Mitochondrial energy metabolism-related gene signature as a prognostic indicator for pancreatic adenocarcinoma.

Background: Pancreatic adenocarcinoma (PAAD) is a highly malignant gastrointestinal tumor and is associated with an unfavorable prognosis worldwide. Considering the effect of mitochondrial metabolism on the prognosis of pancreatic cancer has rarely been investigated, we aimed to establish prognostic gene markers associated with mitochondrial energy metabolism for the prediction of survival probability in patients with PAAD. Methods: Gene expression data were obtained from The Cancer Genome Atlas and Gene Expression Omnibus databases, and the mitochondrial energy metabolism-related genes were obtained from the GeneCards database. Based on mitochondrial energy metabolism score (MMs), differentially expressed MMRGs were established for MMs-high and MMs-low groups using ssGSEA. After the univariate Cox and least absolute and selection operator (LASSO) analyses, a prognostic MMRG signature was used in the multivariate Cox proportional regression model. Survival and immune cell infiltration analyses were performed. In addition, a nomogram based on the risk model was used to predict the survival probability of patients with PAAD. Finally, the expression of key genes was verified using quantitative polymerase chain reaction and immunohistochemical staining. Intro cell experiments were performed to evaluated the proliferation and invasion of pancreatic cancer cells. Results: A prognostic signature was constructed consisting of two mitochondrial energy metabolism-related genes (MMP11, COL10A1). Calibration and receiver operating characteristic (ROC) curves verified the good predictability performance of the risk model for the survival rate of patients with PAAD. Finally, immune-related analysis explained the differences in immune status between the two subgroups based on the risk model. The high-risk score group showed higher estimate, immune, and stromal scores, expression of eight checkpoint genes, and infiltration of M0 macrophages, which might indicate a beneficial response to immunotherapy. The qPCR results confirmed high expression of MMP11 in pancreatic cancer cell lines, and IHC also verified high expression of MMP11 in clinical pancreatic ductal adenocarcinoma tissues. In vitro cell experiments also demonstrated the role of MMP11 in cell proliferation and invasion. Conclusion: Our study provides a novel two-prognostic gene signature-based on MMRGs-that accurately predicted the survival of patients with PAAD and could be used for mitochondrial energy metabolism-related therapies in the future.

Artificial intelligence assisted cytological detection for early esophageal squamous epithelial lesions by using low-grade squamous intraepithelial lesion as diagnostic threshold.

BACKGROUND: Manual cytological diagnosis for early esophageal squamous cell carcinoma (early ESCC) and high-grade intraepithelial neoplasia (HGIN) is unsatisfactory. Herein, we have introduced an artificial intelligence (AI)-assisted cytological diagnosis for such lesions. METHODS: Low-grade squamous intraepithelial lesion or worse was set as the diagnostic threshold for AI-assisted diagnosis. The performance of AI-assisted diagnosis was evaluated and compared to that of manual diagnosis. Feasibility in large-scale screening was also assessed. RESULTS: AI-assisted diagnosis for abnormal cells was superior to manual reading by presenting a higher efficiency for each slide (50.9 ± 0.8 s vs 236.8 ± 3.9 s, p = 1.52 × 10-76 ) and a better interobserver agreement (93.27% [95% CI, 92.76%-93.74%] vs 65.29% [95% CI, 64.35%-66.22%], p = 1.03 × 10-84 ). AI-assisted detection showed a higher diagnostic accuracy (96.89% [92.38%-98.57%] vs 72.54% [65.85%-78.35%], p = 1.42 × 10-14 ), sensitivity (99.35% [95.92%-99.97%] vs 68.39% [60.36%-75.48%], p = 7.11 × 10-15 ), and negative predictive value (NPV) (97.06% [82.95%-99.85%] vs 40.96% [30.46%-52.31%], p = 1.42 × 10-14 ). Specificity and positive predictive value (PPV) were not significantly differed. AI-assisted diagnosis demonstrated a smaller proportion of participants of interest (3.73%, [79/2117] vs.12.84% [272/2117], p = 1.59 × 10-58 ), a higher consistence between cytology and endoscopy (40.51% [32/79] vs. 12.13% [33/272], p = 1.54 × 10- 8), specificity (97.74% [96.98%-98.32%] vs 88.52% [87.05%-89.84%], p = 3.19 × 10-58 ), and PPV (40.51% [29.79%-52.15%] vs 12.13% [8.61%-16.75%], p = 1.54 × 10-8 ) in community-based screening. Sensitivity and NPV were not significantly differed. AI-assisted diagnosis as primary screening significantly reduced average cost for detecting positive cases. CONCLUSION: Our study provides a novel cytological method for detecting and screening early ESCC and HGIN.

Loss of Heterozygosity for KrasG12D Promotes Malignant Phenotype of Pancreatic Ductal Adenocarcinoma by Activating HIF-2α-c-Myc-Regulated Glutamine Metabolism.

Loss of heterozygosity (LOH) for KRAS, in which a wild-type KRAS allele is progressively lost, promotes invasive and migratory abilities of pancreatic ductal adenocarcinoma (PDAC) cells and tissues. Moreover, the occurrence of KrasG12D-LOH activates nonclassical glutamine metabolism, which is related to the malignant behavior of PDAC cells. Herein, we aim to demonstrate the regulatory link between hypoxia-inducible factor-2α (HIF-2α) and glutamine metabolism that mediates malignant phenotypes in KrasG12D-LOH PDAC cells. HIF-2α-shRNA knockdown lentivirus transfection and metabolite analysis were performed in KrasG12D-LOH and KrasG12D cell lines, respectively. Cell proliferation, migration, and invasion were examined using Cell Counting Kit-8, colony formation, and Transwell assays. Cell cycle phase and apoptosis were determined using flow cytometry. Western blotting and real-time quantitative PCR were also performed. Additionally, a subcutaneous xenograft mouse model was established. LOH stimulated HIF-2α activity and transactivated c-Myc, which has a central regulatory effect on glutamine metabolism independent of hypoxia. Meanwhile, HIF-2α silencing repressed KrasG12D-LOH PDAC cell proliferation, invasion, and migration. HIF-2α knockdown inhibited glutamine uptake and GOT1 expression via a c-Myc-dependent pathway. Collectively, KrasG12D-LOH can activate HIF-2α to regulate c-Myc-mediated glutamine metabolism and promote malignant phenotypes. Moreover, targeting HIF-2α-c-Myc regulated nonclassical glutamine metabolism, providing a new therapeutic perspective for KrasG12D-LOH PDAC.

Dosing profiles of concurrent opioid and benzodiazepine use associated with overdose risk among US Medicare beneficiaries: group-based multi-trajectory models.

BACKGROUND AND AIMS: One-third of opioid (OPI) overdose deaths involve concurrent benzodiazepine (BZD) use. Little is known about concurrent opioid and benzodiazepine use (OPI-BZD) most associated with overdose risk. We aimed to examine associations between OPI-BZD dose and duration trajectories, and subsequent OPI or BZD overdose in US Medicare. DESIGN: Retrospective cohort study. SETTING: US Medicare. PARTICIPANTS: Using a 5% national Medicare data sample (2013-16) of fee-for-service beneficiaries without cancer initiating OPI prescriptions, we identified 37 879 beneficiaries (age ≥ 65 = 59.3%, female = 71.9%, white = 87.6%, having OPI overdose = 0.3%). MEASUREMENTS: During the 6 months following OPI initiation (i.e. trajectory period), we identified OPI-BZD dose and duration patterns using group-based multi-trajectory models, based on average daily morphine milligram equivalents (MME) for OPIs and diazepam milligram equivalents (DME) for BZDs. To label dose levels in each trajectory, we defined OPI use as very low (< 25 MME), low (25-50 MME), moderate (51-90 MME), high (91-150 MME) and very high (>150 MME) dose. Similarly, we defined BZD use as very low (< 10 DME), low (10-20 DME), moderate (21-40 DME), high (41-60 DME) and very high (> 60 DME) dose. Our primary analysis was to estimate the risk of time to first hospital or emergency department visit for OPI overdose within 6 months following the trajectory period using inverse probability of treatment-weighted Cox proportional hazards models. FINDINGS: We identified nine distinct OPI-BZD trajectories: group A: very low OPI (early discontinuation)-very low declining BZD (n = 10 598; 28.0% of the cohort); B: very low OPI (early discontinuation)-very low stable BZD (n = 4923; 13.0%); C: very low OPI (early discontinuation)-medium BZD (n = 4997; 13.2%); D: low OPI-low BZD (n = 5083; 13.4%); E: low OPI-high BZD (n = 3906; 10.3%); F: medium OPI-low BZD (n = 3948; 10.4%); G: very high OPI-high BZD (n = 1371; 3.6%); H: very high OPI-very high BZD (n = 957; 2.5%); and I: very high OPI-low BZD (n = 2096; 5.5%). Compared with group A, five trajectories (32.3% of the study cohort) were associated with increased 6-month OPI overdose risks: E: low OPI-high BZD [hazard ratio (HR) = 3.27, 95% confidence interval (CI) = 1.61-6.63]; F: medium OPI-low BZD (HR = 4.04, 95% CI = 2.06-7.95); G: very high OPI-high BZD (HR = 6.98, 95% CI = 3.11-15.64); H: very high OPI-very high BZD (HR = 4.41, 95% CI = 1.51-12.85); and I: very high OPI-low BZD (HR = 6.50, 95% CI = 3.15-13.42). CONCLUSIONS: Patterns of concurrent opioid and benzodiazepine use most associated with overdose risk among fee-for-service US Medicare beneficiaries initiating opioid prescriptions include very high-dose opioid use (MME > 150), high-dose benzodiazepine use (DME > 40) or medium-dose opioid with low-dose benzodiazepine use.

A Rapid Cytological Screening as pre-Endoscopy Screening for Early Esophageal Squamous Cell Lesions: A Prospective Pilot Study from a Chinese Academic Center.

Background: Cytological detection of early esophageal squamous cell carcinoma (ESCC) remains challenging. Therefore, we introduced a rapid cytological screening method and evaluated its efficacy as a pre-endoscopy screening for early ESCC and precursor lesions. Methods: This method consisted of a sponge sample retrieval, automatic liquid-based cytological treatment and slides preparation, computer-assisted screening and manual diagnosis. Efficacy for detection of early ESCC and precursor lesions was evaluated. Also, diagnostic efficiency was compared with manual diagnosis. Results: Eighty-three patients with early ESCC and precursor lesions and 2,090 asymptomatic participants with high risks of ESCC were enrolled. Whole procedure was accomplished within two working days. Abnormal cells were detected in all 83 patients, and in 272 (13.01%) subjects among 2,090 asymptomatic participants. Early ESCC, high-grade intraepithelial neoplasia, low-grade intraepithelial neoplasia and reflux esophagitis and normal endoscopic findings were detected in 8, 13, 11, 187 and 53 participants with abnormal cells, respectively. The calculated sensitivity, specificity, positive predictive value and negative predictive value for detection of early ESCC and precursor lesions were 100%, 88.34%, 11.76%, and 100%, respectively. Compared with manual diagnosis, this method was accomplished in a shorter time duration (5.4 ± 0.45 min vs 320.2 ± 132.4 min, p < 0.001), a higher diagnostic accuracy (96.7% vs74.4%, p = 0.015) and a better inter-observer agreement (93.3% vs66.7%, K = 0.286, p < 0.001). Conclusions: Our study provides a promising methodology as pre-endoscopy screening for early ESCC and precursor lesions.

Candidate oncogene circularNOP10 mediates gastric cancer progression by regulating miR-204/SIRT1 pathway.

BACKGROUND: The role of circular RNA (circRNA) in gastric cancer (GC) is attracting increasing attention. CircNOP10 (hsa_circ-0034351) has been reported to be upregulated in human GC tissue. However, the biological role and mechanism of circNOP10 in GC remain unknown. METHODS: Circular RNA expression profile of GC was detected based on microarray, and circNOP10 was identified for the subsequent investigation. Clinical samples of GC tissue and patient blood were obtained from the Zhongda Hospital, Southeast University. The different degraded GC cell lines were presented in our laboratory. The function and mechanism of circNOP10 in GC were investigated using Western blot, qRT-PCR, flow cytometry, in situ hybridization and pull down experiment. RESULTS: The results indicated that increased circNOP10 in GC tissue was involved in tumor stage and prognosis. In addition, circNOP10 sponged microRNA-24 (miR-204)-mediated biological processes through sirtuin 1 (SIRT1), which further confirmed that the circNOP10/miR-204/SIRT1 pathway promoted proliferation and migration as well as epithelial-mesenchymal transition (EMT) through the NF-κß pathway in GC cell lines. CONCLUSIONS: Candidate oncogene circNOP10 mediated GC cell proliferation, arrest cell cycle in G2/M phase, induced cell apoptosis, enhanced tumor metastasis, as well as EMT by activating the miR-204/SIRT1 pathway, suggesting that it may serve as a potential biomarker in GC therapy.

Significant Gene Biomarker Tyrosine Kinase Non-receptor 2 Mediated Cell Proliferation and Invasion in Colon Cancer.

Objective: This study aimed to investigate the expression and biological functions of TNK2 and miR-125a-3p in colon cancer. Materials and methods: The expression of TNK2 and miR-125a-3p in colon cancer tissues was analyzed using data deposited on public databases including UALCAN and ONCOMINE. We verified their expression in colon cancer cell lines by RT-qPCR and western blotting. By regulating the expression of TNK2 and miR-125a-3p in colon cancer cells, their functions and potential mechanisms were explored. Results: TNK2 was overexpressed in colon cancer cell lines, and it was found to directly bind to miR-125a-3p, which was downregulated in these cell lines. Their expression affected the proliferation and invasion of colon cancer cells. Additionally, colon cancer patients with lower TNK2 expression had better prognoses than those with higher TNK2 expression. Conclusion: Our results indicated that TNK2 and miR-125a-3p play critical roles in colon cancer, and could also serve as biomarkers for the diagnosis and prognosis of this malignant disease.

SLC7A5 serves as a prognostic factor of breast cancer and promotes cell proliferation through activating AKT/mTORC1 signaling pathway.

BACKGROUND: The purpose of our research was to determine if the clinical, pathological, and prognostic functions of SLC7A5 are the same as those of other molecular breast cancer (BC) subgroups. METHODS: We used the Oncomine and The Cancer Genome Atlas (TCGA) online databases to examine the expression and genetic changes of SLC7A5 in BC tissues. Immunohistochemical analysis was used to validate the SLC7A5 protein expression in subtypes of BC, while Kaplan-Meier figures and log-rank tests were used to evaluate the prognostic relevance of SLC7A5. Uni- and multivariate Cox regression models were adapted to analyze hazard ratios (HRs) and the independent prognostic factors. We analyzed the alterations of different malignancies of luminal cells by up-regulation of SLC7A5 in human luminal cell lines MCF-7. SLC7A5 was overexpressed in luminal cells, and then the AKT, mTOR, and p70-S6K phosphorylation and expression were analyzed by western blot analysis and real-time quantitative polymerase chain reaction (qPCR). RESULTS: Our results suggested that SLC7A5 was overexpressed in BC cell lines and in patients' tissues. Elevated SLC7A5 messenger RNA (mRNA) and SLC7A5 protein expression was correlated to a worse clinical prognosis (P<0.001) in luminal subtypes of BC. The multivariate analysis suggested that high level of SLC7A5 expression could be an independent prognostic factor for decreased overall survival (OS). The study also demonstrated that SLC7A5 overexpression increased proliferation of MCF-7 cells by reducing the cell cycle arrest in G1 phase. Our mechanistic study further indicates that SLC7A5 enhances the proliferation of the MCF-7 cell by activation of AKT/mTORC1 pathway through phosphorylation. CONCLUSIONS: Our study demonstrated that SLC7A5 may have a vital function in the biology of BC cells, indicating that SLC7A5 is a potential prognostic biomarker and may be a valuable therapeutic target in BC patients.

CircRHOT1 mediated cell proliferation, apoptosis and invasion of pancreatic cancer cells by sponging miR-125a-3p.

Pancreatic cancer patients are asymptomatic at early stages and leading to late diagnoses. Additionally, pancreatic cancer easily metastasizes and is resistant to radiotherapy and chemotherapy. Therefore, it is critical to understand the underlying molecular mechanisms involved in pancreatic cancer to develop more efficient diagnostic and treatment strategies. In this study, we demonstrated that circRHOT1 was overexpressed in pancreatic cancer tissues and cell lines, and it was found to directly bind to miR-125a-3p, acting as an endogenous sponge to inhibit its activity. Knockdown of circRHOT1 expression significantly inhibited proliferation as well as invasion, and it promoted apoptosis of pancreatic cancer cells via the regulation of E2F3 through the targeting of miR-125a-3p. Taken together, our results showed that circRHOT1 plays critical roles in regulating the biological functions of pancreatic cancer cells, suggesting that circRHOT1 may serve as a potential diagnostic marker and therapeutic target for patients with pancreatic cancer.

Loss of heterozygosity for KrasG12D promotes REDD1-dependent, non-canonical glutamine metabolism in pancreatic ductal adenocarcinoma.

Pancreatic cancer is associated with high mortality, and pancreatic ductal adenocarcinoma (PDAC) is its most common subtype. The rapid growth of PDAC is dependent on the non-canonical pathway of glutamine (Gln) utilization, and loss of heterozygosity for KrasG12D (KrasG12D-LOH) frequently observed in PDAC is associated with an aggressive and invasive phenotype. However, it remains unclear whether KrasG12D-LOH contributes to non-canonical Gln metabolism in PDAC. Here, we showed that KrasG12D-LOH leads to a substantial increase in non-canonical Gln metabolism in PDAC cells. Importantly, we observed elevated expression of regulated in DNA damage and development 1 (REDD1), which is activated in response to hypoxia and nutrient deprivation, in KrasG12D-LOH PDAC, and that REDD1 knockdown efficiently repressed KrasG12D-LOH-regulated Gln metabolism and suppressed proliferation, migration, and invasion of KrasG12D-LOH PDAC cells. These data provide evidence that REDD1 is a downstream target of KrasG12D-LOH and is involved in promoting non-canonical Gln metabolism in PDAC.

Single high dose irradiation induces cell cycle arrest and apoptosis in human hepatocellular carcinoma cells through the Ras/Raf/MEK/ERK pathways.

Purpose: Stereotactic body radiation therapy (SBRT) is emerging as a new noninvasive treatment in patients with primary liver carcinoma or liver-confined metastatic cancer. However, the radiobiological targets remain a subject of debate. Here, we investigated the potential biological effects of the radiation on the human hepatocellular carcinoma HepG2 cells.Materials and methods: Firstly, HepG2 cells were divided into three groups: control group, 3.5 Gy*8f group (L group), and 15 Gy*1f group (H group). After treatment, cell proliferation was examined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide and plate colony formation assays. Cell cycle and apoptosis were assessed using propidium iodide and Hoechst 33258 staining, respectively. Furthermore, the mechanisms underlying irradiation-induced cell cycle arrest and cell apoptosis were investigated by Western blot assay.Results: Irradiation could effectively inhibit the proliferation and colony formation of HepG2 cells, and the single high dose irradiation showed stronger inhibitory effects. Irradiation-induced cell cycle arrest at G2/M phase in HepG2 cell, during which the expression levels of cyclin B1, CDK1, and p-CDK1 proteins were downregulated, whereas expression of p21 was upregulated in the irradiated HepG2 cells. After irradiation, typical morphological changes of apoptosis in HepG2 cells were observed; the number of cell apoptosis and the expression of apoptosis associated proteins were significantly increased in HepG2 cells by high dose irradiation compared with low dose irradiation. Additionally, compared with low dose irradiation, high dose irradiation significantly downregulated the phosphorylated proteins in the Ras/Raf/MEK/ERK signaling pathway.Conclusions: Our results suggest that irradiation applied in SBRT, particularly single high dose irradiation, mediates its anti-tumor effects by inducing cell cycle arrest and apoptosis via modulation of the Ras/Raf/MEK/ERK signaling pathway.

Long non-coding RNA HOTAIR promotes cancer cell energy metabolism in pancreatic adenocarcinoma by upregulating hexokinase-2.

Hox transcript antisense RNA (HOTAIR) is a long non-coding RNA (lncRNA) that serves a key role in the pathogenesis of various types of cancer, including pancreatic adenocarcinoma. However, the diagnostic and prognostic values of HOTAIR in pancreatic adenocarcinoma, as well as its involvement in cancer cell energy metabolism, remain unclear. In the present study, tumor tissues and adjacent healthy tissues were collected from patients with pancreatic adenocarcinoma, and blood samples were collected from patients and healthy controls. Expression levels of HOTAIR and hexokinase-2 (HK2) were detected by reverse transcription-quantitative polymerase chain reaction. All patients were followed up for 5 years, and the diagnostic and prognostic values of serum HOTAIR levels were investigated by receiver operating characteristic curve and survival analyses, respectively. Pancreatic adenocarcinoma cell lines overexpressing HOTAIR and HK2 were established, and the effects on cell proliferation, lactate production, glucose uptake and ATP production were detected by Cell Counting Kit-8, lactate, glucose uptake and ATP assays, respectively. The protein expression was detected by western blot analysis. The results revealed that HOTAIR and HK2 expression levels were increased in tumor tissues compared with adjacent healthy tissues. The serum levels of HOTAIR and HK2 were higher in patients with pancreatic cancer compared with healthy controls. The serum levels of these two factors may be used to accurately predict pancreatic adenocarcinoma and its prognosis. HOTAIR and HK2 overexpression led to the promotion of tumor cell proliferation. HOTAIR overexpression increased lactate production, glucose uptake and ATP production. Furthermore, it promoted HK2 expression, however HK2 overexpression displayed no significant effects on HOTAIR expression levels. Therefore, it was concluded that the lncRNA HOTAIR may promote cancer cell energy metabolism in pancreatic adenocarcinoma by upregulating HK2.

AMPK Inhibition Suppresses the Malignant Phenotype of Pancreatic Cancer Cells in Part by Attenuating Aerobic Glycolysis.

Pancreatic cancer is a highly aggressive tumor characterized by enhanced aerobic glycolysis. AMP-activated protein kinase (AMPK), which is identified as a well-known regulator of glycolysis, plays an essential role in tumorigenesis. In the present study, we aim to explore the function of AMPK in pancreatic cancer cells and attempt to clarify the possible underlying mechanism. The Cancer Genome Atlas (TCGA) data showed that elevated AMPK expression highly correlated with lower median survival time. In an in vitro study, inhibition of AMPK blocked the proliferation, migration, and invasion ability of four cell lines under normoxia and hypoxia. Additionally, AMPK suppression led to cell cycle arrest and remarkably induced apoptosis. Furthermore, the lactic acid content, ATP content, and the glucose consumption rate were significantly reduced in all four cell lines under different conditions, accompanied by down-regulation of glycolytic biomarkers including phosphorylated mammalian target of rapamycin (p-mTOR)/total mTOR (t-mTOR), Pyruvate kinase M2 (Pkm2), and Hexokinase 2 (Hk2). Collectively, our data showed that AMPK activation is highly involved in pancreatic cancer progression and exerts its pro-tumorigenic functions partly by sustaining glycolytic activity. Hence, AMPK is expected to be a potential therapeutic target for pancreatic cancer.

Dietary fiber intake and risks of proximal and distal colon cancers: A meta-analysis.

The purpose of this study was to conduct a systematic review and meta-analysis of studies investigating the relationship between dietary fiber intake and subsite-specific colon cancer.The PubMed database was searched to identify relevant cohort studies published from inception to August 2016 in order to examine individually the association between dietary fiber intake and the risk of proximal colon cancer (PCC), and that between dietary fiber intake and the risk of distal colon cancer (DCC). We searched the reference lists of the studies included in our analysis as well as those listed in the published meta-analyses. A random-effects model was used to compute summary risk estimates. Heterogeneity was assessed using I and Q statistics. Publication bias was assessed with the Egger's and Begg's tests, with a P value of P < .10 indicating publication bias. All statistical tests were 2-sided.We identified and included 11 prospective cohort studies in the final meta-analysis. The risks of PCC and DCC among individuals in the highest dietary fiber intake quartile/quintile were 14% (relative risk [RR] = 0.86, 95% confidence interval [CI] = 0.78-0.95) and 21% (RR = 0.79, 95% CI = 0.71-0.87) lower, respectively, than those among individuals with the lowest dietary fiber intake. In a subgroup analysis, the inverse association observed in the sex-based subgroup was apparent only for men with PCC. Dietary fiber intake was inversely associated with DCC for both men and women. In addition, dietary fiber intake appeared to be inversely associated with PCC only in European countries, whereas this association was observed for DCC in both European countries and the United States.Our findings reveal that dietary fiber intake is associated inversely with the risk of both PCC and DCCs.

Glycemic Variability Promotes Both Local Invasion and Metastatic Colonization by Pancreatic Ductal Adenocarcinoma.

Background & Aims: Although nearly half of pancreatic ductal adenocarcinoma (PDAC) patients have diabetes mellitus with episodes of hyperglycemia, its tumor microenvironment is hypoglycemic. Thus, it is crucial for PDAC cells to develop adaptive mechanisms dealing with oscillating glucose levels. So far, the biological impact of such glycemic variability on PDAC biology remains unknown. Methods: Murine PDAC cells were cultured in low- and high-glucose medium to investigate the molecular, biochemical, and metabolic influence of glycemic variability on tumor behavior. A set of in vivo functional assays including orthotopic implantation and portal and tail vein injection were used. Results were further confirmed on tissues from PDAC patients. Results: Glycemic variability has no significant effect on PDAC cell proliferation. Hypoglycemia is associated with local invasion and angiogenesis, whereas hyperglycemia promotes metastatic colonization. Increased metastatic colonization under hyperglycemia is due to increased expression of runt related transcription factor 3 (Runx3), which further activates expression of collagen, type VI, alpha 1 (Col6a1), forming a glycemic pro-metastatic pathway. Through epigenetic machinery, retinoic acid receptor beta (Rarb) expression fluctuates according to glycemic variability, acting as a critical sensor relaying the glycemic signal to Runx3/Col6a1. Moreover, the signal axis of Rarb/Runx3/Col6a1 is pharmaceutically accessible to a widely used antidiabetic substance, metformin, and Rar modulator. Finally, PDAC tissues from patients with diabetes show an increased expression of COL6A1. Conclusions: Glycemic variability promotes both local invasion and metastatic colonization of PDAC. A pro-metastatic signal axis Rarb/Runx3/Col6a1 whose activity is controlled by glycemic variability is identified. The therapeutic relevance of this pathway needs to be explored in PDAC patients, especially in those with diabetes.

Circulating tumor cell detection: A direct comparison between negative and unbiased enrichment in lung cancer.

Circulating tumor cells (CTCs), isolated as a 'liquid biopsy', may provide important diagnostic and prognostic information. Therefore, rapid, reliable and unbiased detection of CTCs are required for routine clinical analyses. It was demonstrated that negative enrichment, an epithelial marker-independent technique for isolating CTCs, exhibits a better efficiency in the detection of CTCs compared with positive enrichment techniques that only use specific anti-epithelial cell adhesion molecules. However, negative enrichment techniques incur significant cell loss during the isolation procedure, and as it is a method that uses only one type of antibody, it is inherently biased. The detection procedure and identification of cell types also relies on skilled and experienced technicians. In the present study, the detection sensitivity of using negative enrichment and a previously described unbiased detection method was compared. The results revealed that unbiased detection methods may efficiently detect >90% of cancer cells in blood samples containing CTCs. By contrast, only 40-60% of CTCs were detected by negative enrichment. Additionally, CTCs were identified in >65% of patients with stage I/II lung cancer. This simple yet efficient approach may achieve a high level of sensitivity. It demonstrates a potential for the large-scale clinical implementation of CTC-based diagnostic and prognostic strategies.

Enhancement of scutellarin oral delivery efficacy by vitamin B12-modified amphiphilic chitosan derivatives to treat type II diabetes induced-retinopathy.

BACKGROUND: Diabetic retinopathy is the most common complication in diabetic patients relates to high expression of VEGF and microaneurysms. Scutellarin (Scu) turned out to be effective against diabetes related vascular endothelial cell dysfunction. However, its clinical applications have been limited by its low bioavailability. In this study, we formulated and characterized a novel intestinal target nanoparticle carrier based on amphiphilic chitosan derivatives (Chit-DC-VB12) loaded with scutellarin to enhance its bioavailability and then evaluated its therapeutic effect in experimental diabetic retinopathy model. RESULTS: Chit-DC-VB12 nanoparticles showed low toxicity toward the human colon adenocarcinoma (Caco-2) cells and zebra fish within concentration of 250 µg/ml, owing to good biocompatibility of chitosan. The scutellarin-loaded Chit-DC-VB12 nanoparticles (Chit-DC-VB12-Scu) were then prepared by self-assembly in aqueous solution. Scanning electron microscopy and dynamic light scattering analysis indicated that the Chit-DC-VB12-Scu nanoparticles were spherical particles in the sizes ranging from 150 to 250 nm. The Chit-DC-VB12-Scu nanoparticles exhibited high permeation in Caco-2 cell, indicated it could be beneficial to be absorbed in humans. We also found that Chit-DC-VB12 nanoparticles had a high cellular uptake. Bioavailability studies were performed in Sprague-Dawley rats, which present the area under the curve of scutellarin of Chit-DC-VB12-Scu was two to threefolds greater than that of free scutellarin alone. Further to assess the therapeutic efficacy of diabetic retinopathy, we showed Chit-DC-VB12-Scu down-regulated central retinal artery resistivity index and the expression of angiogenesis proteins (VEGF, VEGFR2, and vWF) of retinas in type II diabetic rats. CONCLUSIONS: Chit-DC-VB12 nanoparticles loaded with scutellarin have better bioavailability and cellular uptake efficiency than Scu, while Chit-DC-VB12-Scu nanoparticles alleviated the structural disorder of intraretinal neovessels in the retina induced by diabetes, and it also inhibited the retinal neovascularization via down-regulated the expression of angiogenesis proteins. In conclusion, the Chit-DC-VB12 nanoparticles enhanced scutellarin oral delivery efficacy and exhibited potential as small intestinal target promising nano-carriers for treatment of type II diabetes induced-retinopathy.

Loss of fatty acid synthase suppresses the malignant phenotype of colorectal cancer cells by down-regulating energy metabolism and mTOR signaling pathway.

PURPOSE: Altered cellular metabolism has received increased attention as an important hallmark of cancer. Activation of FASN has been found to be involved in many human tumors. Despite extensive research in FASN function on cancer, the underlying mechanism is not entirely understood yet. METHODS: Cerulenin was used to suppress the FASN expression in human colorectal cancer cell lines (HT29 and LoVo). Expression of PI3K, Akt, p-Akt, mTOR, p-mTOR, FASN, and AZGP1 was measured using western blotting and qPCR. ATP and lactic acid were assessed to investigate the activation of energy metabolism. Cell cytotoxicity assay was studied by cell counting kit-8 assay. The capacity of cell proliferation and migration was investigated by clonogenic and invasion assay. Analysis of apoptosis and the cell cycle was detected by flow cytometry. RESULTS: We found that the expression of FASN was down-regulated, while the expression of PI3K, p-Akt, p-mTOR, and AZGP1 was down-regulated in HT29 and LoVo cells treated with FASN inhibitor. Proliferation was reduced in FASN inhibitor-treated cells, which is consistent with an increased apoptosis rate. Furthermore, the migration of FASN inhibitor-treated cells was decreased and the content of ATP and lactic acid was also dropped. CONCLUSION: These findings suggest that inhibited FASN suppresses the malignant phenotype of colorectal cancer cells by down-regulating energy metabolism and mTOR signaling pathway. The results have paved the way to understand the relations of FASN, mTOR signaling pathway, and energy metabolism in colorectal cancer cells.

Novel drug delivery nanosystems based on out-inside bifunctionalized mesoporous silica yolk-shell magnetic nanostars used as nanocarriers for curcumin.

Mesoporous silica has aroused lots of interest in biomedical fields, in which novel kinds of mesoporous silica with tunable mesoporosity and size have attracted much attention, but functionalization or multi-functionalization inside the mesopores of these structures is still rarely detected. Among all the functionalized molecules, beta cyclodextrin hydrate (ß-CD), a kind of hydrophilic non-toxic carrier for hydrophobic drugs, can increase the solubility and bioavailability of drugs, acting as a pH responsive "gate" when functionalized on the surface of mesoporous silica. Herein, we functionalized ß-CD inside novel kinds of mesoporous silica or magnetic mesoporous silica as the physical binding sites for the model drug curcumin through an out-inside two step bifunctionalization process including a vacuum pumping recrystallization drug loading process. According to the physical characterization with XRD, FT-IR, XPS, solid state NMR, BET, TG, DTA, TEM and DLS, the drug delivery nanosystems were successfully fabricated and contained nano-formulations of curcumin. In vitro cell testing, including Cell Counting Kit-8, Hoechst 33342 staining, hemolysis experiments and cell apoptosis, indicated that through the bifunctionalization, the biocompatibility of the mesoporous silica with cells could be improved, and the toxicity of the drug delivery nano-formulations towards two kinds of cancer cells, SK-HEP1 and HepG2, was significantly increased compared with free curcumin. In vitro release studies revealed that the nano-formulations contained more suspended curcumin molecules as described by the Higuchi models and showed enhanced pH responsive release properties, and that the magnetic nano-formulations exhibited alternating magnetically controlled release properties. The confocal microscopy analysis results revealed the obviously increasing intercellular release and uptake of both nano-formulations of curcumin by SK-HEP1 cells and the "on" and "off" stages of the alternating magnetism responsive intercellular release properties of the fabricated magnetic nano-formulations. The bifunctionalization process combined with the nanostar structure has immense potential in the drug delivery field, especially for hydrophobic drugs.

Expressions of fatty acid synthase and HER2 are correlated with poor prognosis of ovarian cancer.

The present study was designed to explore the cross talk between fatty acid synthase (FASN) and HER2 (ErbB2) in ovarian cancer. A total of 60 ovarian cancer patients and 15 normal ovarian tissues were enrolled. Tissue array was conducted by using a tissue microarray instrument. Immunohistochemistry was performed to quantify the expressions of HER2 and FASN. The FASN was detected to be distributed in the cell cytoplasm and was significantly correlated with cancer grade (p = 0.000) and FIGO staging (p = 0.000). Patients with FASN overexpression in ovarian cancer tend to have a worse overall survival rate (p = 0.000). HER2 was also stained to be distributed in the cell cytoplasm associated with higher expression in high-grade cancer. It was also disclosed that FASN expression level is not correlated with HER2 status in ovarian cancer. These results for the first time indicated that a cross talk in FASN and HER2 expressions might be associated with prognosis in malignant ovarian cancer.