Many traditional pharmacopeias include Aristolochia and related plants, which contain nephrotoxins and mutagens in the form of aristolochic acids and similar compounds (collectively, AA). AA is implicated in multiple cancer types, sometimes with very high mutational burdens, especially in upper tract urothelial cancers (UTUCs). AA-associated kidney failure and UTUCs are prevalent in Taiwan, but AA’s role in hepatocellular carcinomas (HCCs) there remains unexplored. Therefore, we sequenced the whole exomes of 98 HCCs from two hospitals in Taiwan and found that 78% showed the distinctive mutational signature of AA exposure, accounting for most of the nonsilent mutations in known cancer driver genes. We then searched for the AA signature in 1400 HCCs from diverse geographic regions. Consistent with exposure through known herbal medicines, 47% of Chinese HCCs showed the signature, albeit with lower mutation loads than in Taiwan. In addition, 29% of HCCs from Southeast Asia showed the signature. The AA signature was also detected in 13 and 2.7% of HCCs from Korea and Japan as well as in 4.8 and 1.7% of HCCs from North America and Europe, respectively, excluding one U.S. hospital where 22% of 87 “Asian” HCCs had the signature. Thus, AA exposure is geographically widespread. Asia, especially Taiwan, appears to be much more extensively affected, which is consistent with other evidence of patterns of AA exposure. We propose that additional measures aimed at primary prevention through avoidance of AA exposure and investigation of possible approaches to secondary prevention are warranted.

High invasiveness is a hallmark of human hepatocellular carcinoma (HCC). Large tumors predict invasion and metastasis. Epithelial‐mesenchymal transition (EMT) is crucial for cancer invasion and metastasis. However, the mechanisms whereby large tumors tend to undergo EMT remain unclear. We conducted a subgenome‐wide screen and identified KLHL23 as an HCC invasion suppressor by inhibiting EMT. KLHL23 binds to actin and suppresses actin polymerization. KLHL23 silencing induced filopodium and lamellipodium formation. Moreover, EMT was suppressed by KLHL23 through its action on actin dynamics. Traditionally, actin cytoskeleton remodeling is downstream of EMT reprogramming. It is therefore intriguing to ask why and how KLHL23 inversely regulates EMT. Activation of actin cytoskeleton remodeling by either KLHL23 silencing or treatment with actin cytoskeleton modulators augmented cellular hypoxic responses in a cell‐density–dependent manner, resulting in hypoxia‐inducible factor (HIF) and Notch signals and subsequent EMT. Environmental hypoxia did not induce EMT unless actin cytoskeleton remodeling was simultaneously activated and only when cells were at high density. The resulting EMT was reversed by either adenosine 5′‐triphosphate supplementation or actin polymerization inhibitors. Down‐regulation of KLHL23 was associated with invasion, metastasis, and poor prognosis of HCC and pancreatic cancer. Correlations of tumor size with EMT and inverse association of expression of KLHL23 with HIF/Notch signals were further validated in patient‐derived xenograft HCCs in mice. Conclusion: Simultaneously activation of actin cytoskeleton remodeling by intrinsic (such as KLHL23 down‐regulation) or microenvironment cues is crucial for cell‐density–dependent and hypoxia‐mediated EMT, providing a mechanistic link between large tumor size and invasion/metastasis. Our findings provide a means of developing the prevention and treatment strategies for tumor invasion and metastasis. (Hepatology 2018;67:2226‐2243).

A novel electrochemical sensor based on graphene oxide (GO) modified glassy carbon electrode (GCE) has been successfully developed for the determination of anti-cancer drug flutamide for the first time. The morphology and structure of the prepared GO were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), UV-visible spectroscopy, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), field emission-scanning electron microscopy (FE-SEM), and transmission electron microscopy (TEM). The electrochemical properties of the GO were studied by electrochemical impedance spectroscopy (EIS). GO modified GCE was fabricated and utilized to study the electrochemical performance of flutamide by cyclic voltammetry (CV) and linear sweep voltammetry (LSV). As an electrochemical sensor, GO modified GCE exhibited strong electrocatalytic activity towards the reduction of flutamide. Interestingly, the electrochemical sensor displayed an excellent current response for the detection of flutamide with wide linear response range, excellent limit of detection and good sensitivity of 0.009 to 1.9 μM, 6 nM and 29.55 μA μM⁻¹ cm⁻², respectively. The proposed sensor has good repeatability, reproducibility, stability and selectivity even in the presence of biologically co-interfering substances. GO modified GCE sensor was successfully used for the detection of flutamide (spiked) in various blood serum samples.

Introduction: serum alpha-fetoprotein (AFP) was routinely employed as a tumor marker for screening, diagnosis, and treatment follow-up of hepatocellular carcinoma (HCC). However, a substantial proportion of HCC patients had normal AFP level even at an advanced disease status. Few studies to date had tried to explore the nature and behavior of this normal AFP HCC (N-HCC). The purpose of this study was to investigate the clinicopathological characteristics and survival outcome of N-HCC after operation. In addition, potential tumor markers for N-HCC were also sought in an attempt to augment diagnostic ability. Methods: between 2005 and 2015, patients with hepatocellular carcinoma who were treated with hepatectomy in Chang Gung Memorial Hospital Linkou branch were divided into two groups according to their preoperative serum AFP level (<15 ng/mL: NHCC; ≥15 ng/mL: abnormal AFP HCC (A-HCC)). Patient demographic data and clinicopathological variables were collected. Kaplan–Meier and Cox regression multivariate analyses were performed to identify significant risk factors for disease-free survival (DFS) and overall survival (OS) for N-HCC. ELISA and immunohistochemical (IHC) studies were employed to determine the diagnostic accuracy of various tumor markers. Results: a total of 1616 patients (78% male) who underwent liver resection for HCC were included in this study. Of them, 761 patients (47.1%) were N-HCC. N-HCC patients were significantly older with more comorbidities and less hepatitis virus infections. Furthermore, N-HCC had fewer early recurrences (49.6% vs. 60.8%, p < 0.001) and better DFS (44.6 months vs. 23.6 months, p < 0.001) and OS (94.5 months vs. 81.7 months, p < 0.001). Both ELISA and IHC studies demonstrated that glypican-3 (GPC3) would be a promising diagnostic tumor marker for N-HCC. Conclusion: N-HCC patients were significantly older and had less hepatitis virus infections or cirrhosis. Their tumors tended to be smaller, less vascular invaded, and well-differentiated. The carcinogenesis of N-HCC may thus not be identical to that of typical HCC. GPC3 would be a promising tumor marker for diagnosing N-HCC. Further study is warranted to validate our findings.

Acute severe pancreatitis caused high mortality, and several scoring systems for predicting mortality are available. We evaluated the effectiveness of serial measurement of several scoring systems in patients with acute severe pancreatitis. We retrospectively obtained serial measurements of Ranson, Acute Physiology and Chronic Health Assessment (APACHE) II, and Sequential Organ Failure Assessment (SOFA) scores of 159 patients with acute severe pancreatitis. The overall mortality rate was 20%, and early mortality (in the first 2 weeks) occurred in 10 (7.4%) patients, while late mortality occurred in 17 (12.6%). All scoring systems were reliable for predicting overall and intensive care unit mortality, while the SOFA score on day 7 presented the largest area under the receiver operator characteristic (ROC) curve (0.858, SE 0.055). Changes in scores over time were evaluated for predicting the progression of organ failure, and the change in SOFA score on hospital day 7 or no interval change in SOFA score was associated with higher mortality rates. APACHE II and SOFA scores are both sensitive for predicting mortality in acute pancreatitis. The serial SOFA scores showed reliable for predicting mortality. Hospital day 7 is a reasonable time for SOFA score reassessment to predict late mortality in acute severe pancreatitis.

Membrane protrusion and adhesion to the extracellular matrix, which involves the extension of actin filaments and formation of adhesion complexes, are the fundamental processes for cell migration, tumor invasion, and metastasis. How cancer cells efficiently coordinate these processes remains unclear. Here, we showed that membrane-targeted chloride intracellular channel 1 (CLIC1) spatiotemporally regulates the formation of cell-matrix adhesions and membrane protrusions through the recruitment of PIP5Ks to the plasma membrane. Comparative proteomics identified CLIC1 upregulated in human hepatocellular carcinoma (HCC) and associated with tumor invasiveness, metastasis, and poor prognosis. In response to migration-related stimuli, CLIC1 recruited PIP5K1A and PIP5K1C from the cytoplasm to the leading edge of the plasma membrane, where PIP5Ks generate a phosphatidylinositol 4,5-bisphosphate–rich (PIP₂-rich) microdomain to induce the formation of integrin-mediated cell-matrix adhesions and the signaling for cytoskeleon extension. CLIC1 silencing inhibited the attachment of tumor cells to culture plates and the adherence and extravasation in the lung alveoli, resulting in suppressed lung metastasis in mice. This study reveals what we believe is an unrecognized mechanism that spatiotemporally coordinates the formation of both lamellipodium/invadopodia and nascent cell-matrix adhesions for directional migration and tumor invasion/metastasis. The unique traits of upregulation and membrane targeting of CLIC1 in cancer cells make it an excellent therapeutic target for tumor metastasis.