclusion: Our findings indicated the potential value of the IRGPI risk model for predicting the prognosis of EC patients.

The long non-coding RNA MIAT/miR-139-5p/MMP2 axis regulates cell migration and invasion in non-small-cell lung cancer.

Non-small-cell lung cancer (NSCLC) is a complex disease which is influenced by multiple factors. Recent studies demonstrated that long non-coding RNA (lncRNA) MIAT was involved in tumor metastasis. However, the underlying mechanism of MIAT in NSCLC remains largely unknown. In this study, MIAT, miR-139-5p and MMP2 expression were measured by quantitative reverse transcriptase PCR (QRT-PCR) or Western blotting, respectively, and we found the expression of MIAT and MMP2 were elevated, while miR-139-5p was decreased in NSCLC tissues and cell lines. Transwell assay showed MIAT and MMP2 functioned as an oncogene to induce cell migration and invasion in NSCLC, but miR-139-5p served as a tumor suppressor in NSCLC to inhibit cell migration and invasion. Besides that, in vivo experiments also indicated MIAT deletion inhibited tumor growth. The relationship between miR-139-5p and MIAT or MMP2 was then confirmed by Luciferase reporter assay, and the results showed that MIAT directly interacted with miR-139-5p and miR-139- 5p targetedly suppressed MMP2 in NSCLC cells. Furthermore, expression analysis showed that MIAT indirectly regulated MMP2 by sponging miR-139-5p. Finally, rescue assay suggested that miR-139-5p restoration reversed MIAT-overexpression-induced promotion on the migration and invasion of NSCLC cells. In conclusion, our results demonstrated that lncRNA MIAT modulated the migration and invasion of NSCLC by regulating miR-139-5p and MMP2.

miR-485-5p inhibits the progression of breast cancer cells by negatively regulating MUC1.

OBJECTIVE: To investigate the mechanism of miR-485-5p inhibiting breast cancer cells by targeting MUC1. METHODS: Differentially expressed genes (DEGs) in breast cancer tissues were analyzed using breast cancer tissue microarrays (TMA) in the GEO database. Differential expression of MUC1 in breast cancer tissue samples was detected by TCGA database. qRT-PCR was used to detect the expression of MUC1 and miR-485-5p in human normal breast epithelial cell lines and human breast cancer cell lines. Bioinformatics was applied to analyze targeted binding site of miR-485-5p and MUC1 and their targeted relationship was identified by dual luciferase assay. The proliferation ability of breast cancer cells was detected by CCK-8 assay. Cell apoptosis was detected by flow cytometry. The ability of cell migration was measured by scratch healing test. Transwell assay was used to detect the invasion ability of cells. The protein expression levels of MUC1 and EMT-related molecules (E-cadherin, N-cadherin and Vimentin) were detected by Western blot. RESULTS: MUC1 was highly expressed in breast cancer tissue samples and breast cancer cell lines, while miR-485-5p was lowly expressed. Overexpression of miR-485-5p inhibits cell viability and invasion and migration of breast cancer cell line MCF-7 and promotes apoptosis. The same results were obtained by silencing the expression of MUC1. MiR-485-5p targets to bind to the 3'-UTR region of MUC1 and negatively regulates the expression of MUC1. Overexpressing MUC1 while overexpressing miR-485-5p reversed the inhibitory effect of miR-485-5p on breast cancer and inhibited EMT. CONCLUSION: MiR-485-5p can down-regulate the expression of MUC1, thus inhibit the proliferation, invasion and migration of breast cancer cells and promote cell apoptosis.

Curcumin Reverses 5-Fluorouracil Resistance by Promoting Human Colon Cancer HCT-8/5-FU Cell Apoptosis and Down-regulating Heat Shock Protein 27 and P-Glycoprotein.

OBJECTIVE: To investigate the potential mechanisms that curcumin reverses 5-fluorouracil (5-FU) multidrug resistance (MDR). METHODS: Cell growth and the inhibitory rate of curcumin (2-25 µg/mL) and/or 5-FU (0.05-1000 µg/mL) on human colon cancer HCT-8 and HCT-8/5-FU (5-FU-resistant cell line) were determined using cell counting kit-8 (CCK-8) assay. Apoptosis and cell cycle after 5-FU and/or curcumin treatment were detected by flow cytometry (FCM) and transmission electron microscopy (TEM). The expression of the multidrug resistance related factors p-glycoprotein (P-gp) and heat shock protein 27 (HSP-27) genes and proteins were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting (WB), respectively. RESULTS: The inhibitory rate of curcumin or 5-FU on HCT-8 and HCT-8/5-FU cells proliferation at exponential phase were in a dosedependent manner, HCT-8 cell line was more sensitive to curcumin or 5-FU when compared the inhibitory rate of HCT-8/5-FU. The 50% inhibitory concentration (IC50) of combination 5-FU and curcumin (4.0 µg/mL) in HCT-8/5-FU was calculated as 179.26 µg/mL, with reversal fold of 1.85. Another IC50 of combination 5-FU and curcumin (5.5 µg/mL) in HCT-8/5-FU was calculated as 89.25 µg/mL, with reversal fold of 3.71. Synergistic effect of 5-FU and curcumin on HCT-8 and HCT-8/5-FU cells were found. The cell cycle analysis performed by FCM showed that HCT-8 and HCT-8/5-FU cells mostly accumulated at G0/G1 phase, which suggested a synergistic effect of curcumin and 5-FU to induce apoptosis. FCM analysis found that the percentage of apoptosis of cells treated with curcumin, 5-FU and their combination were significantly increased compared to the control group (P<0.05), and the percentage of apoptosis of the combination groups were slightly higher than other groups (P<0.05). The mRNA levels of P-gp (0.28±0.02) and HSP-27 (0.28±0.09) in HCT-8/5-FU cells treated with combination drugs were lower than cells treated with 5-FU alone (P-gp, 0.48±0.07, P=0.009; HSP-27, 0.57±0.10, P=0.007). The protein levels of P-gp (0.25±0.06) and HSP-27 (0.09±0.02) in HCT-8/5-FU cells treated with combination drugs were decreased when compared to 5-FU alone (P-gp, 0.46±0.02, P=0.005; HSP-27, 0.43±0.01, P=0.000). CONCLUSIONS: Curcumin can inhibit the proliferation of human colon cancer cells. Curcumin has the ability of reversal effects on the multidrug resistance of human colon cancer cells lines HCT-8/5-FU. Down-regulation of P-gp and HSP-27 may be the mechanism of curcumin reversing the drug resistance of HCT-8/5-FU to 5-FU.

Prognostic value of lymph node metastasis in patients with T1-stage colorectal cancer from multiple centers in China.

AIM: To explore the features and prognostic value of lymph node metastasis in patients with T1-stage colorectal cancer (CRC). METHODS: In all, 321 cases of T1-stage CRC were selected from 10132 patients with CRC who received surgical therapy in six large-scale hospitals in China and were retrospectively analyzed. Univariate and multivariate analyses were performed to analyze the risk factors for lymphatic metastasis. A survival analysis was then performed to analyze the prognostic value of lymph node metastasis. RESULTS: The occurrence rate of T1 stage was 3.17% (321/10132); of these patients, the lymph node metastasis rate was 8.41% (27/321), and the non-lymph node metastasis rate was 91.59% (294/321). Univariate analysis showed that preoperative serum CEA, preoperative serum CA199, preoperative serum CA724, vascular invasion, and degree of differentiation were associated with lymph node metastasis in T1-stage CRC (P < 0.05 for all). Multivariate analysis indicated that preoperative serum CA724, vascular invasion, and degree of differentiation were closely related to lymph node metastasis (P < 0.05 for all). Log-rank survival analysis showed that age, preoperative serum CEA, preoperative serum CA199, vascular invasion, degree of differentiation, and lymph node metastasis (χ2 = 24.180, P < 0.001) were predictors of 5-year overall survival (OS) (P < 0.05 for all). COX regression analysis demonstrated that preoperative serum CA199 and lymph node metastasis (HR = 5.117; P < 0.05; 95%CI: 0.058-0.815) were independent prognostic indicators of 5-year OS in patients with T1-stage CRC (P < 0.05 for both). CONCLUSION: The morbidity of T1-stage CRC was 3.17% for all CRC cases. Preoperative serum CA724, vascular invasion, and degree of differentiation are independent risk factors for lymph node metastasis. Lymph node metastasis is an independent prognostic factor for OS in patients with T1-stage CRC.

Cytotoxic effects of ß-carboline alkaloids on human gastric cancer SGC-7901 cells.

To investigate the cytotoxic effects of ß-carboline alkaloids on human gastric cancer SGC-7901 cells. Human gastric cancer SGC-790s1 cells were treated with ß-carboline alkaloids at the concentration of 0, 10, 20, 30 and 40 µg/ml for 48 hr. Cell viability was measured by Cell Counting Kit-8 assay. Cell apoptosis was detected by Hoechst 33258 staining and DNA fragmentation analysis. The expression of phosphatase and tensin homolog (PTEN) and extracellular signal-regulated kinase (ERK) was examined by quantitative real-time PCR (qRT-PCR) assay and western blot analysis. ß-carboline alkaloids inhibited the growth of SGC-7901 cells concentration dependently. ß-carboline alkaloids treated SGC-7901 cells displayed apoptotic nuclei as detected using Hoechst 33258 staining. ß-carboline alkaloids also induced DNA ladder, indicative of apoptosis in SGC-7901 cells concentration-dependently. Furthermore, ß-carboline alkaloids increased PTEN and decreased ERK mRNA expression in SGC-7901 cells in a concentration dependent manner. They also increased PTEN and decreased ERK protein expression. ß-carboline alkaloids inhibit the growth and induce apoptosis of SGC-7901 cells. The cytotoxic effects of ß-carboline alkaloids might correlate with increased PTEN expression and decreased ERK expression in SGC-7901 cells.

The clinicopathological significance of CDH1 in gastric cancer: a meta-analysis and systematic review.

BACKGROUND: CDH1 is a protein encoded by the CDH1 gene in humans. Loss of CDH1 function contributes to cancer progression by increasing proliferation, invasion, and/or metastasis. However, the association and clinicopathological significance between CDH1 hypermethylation and gastric cancer (GC) remains unclear. In this study, we systematically reviewed the studies of CDH1 hypermethylation and GC, and evaluated the association between CDH1 hypermethylation and GC using meta-analysis methods. METHODS: A comprehensive search of the PubMed and Embase databases was performed for publications up to July 2014. Methodological quality of the studies was also evaluated. The data were extracted and assessed by two reviewers independently. Analyses of pooled data were performed. Odds ratios (ORs) were calculated and summarized. RESULTS: A final analysis of 1,079 GC patients from 14 eligible studies was performed. CDH1 hypermethylation level in the cancer group was significantly higher compared to the normal gastric mucosa (OR =8.55, 95% confidence interval [CI]: 2.39-33.51, Z=5.47, P<0.00001). CDH1 hypermethylation was not significantly higher in GC than in adjacent gastric mucosa (OR =3.68, 95% CI: 0.96-14.18, Z=1.90, P=0.06). However, CDH1 hypermethylation was higher in adjacent gastric mucosa compared to that in normal gastric mucosa (OR =2.55, 95% CI: 1.22-5.32, Z=2.49, P<0.01). In addition, CDH1 hypermethylation was correlated with Helicobacter pylori (HP) status in GC. The pooled OR from six studies including 280 HP-positive GCs and 193 HP-negative GCs is 1.72 (95% CI: 1.13-2.61, Z=2.55, P=0.01). CONCLUSION: The results of this meta-analysis reveal that CDH1 hypermethylation levels in cancer and adjacent gastric mucosa are significantly higher compared to normal gastric mucosa. Thus, CDH1 hypermethylation is significantly correlated with GC risk. CDH1 hypermethylation is correlated with HP status, indicating that it plays a more important role in the pathogenesis of HP-positive GC and might be an interesting potential drug target for GC patients.