Reappraisal of the Roles of the Sonic Hedgehog Signaling Pathway in Hepatocellular Carcinoma.

HCC remains one of the leading causes of cancer-related death globally. The main challenges in treatments of hepatocellular carcinoma (HCC) primarily arise from high rates of postoperative recurrence and the limited efficacy in treating advanced-stage patients. Various signaling pathways involved in HCC have been reported. Among them, the Sonic hedgehog (SHH) signaling pathway is crucial. The presence of SHH ligands is identified in approximately 60% of HCC tumor tissues, including tumor nests. PTCH-1 and GLI-1 are detected in more than half of HCC tissues, while GLI-2 is found in over 84% of HCC tissues. The SHH signaling pathway (including canonical and non-canonical) is involved in different aspects of HCC, including hepatocarcinogenesis, tumor growth, tumor invasiveness, progression, and migration. The SHH signaling pathway also contributes to recurrence, metastasis, modulation of the cancer microenvironment, and sustaining cancer stem cells. It also affects the resistance of HCC cells to chemotherapy, target therapy, and radiotherapy. Reappraisal of the roles of the SHH signaling pathway in HCC may trigger some novel therapies for HCC.

Cellular and Molecular Biology of Cancer Stem Cells of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death globally. The cancer stem cells (CSCs) of HCC are responsible for tumor growth, invasion, metastasis, recurrence, chemoresistance, target therapy resistance and radioresistance. The reported main surface markers used to identify liver CSCs include epithelial cell adhesion/activating molecule (EpCAM), cluster differentiation 90 (CD90), CD44 and CD133. The main molecular signaling pathways include the Wnt/ß-catenin, transforming growth factors-ß (TGF-ß), sonic hedgehog (SHH), PI3K/Akt/mTOR and Notch. Patients with EpCAM-positive alpha-fetoprotein (AFP)-positive HCC are usually young but have advanced tumor-node-metastasis (TNM) stages. CD90-positive HCCs are usually poorly differentiated with worse prognosis. Those with CD44-positive HCC cells develop early metastases. Those with CD133 expression have a higher recurrence rate and a shorter overall survival. The Wnt/ß-catenin signaling pathway triggers angiogenesis, tumor infiltration and metastasis through the enhancement of angiogenic factors. All CD133+ liver CSCs, CD133+/EpCAM+ liver CSCs and CD44+ liver CSCs contribute to sorafenib resistance. SHH signaling could protect HCC cells against ionizing radiation in an autocrine manner. Reducing the CSC population of HCC is crucial for the improvement of the therapy of advanced HCC. However, targeting CSCs of HCC is still challenging.

Ubiquitin-proteasome system in diabetic retinopathy.

Diabetic retinopathy (DR) is the most common complication of diabetes, being the most prevalent reason for blindness among the working-age population in the developed world. Despite constant improvement of understanding of the pathogenesis of DR, identification of novel biomarkers of DR is needed for improvement of patient risk stratification and development of novel prevention and therapeutic approaches. The ubiquitin-proteasome system (UPS) is the primary protein quality control system responsible for recognizing and degrading of damaged proteins. This review aims to summarize literature data on modifications of UPS in diabetes and DR. First, we briefly review the structure and functions of UPS in physiological conditions. We then describe how UPS is involved in the development and progression of diabetes and touch upon the association of UPS genetic factors with diabetes and its complications. Further, we focused on the effect of diabetes-induced hyperglycemia, oxidative stress and hypoxia on UPS functioning, with examples of studies on DR. In other sections, we discussed the association of several other mechanisms of DR (endoplasmic reticulum stress, neurodegeneration etc) with UPS modifications. Finally, UPS-affecting drugs and remedies are reviewed. This review highlights UPS as a promising target for the development of therapies for DR prevention and treatment and identifies gaps in existing knowledge and possible future study directions.

Upper Gastrointestinal Cancer and Liver Cirrhosis.

The extended scope of upper gastrointestinal cancer can include esophageal cancer, gastric cancer and pancreatic cancer. A higher incidence rate of gastric cancer and esophageal cancer in patients with liver cirrhosis has been reported. It is attributable to four possible causes which exist in cirrhotic patients, including a higher prevalence of gastric ulcers and congestive gastropathy, zinc deficiency, alcohol drinking and tobacco use and coexisting gut microbiota. Helicobacter pylori infection enhances the development of gastric cancer. In addition, Helicobacter pylori, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans also contribute to the development of pancreatic cancer in cirrhotic patients. Cirrhotic patients (especially those with alcoholic liver cirrhosis) who undergo liver transplantation have a higher overall risk of developing de novo malignancies. Most de novo malignancies are upper gastrointestinal malignancies. The prognosis is usually poor. Considering the surgical risk of upper gastrointestinal cancer among those with liver cirrhosis, a radical gastrectomy with D1 or D2 lymph node dissection can be undertaken in Child class A patients. D1 lymph node dissection can be performed in Child class B patients. Endoscopic submucosal dissection for gastric cancer or esophageal cancer can be undertaken safely in selected cirrhotic patients. In Child class C patients, a radical gastrectomy is potentially fatal. Pancreatic radical surgery should be avoided in those with liver cirrhosis with Child class B or a MELD score over 15. The current review focuses on the recent reports on some factors in liver cirrhosis that contribute to the development of upper gastrointestinal cancer. Quitting alcohol drinking and tobacco use is important. How to decrease the risk of the development of gastrointestinal cancer in those with liver cirrhosis remains a challenging problem.

Effect of anticoagulant/antiplatelet therapy on the development and progression of diabetic retinopathy.

BACKGROUND: We investigated whether antiplatelet/anticoagulant (APAC) therapy can protect patients with type 2 diabetes mellitus (T2DM) from the development or progression of diabetic retinopathy (DR). METHODS: This is a retrospective cohort study using Longitudinal Health Insurance Database in Taiwan. A total of 73,964 type 2 diabetic patients older than 20 years old were included. Hazard ration (HR) of non-proliferative DR (NPDR), proliferative DR (PDR), and diabetic macular edema (DME) were analyzed with APAC usage as a time-dependent covariate. Age, sex, comorbidities, and medicines were further adjusted in a multi-variable model. Contributions of respective APAC was investigated with sensitivity analysis. RESULTS: Compared with nonusers, APAC users had a lower cumulative incidence of NPDR (P < 0.001), overall incidence of NPDR (10.7 per 1000 person-years), and risk of developing NPDR (adjusted HR = 0.78, 95% CI = 0.73-0.83). However, no significant differences were observed between APAC users and nonusers in the risks of PDR or DME. Hypertension, diabetic nephropathy and diabetic neuropathy were risk factors for NDPR development, while heart disease, cardiovascular disease, peripheral arterial occlusive disease, and statin usage were covariates decreasing NPDR development. Aspirin and Dipyridamole showed significant protection against NPDR development. Clopidogrel, Ticlopidine, and warfarin showed enhanced protection in combination with aspirin usage. CONCLUSIONS: APAC medications have a protective effect against NPDR development. Diabetic patients benefit from single use of aspirin or dipyridamole on prevention of NPDR.

Sonic Hedgehog signaling pathway as a potential target to inhibit the progression of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-associated mortality worldwide. Hepatocarcinogenesis involves numerous interlinked factors and processes, including the Sonic hedgehog (Shh) signaling pathway, which participates in the carcinogenesis, progression, invasiveness, recurrence and cancer stem cell maintenance of HCC. The Shh signaling pathway is activated by ligands that bind to their receptor protein, Protein patched homolog (Ptch). The process of Shh ligand binding to Ptch weakens the inhibition of smoothened homolog (SMO) and activates signal transduction via glioma-associated oncogene homolog (Gli) transcription factors. The overexpression of Shh pathway molecules, including Shh, Ptch-1, Gli and SMO has been indicated in patients with HCC. It has also been suggested that the Shh signaling pathway exhibits cross-talk between numerous other signaling pathways. The inactivation of the Shh signaling pathway reduces HCC growth, increases radio-sensitivity and increases the beneficial effect of chemotherapy in HCC treatment. Therefore, inhibition of the Shh pathway may be an effective target therapy that can be used in the treatment of HCC.

Development of diabetic retinopathy after cataract surgery.

This study explored whether cataract surgery precipitates diabetic retinopathy (DR) development in diabetic patients without previous DR. Patients with the diagnosis of type II diabetes but without DR were selected from the Longitudinal Health Insurance Database 2000. Patients who received cataract surgery between January 1, 2000, and December 31, 2010, were included in the case group, and the control group was matched to the case group by age, sex, and index year. The postoperative incidence rates of nonproliferative diabetic retinopathy (NPDR), proliferative diabetic retinopathy (PDR), and diabetic macular edema (DME) were the main outcomes studied and were adjusted by age, sex, comorbidities, and statin, fibrate, angiotensin-converting-enzyme inhibitor (ACEI), oral hypoglycemic agents (OHA), and insulin use. In our cohort, patients who had dyslipidemia and used statins were more likely to undergo cataract surgery. Among diabetic patients without previous DR, patients receiving cataract surgery had a higher risk of NDPR development (adjusted hazard ratio = 1.48, 95% confidence interval = 1.15-1.91). No statistical difference was observed in PDR or DME development between operative and nonoperative groups. In additional stratified analyses, female sex, older age, comorbidities, surgery within 5 years, statin, ACEI, OHA, and insulin use increased the risk of NPDR development. In an adjusted Cox regression model, cataract surgery, OHA and insulin use were found to be risk factors for NPDR development. Cataract surgery with complications increased post-operative risks for NPDR were even higher, and the significant influence from cataract surgery persisted 5 years after surgery.

Glioma-Associated Oncogene Homolog Inhibitors Have the Potential of Suppressing Cancer Stem Cells of Breast Cancer.

Overexpression of Sonic Hedgehog signaling (Shh) pathway molecules is associated with invasiveness and recurrence in breast carcinoma. Therefore, inhibition of the Shh pathway downstream molecule Glioma-associated Oncogene Homolog (Gli) was investigated for its ability to reduce progression and invasiveness of patient-derived breast cancer cells and cell lines. Human primary breast cancer T2 cells with high expression of Shh signaling pathway molecules were compared with breast cancer line MDA-MB-231 cells. The therapeutic effects of Gli inhibitors were examined in terms of the cell proliferation, apoptosis, cancer stem cells, cell migration and gene expression. Blockade of the Shh signaling pathway could reduce cell proliferation and migration only in MDA-MB-231 cells. Hh pathway inhibitor-1 (HPI-1) increased the percentages of late apoptotic cells in MDA-MB-231 cells and early apoptotic cells in T2 cells. It reduced Bcl2 expression for cell proliferation and increased Bim expression for apoptosis. In addition, Gli inhibitor HPI-1 decreased significantly the percentages of cancer stem cells in T2 cells. HPI-1 worked more effectively than GANT-58 against breast carcinoma cells. In conclusion, HPI-1 could inhibit cell proliferation, reduce cell invasion and decrease cancer stem cell population in breast cancer cells. To target Gli-1 could be a potential strategy to suppress breast cancer stem cells.

Diabetic Retinopathy in Patients with Dyslipidemia: Development and Progression.

PURPOSE: To investigate the association of dyslipidemia with the development of diabetic retinopathy (DR). DESIGN: A prospective cohort from the Longitudinal Health Insurance Database in Taiwan. PARTICIPANTS: Patients with diabetes mellitus (DM) aged ≥18 years from this cohort. METHODS: A logistic regression model considering age, sex, and adapted Diabetes Complication Severity Index (aDCSI) including diabetic retinopathy, diabetic neuropathy, diabetic nephropathy cardiovascular disease, cerebrovascular disease, peripheral arteriolar disease, and metabolic disease. We estimated the propensity score for disease assignment probability for each included patient with DM and dyslipidemia. For each patient, a comparison patient without dyslipidemia was matched with a propensity score using a greedy algorithm. The standardized mean differences method was used to measure the difference in means or proportions divided by the pooled standard deviation of a variable. We calculated the incidence densities of nonproliferative DR (NPDR), diabetic macular edema (DME), and proliferative DR (PDR) as total events divided by the sum of follow-up duration, and the incidence curves were measured using the Kaplan-Meier method. The log-rank test was applied to test the differences of incidence curves. MAIN OUTCOME MEASURES: Hazard ratios (HRs) for DR. RESULTS: Our results demonstrated that the cumulative incidence of NPDR, DME, and PDR significantly increased in patients with DM and dyslipidemia compared with those without before adjustment for covariates (P < 0.001). Adjusted HRs for NPDR, DME, and PDR in patients with dyslipidemia were 1.77 (95% confidence interval [CI] = 1.63-1.92), 2.34 (95% CI = 1.24-4.41), and 1.07 (95% CI = 0.91-1.27), respectively. The risks of NPDR, DME, and PDR increased in patients who had underlying complications according to the aDCSI. Only statin use had a protective effect against the development of NPDR (HR = 0.83; 95% CI = 0.76-0.90), but it had no effect on DME and PDR. The protective effect was not significantly different between patients with and without dyslipidemia. CONCLUSION: Dyslipidemia is involved in the development of DR at an earlier stage, but the role of lipid-modulating agents in DR requires additional study.

Tropism of liver epithelial cells toward hepatocellular carcinoma in vitro and in vivo with altering gene expression of cancer stem cells.

BACKGROUND: Rat liver epithelial (RLE) cells could inhibit the proliferation and invasiveness of hepatoma cells in vitro. This study is to understand the tropism and the effect of RLE cells on mouse hepatoma cells both in vitro and in vivo. METHODS: RLE cells were isolated from new-born rats and characterized their stem cell markers. Co-culture and HCC mouse model was established to detect therapeutic effect of RLE cells. RESULTS: RLE cells (including Thy-1+ RLE cells, Thy-1- RLE cells, RLE cells) displayed a selective tropism toward ML-1 hepatoma cells both in vitro and in vivo. They altered the gene expression of some cancer stem cell markers in the liver tumor. CONCLUSION: Liver epithelial cells have a selective tropism toward HCC in vitro and in vivo. They could alter the gene expression of cancer stem cells.

Role of C-X-C chemokine ligand 12/C-X-C chemokine receptor 4 in the progression of hepatocellular carcinoma.

The efficacy of the current non-surgical treatments for advanced hepatocellular carcinoma (HCC) remains limited and novel treatments are required to improve patient outcomes. The majority of HCCs develop from chronically damaged tissue that contains a high degree of inflammation and fibrosis, which promotes tumor progression and resistance to therapy. Understanding the interaction between stromal components and cancer cells (and the signaling pathways involved in this interaction) could aid the identification of novel therapeutic targets. Numerous studies have demonstrated a marked association between high C-X-C chemokine receptor 4 (CXCR4) expression and the invasiveness, progression and metastasis of HCC. The present review will investigate the different roles of CXCR4 in the progression of HCC and discuss possible future treatments. Through the C-X-C chemokine ligand 12 (CXCL12)/CXCR4 signaling pathway, ephrin A1 activation enhances the migration of endothelial progenitor cells to HCC to enable the neovascularization of tumors. There is an association between nuclear CXCR4 expression and the lymph node metastasis of HCC to distant areas. CXCR4 enhances cell migration in vitro and cell homing in vivo. CXCR4 levels are concentrated at the border of a tumor and in perivascular areas, inducing invasive behavior. The binding of CXCL12 to CXCR4 activates intracellular signaling pathways and induces crosstalk with transforming growth factor-ß signaling, which enhances the migration of cancer cells. The CXCL12/CXCR4 axis also activates expression of matrix metalloproteinase 10, which further stimulates migration. CXCR4 is likely to crosstalk with the sonic hedgehog signaling pathway, contributing to tumor invasiveness and supporting the cancer stem-cell population; as a result, CXCR4 can be regarded as a cancer stem-cell marker. CXCR4 influences interstitial fluid flow-induced invasion. CXCR4 expression and HCC cell migration are promoted by α-fetoprotein, which activates AKT/mechanistic target of rapamycin signaling. CXCR4 also has the potential to affect sorafenib treatment for HCC. Targeting the CXCL12/CXCR4 signaling pathway may, therefore, be a promising strategy in HCC treatment.

Diabetic Retinopathy in Patients with Diabetic Nephropathy: Development and Progression.

The purpose of current study aims to investigate the development and progression of diabetic retinopathy (DR) in patients with diabetic nephropathy (DN) in a nationwide population-based cohort in Taiwan. Newly diagnosed DN patients and age- and sex-matched controls were identified from the Taiwanese Longitudinal Health Insurance Database from 2000 to 2010. We studied the effects of age, sex, hypertension, dyslipidemia, diabetic polyneuropathy (DPN), and medications on the development of nonproliferative DR (NPDR), proliferative DR (PDR), and diabetic macular edema (DME) in patients with DN. Cox proportional hazard regression analyses were used to estimate the adjusted hazard ratios (HRs) of the development of DR. Our results show that the adjusted HRs of NPDR and PDR were 5.01 (95% confidence interval (CI) = 4.68-5.37) and 9.7 (95% CI = 8.15-11.5), respectively, in patients with DN as compared with patients in the non-DN cohort. At 5-year follow-up, patients with DN showed an increased HR of NPDR progression to PDR (HR = 2.26, 95% CI = 1.68-3.03), and the major comorbidities were hypertension (HR = 1.23, 95% CI = 1.10-1.38 with NPDR; HR = 1.33, 95% CI = 1.02-1.72 with PDR) and DPN (HR = 2.03, 95% CI = 1.72-2.41 in NPDR; HR = 2.95, 95% CI = 2.16-4.03 in PDR). Dyslipidemia increased the HR of developing NPDR but not PDR or DME. Moreover, DN did not significantly affect DME development (HR = 1.47, 95% CI = 0.87-2.48) or progression (HR = 0.37, 95% CI = 0.11-1.20). We concluded that DN was an independent risk factor for DR development and progression; however, DN did not markedly affect DME development in this study, and the potential association between these disorders requires further investigation.

Liver epithelial cells inhibit proliferation and invasiveness of hepatoma cells.

Hepatocellular carcinoma (HCC) is a worldwide malignancy with poor prognosis. Liver progenitors or stem cells could be a potential therapy for HCC treatment since they migrate toward tumors. Rat liver epithelial (RLE) cells have both progenitor and stem cell-like properties. Therefore, our study elucidated the therapeutic effect of RLE cells in rat hepatoma cells. RLE cells were isolated from 10-day old rats and characterized for stem cell marker expression. RLE cells and rat hepatoma cells (H4-IIE-C3 cells) were co-cultured and divided into four groups with different ratios of RLE and hepatoma cells. Group A had only rat hepatoma cells as a control group. The ratios of rat hepatoma and RLE cells in group B, C and D were 5:1, 1:1 and 1:5, respectively. Effective inhibition of cell proliferation and migration was found in group D when compared to group A. There was a significant decrease in Bcl2 expression and increase in late apoptosis of rat hepatoma cells when adding more RLE cells. RLE cells reduced cell proliferation and migration of rat hepatoma cells. These results suggested that RLE cells could be used as a potential cell therapy.

Sonic hedgehog pathway inhibitor mitigates mouse hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a leading cause of death in Asian countries. Sonic hedgehog (Shh) pathway plays a role in hepatocarcinogenesis. We investigated the treatment effect of mouse HCC with Shh inhibitor GDC-0449. METHODS: Mouse hepatoma ML-1 cells were implanted in B6 mice. Fifteen days later, GDC-0449 (vismodegib), antagonist of smoothened, was used to treat HCC-bearing mice. The tumor size and liver histopathological features were analyzed, as well as gene expression in Shh pathways. RESULTS: GDC-0449 treatment effectively reduced tumor size and cell infiltration of the HCC in mice. Gene expression of Shh pathway molecules was altered, including upregulated Shh expression and downregulated smoothened expression in tumor fractions after GDC-0449 treatment. CONCLUSION: GDC-0449 could effectively mitigate HCC growth in vivo.

Heterogeneity of hepatocellular carcinoma contributes to cancer progression.

Hepatocellular carcinoma (HCC) is a highly heterogeneous disease displaying differences in angiogenesis, extracellular matrix proteins, the immune microenvironment and tumor cell populations. Additionally, genetic variations and epigenetic changes of HCC cells could lead to aberrant signaling pathways, induce cancer stem cells and enhance tumor progression. Thus, the heterogeneity in HCC contributes to disease progression and a better understanding of its heterogeneity will greatly aid in the development of strategies for the HCC treatment.

Fumagillin treatment of hepatocellular carcinoma in rats: an in vivo study of antiangiogenesis.

AIM: To investigate the effect and possible mechanisms of antiangiogenesis therapy for HCC in rats. METHODS: Adult male LEW/SsN rats were divided into 3 groups, 25 animals each. Group A was the control group. Groups B and C were given diethylnitrosamine, 5 mg/kg/d. In addition, group C rats received an intraperitoneal injection of fumagillin, 30 mg/(kg x d). Five animals in each group were killed at 6th, 12th, 18th, 20th and 24th wk to evaluate the development of HCC and metastasis. Weight of the rats, liver tumors, and number of organs involved by HCC were measured at each stage. We compared methionine aminopeptidase-2 (MetAP-2) mRNA, Bcl-2 mRNA, telomerase mRNA, and telomerase activity at 24th wk in the liver tissue of group A rats and tumor tissue of HCC from group B and C rats. RESULTS: No HCC developed in group A, but tumors were present in group B and C rats by the 18th wk. At wk 20 and 24, the median liver weight in group B was 0.64 g (range: 0.58-0.70 g) and 0.79 g (range: 0.70-0.90 g) (P = 0.04), and that in group C was 0.37 g (range: 0.35-0.42 g) and 0.39 g (range: 0.35-0.47 g) (P = 0.67). The liver weight in group C rats was significantly lower than that in group B rats (P = 0.009). At the same time, the median metastasis score (number of organ systems involved) was 3 (range2-3) in group B, and 1 (range 1-2) in group C, a significant difference between the groups (P = 0.007, 0.004). The levels of MetAP-2 mRNA were significantly higher in groups B and C than in group A (P = 0.025), and significantly higher in group C than in group B (P = 0.047). The level of Bcl-2 mRNA was significantly higher in group B than in group A (P = 0.024), but lower in group C than in group B, although not significantly (P = 0.072). Telomerase mRNA was significantly higher in group B than in group A (P = 0.025), but significantly lower in group C than in group B (P = 0.016). The same inter-group relationship was also true for telomerase activity (P = 0.025 and 0.046). CONCLUSION: Fumagillin effectively inhibits both liver tumor growth and metastasis in rats in vivo. A possible mechanism is fumagillin-induced inhibition of MetAP-2, which plays an essential role in endothelial cell proliferation. Inhibition of MetAP-2 also results in inhibition of Bcl-2 and telomerase activity.