Influence of surgical margin width on survival rate after resection of intrahepatic cholangiocarcinoma: a systematic review and meta-analysis.

OBJECTIVES: Hepatectomy is the best treatment for patients with intrahepatic cholangiocarcinoma (ICC) at present, but there has been controversy about the width of surgical margins. In this study, we systematically investigated the effects of different surgical margin widths on the prognosis of patients with ICC undergoing hepatectomy. DESIGN: Systematic review and meta-analysis. DATA SOURCES: PubMed, Embase and Web of Science databases were systematically searched from inception to June 2022. ELIGIBILITY CRITERIA: Cohort studies reported in English with patients who underwent negative marginal (R0) resection were included. The effects of surgical margin width on overall survival (OS), disease-free survival (DFS) and recurrence-free survival (RFS) in patients with ICC were assessed. DATA EXTRACTION AND SYNTHESIS: Two investigators independently conducted literature screening and data extraction. Risk of bias was assessed using funnel plots and quality was assessed by the Newcastle-Ottawa Scale. Forest plots of HRs and their 95% CIs for outcome indicators were plotted. Heterogeneity was assessed and determined quantitatively using I2, and the stability of the study results was evaluated using sensitivity analysis. Analyses were performed using Stata software. RESULTS: Nine studies were included. With the wide margin group (≥10 mm) as the control, pooled HR of OS in the narrow margin group (<10 mm) was 1.54 (95% CI 1.34 to 1.77). HRs of OS in three subgroups where the margin was less than 5 mm ranged from 5 mm to 9 mm, or was less than 10 mm in length were 1.88 (1.45 to 2.42), 1.33 (1.03 to 1.72) and 1.49 (1.20 to 1.84), respectively. Pooled HR of DFS in the narrow margin group (<10 mm) was 1.51 (1.14 to 2.00). Pooled HR of RFS in the narrow margin group (<10 mm) was 1.35 (1.19 to 1.54). HRs of RFS in three subgroups where the margin was less than 5 mm ranged from 5 mm to 9 mm, or was less than 10 mm in length were 1.38 (1.07 to 1.78), 1.39 (1.11 to 1.74) and 1.30 (1.06 to 1.60), respectively. Neither lymph node lesions (HR 1.44, 95% CI 1.22 to 1.70) nor lymph node invasion (2.14, 1.39 to 3.28) was favourable for postoperative OS in patients with ICC. Lymph node metastasis (1.31, 1.09 to 1.57) was unfavourable for RFS in patients with ICC. CONCLUSION: Patients with ICC who underwent curative hepatectomy with a negative margin ≥10 mm may have a long-term survival advantage, but lymph node dissection also needs to be considered. In addition, tumour-related pathological features need to be explored to see if they affect the surgical outcome of R0 margins.

Torin2 overcomes sorafenib resistance via suppressing mTORC2-AKT-BAD pathway in hepatocellular carcinoma cells.

BACKGROUND: Sorafenib is an oral multi-kinase inhibitor that was approved by the US Food and Drug Administration for the treatment of patients with advanced hepatocellular carcinoma (HCC). However, resistance to sorafenib is an urgent problem to be resolved to improve the therapeutic efficacy of sorafenib. As the activation of AKT/mTOR played a pivotal role in sorafenib resistance, we evaluated the effect of a dual mTOR complex 1/2 inhibitor Torin2 on overcoming the sorafenib resistance in HCC cells. METHODS: The sorafenib-resistant Huh7 and Hep3B cell lines were established from their parental cell lines. The synergistic effect of sorafenib and Torin2 on these cells was measured by cell viability assay and quantified using the Chou-Talalay method. Apoptosis induced by the combination of sorafenib and Torin2 and the alteration in the specific signaling pathways of interest were detected by Western blotting. RESULTS: Sorafenib treatment inversely inhibited AKT in parental but activated AKT in sorafenib-resistant Huh7 and Hep3B HCC cells, which underscores the significance of AKT activation. Torin2 and sorafenib synergistically suppressed the viability of sorafenib-resistant cells via apoptosis induction. Torin2 successfully suppressed the sorafenib-activated mTORC2-AKT axis, leading to the dephosphorylation of Ser136 in BAD protein, and increased the expression of total BAD, which contributed to the apoptosis in sorafenib-resistant HCC cells. CONCLUSIONS: In this study, Torin2 and sorafenib showed synergistic cytostatic capacity in sorafenib-resistant HCC cells, via the suppression of mTORC2-AKT-BAD pathway. Our results suggest a novel strategy of drug combination for overcoming sorafenib resistance in HCC.

The clinical significance of lipopolysaccharide binding protein in hepatocellular carcinoma.

Lipopolysaccharide binding protein (LBP) has been reported to be associated with prognosis in colorectal carcinoma and renal cell carcinoma; however, the clinical significance of LBP in human primary hepatocellular carcinoma (HCC) is inconclusive. We aimed to investigate the clinical significance and prognostic value of LBP in human primary HCC. In the present study, 346 patients with HCC who underwent curative resection were retrospectively analyzed. LBP protein expression was evaluated using western blot analysis and immunohistochemistry. LBP scores collected from immunohistochemical analysis were obtained by multiplying staining intensity and the percentage of positive cells. An outcome-based best cutoff-point was calculated by X-tile software. Moreover, Kaplan-Meier curves and Cox regressions were used for prognosis evaluation. LBP was frequently overexpressed in HCC compared with that in peritumor tissues (five pairs by western blot analysis, P=0.0533; 77 pairs by immunohistochemistry, P=0.0171), and LBP expression was positively associated with tumor-node-metastasis stage and tumor differentiation. Patients who had high LBP expression had decreased overall survival and time to recurrence compared with patients with low LBP expression. Furthermore, patients who were both serum α-fetoprotein positive and had high LBP expression had poor prognoses. Univariate and multivariate Cox analyses indicated that this combination was an independent prognostic factor [overall survival: Hazard ratio (HR), 1.458; 95% confidence interval (CI), 1.158-1.837; P=0.001; time to recurrence: HR,1.382; 95% Cl, 1.124-1.700; P=0.002]. In conclusion, LBP is highly expressed in HCC, and high LBP expression combined with serum α-fetoprotein may predict poor outcomes in patients with HCC following curative resection.

Glycochenodeoxycholate promotes hepatocellular carcinoma invasion and migration by AMPK/mTOR dependent autophagy activation.

Metastasis and recurrence severely impact the treatment effect of hepatocellular carcinoma (HCC). HCC complicated with cholestasis is more prone to recurrence and metastasis. Previous studies have implicated pathogenesis of HCC by bile acid; however, the underlying mechanism is unknown yet. Glycochenodeoxycholate (GCDC) is one of most important component of bile acid (BA). In the present study, the role of GCDC in HCC cells invasion was detected by in vitro and in vivo assays. GCDC was found to significantly enhance the invasive potential of HCC cells; Further studies showed that GCDC could induce autophagy activation and higher invasive capability in HCC cells. Interestingly, inhibition of autophagy by chloroquine (CQ) reversed this phenomenon. Subsequently, the correlation between TBA expression level and clinicopathological characteristics was analyzed in HCC patients. Clinically, high TBA level in HCC tissue was found to be associated with more invasive and poor survival in HCC patients. Mechanistic study showed that bile acid induced autophagy by targeting the AMPK/mTOR pathway in HCC cells. Therefore, our results suggest that bile acid may promote HCC invasion via activation of autophagy and the level of bile acid may serve as a potential useful indicator for prognosis of HCC patients.

Hepatocyte nuclear factor-1beta enhances the stemness of hepatocellular carcinoma cells through activation of the Notch pathway.

Hepatocyte nuclear factor-1beta plays an important role in the development and progression of liver cancer. In recent years, the expression of HNF-1ß has been reported to be associated with risk for a variety of cancers. The purpose of this study is to investigate whether the expression of HNF-1ß promotes the malignancy of HCC and its mechanism. We retrospectively investigated the expression of HNF-1ß in 90 patients with hepatocellular carcinoma and found that the high expression of HNF-1ß indicated poor prognosis. We overexpressed HNF-1ß in liver cancer cell lines and found the expression of liver progenitor cell markers and stemness were upregulated. The invasion ability and epithelial-mesenchymal transition (EMT)-associated genes were also significantly higher in liver cancer cells overexpressing HNF-1ß than in the control group. A mechanistic study suggested the activation of the Notch signalling pathway probably plays a key role downstream of HNF-1ß. More importantly, HNF-1ß promoted tumourigenesis of HCC cells in vivo. In conclusion, high expression of HNF-1ß not only promoted the de-differentiation of HCC cells into liver cancer stem cells through activating the Notch pathway but also enhanced the invasive potential of HCC cells and EMT occurrence, which would contribute to the enhancement of cell migration and invasion.

Overexpression of SIRT1 promotes metastasis through epithelial-mesenchymal transition in hepatocellular carcinoma.

BACKGROUND: SIRT1 is a member of the mammalian sirtuin family with the ability to deacetylate histone and nonhistone proteins. The correlation between SIRT1 expression and tumor metastasis in several types of cancer has aroused widespread concern. This study investigated SIRT1 expression and its prognostic value in hepatocellular carcinoma (HCC). The function of SIRT1 in hepatocarcinogenesis was further investigated in cell culture and mouse models. METHODS: Western blotting and immunohistochemistry were used to explore SIRT1 expression in HCC cell lines and primary HCC clinical specimens. The functions of SIRT1 in the migration and invasion in the HCC cell line were analyzed by infecting cells with adenovirus containing full-length SIRT1 or sh-RNA. The effect of SIRT1 on tumorigenicity in nude mice was also investigated. RESULTS: SIRT1 expression was significantly overexpressed in the tumor tissues and HCC cell lines. SIRT1 significantly promoted the ability of migration and invasion in HCC cells. In addition, experiments with a mouse model revealed that SIRT1 overexpression enhanced HCC tumor metastasis in vivo. Furthermore, we demonstrated that SIRT1 significantly enhanced the invasive and metastatic potential by inducing epithelial-mesenchymal transition in HCC cells. A clinicopathological analysis showed that SIRT1 expression was significantly correlated with tumor size, tumor number, and TNM staging. Kaplan-Meier survival curves revealed that positive SIRT1 expression was associated with poor prognosis in patients with HCC. CONCLUSIONS: Our data suggest that SIRT1 may play an important role in HCC progression and could be a potential molecular therapy target for HCC.

Magnolia polyphenols attenuate oxidative and inflammatory responses in neurons and microglial cells.

BACKGROUND: The bark of magnolia has been used in Oriental medicine to treat a variety of remedies, including some neurological disorders. Magnolol (Mag) and honokiol (Hon) are isomers of polyphenolic compounds from the bark of Magnolia officinalis, and have been identified as major active components exhibiting anti-oxidative, anti-inflammatory, and neuroprotective effects. In this study, we investigate the ability of these isomers to suppress oxidative stress in neurons stimulated by the ionotropic glutamate receptor agonist N-methyl-D-aspartate (NMDA) and oxidative and inflammatory responses in microglial cells activated by interferon-γ (IFNγ) and lipopolysaccharide (LPS). We also attempt to elucidate the mechanism and signaling pathways involved in cytokine-induced production of reactive oxygen species (ROS) in microglial cells. METHODS: Dihydroethidium (DHE) was used to assay superoxide production in neurons, while CM-H2DCF-DA was used to test for ROS production in murine (BV-2) and rat (HAPI) immortalized microglial cells. NADPH oxidase inhibitors (for example, diphenyleneiodonium (DPI), AEBSF, and apocynin) and immunocytochemistry targeting p47phox and gp91phox were used to assess the involvement of NADPH oxidase. Western blotting was used to assess iNOS and ERK1/2 expression, and the Griess reaction protocol was employed to determine nitric oxide (NO) concentration. RESULTS: Exposure of Hon and Mag (1-10 µM) to neurons for 24 h did not alter neuronal viability, but both compounds (10 µM) inhibited NMDA-stimulated superoxide production, a pathway known to involve NADPH oxidase. In microglial cells, Hon and Mag inhibited IFNγ±LPS-induced iNOS expression, NO, and ROS production. Studies with inhibitors and immunocytochemical assay further demonstrated the important role of IFNγ activating the NADPH oxidase through the p-ERK-dependent pathway. Hon and, to a lesser extent, Mag inhibited IFNγ-induced p-ERK1/2 and its downstream pathway for ROS and NO production. CONCLUSION: This study highlights the important role of NADPH oxidase in mediating oxidative stress in neurons and microglial cells and has unveiled the role of IFNγ in stimulating the MAPK/ERK1/2 signaling pathway for activation of NADPH oxidase in microglial cells. Hon and Mag offer anti-oxidative or anti-inflammatory effects, at least in part, through suppressing IFNγ-induced p-ERK1/2 and its downstream pathway.

Effects of bee venom on glutamate-induced toxicity in neuronal and glial cells.

Bee venom (BV), which is extracted from honeybees, is used in traditional Korean medical therapy. Several groups have demonstrated the anti-inflammatory effects of BV in osteoarthritis both in vivo and in vitro. Glutamate is the predominant excitatory neurotransmitter in the central nervous system (CNS). Changes in glutamate release and uptake due to alterations in the activity of glutamate transporters have been reported in many neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis. To assess if BV can prevent glutamate-mediated neurotoxicity, we examined cell viability and signal transduction in glutamate-treated neuronal and microglial cells in the presence and absence of BV. We induced glutamatergic toxicity in neuronal cells and microglial cells and found that BV protected against cell death. Furthermore, BV significantly inhibited the cellular toxicity of glutamate, and pretreatment with BV altered MAP kinase activation (e.g., JNK, ERK, and p38) following exposure to glutamate. These findings suggest that treatment with BV may be helpful in reducing glutamatergic cell toxicity in neurodegenerative diseases.

Anti-inflammatory effects of electroacupuncture in the respiratory system of a symptomatic amyotrophic lateral sclerosis animal model.

BACKGROUND: Because amyotrophic lateral sclerosis (ALS) is a progressive inflammatory disease, treatment of the pulmonary system plays a key role in ALS patients' care. Previous studies have mainly examined the pathological mechanism of ALS in the central nervous system; however, there has been relatively little research regarding the pulmonary system in ALS animal models. In inflammatory diseases, including asthma and arthritis, electroacupuncture (EA) is commonly used for its anti-inflammatory effects. The goal of this study was to determine whether EA treatment affects inflammation in the pulmonary system in an ALS animal model. METHODS: EA treatment at ST36 (Zusanli) acupoint was performed with 14-week-old hSOD1(G93A) transgenic mice. Immunohistochemical analysis was performed using anti-ionized calcium binding adaptor molecule 1 (Iba-1) and anti-tumor necrosis factor alpha (TNF-α) antibodies. To investigate the expression level of inflammatory proteins, Western blot analyses were performed using anti-Iba-1, anti-TNF-α, anti-nuclear factor kappa B (NF-κB), and anti-interleukin 6 (IL-6) antibodies. The activation of Ser435-phospho-specific RAC-alpha serine/threonine-protein kinase 1 (pAKT) and the increase of phosphorylated extracellular-signal-regulated kinases (pERK) protein in lung tissues of EA-treated and untreated hSOD1(G93A) mice were also evaluated by Western blot. RESULTS: EA treatment decreased the expression of the proinflammatory proteins such as TNF-α and IL-6, pNF-κB, and Iba-1 and increased the level of activated pAKT and pERK compared to control hSOD1(G93A) mice. CONCLUSIONS: Our findings suggest that EA could be an effective anti-inflammatory treatment for the respiratory impairment that occurs in ALS animal models.

Bee venom attenuates neuroinflammatory events and extends survival in amyotrophic lateral sclerosis models.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a disease affecting the central nervous system that is either sporadic or familial origin and causing the death of motor neurons. One of the genetic factors contributing to the etiology of ALS is mutant SOD1 (mtSOD1), which induces vulnerability of motor neurons through protein misfolding, mitochondrial dysfunction, oxidative damage, cytoskeletal abnormalities, defective axonal transport, glutamate excitotoxicity, inadequate growth factor signaling, and neuroinflammation. Bee venom has been used in the practice of Oriental medicine and evidence from the literature indicates that BV plays an anti-inflammatory or anti-nociceptive role against inflammatory reactions associated with arthritis and other inflammatory diseases. The purpose of the present study was to determine whether bee venom suppresses motor neuron loss and microglial cell activation in hSOD1G93A mutant mice. METHODS: Bee venom (BV) was bilaterally injected (subcutaneously) into a 14-week-old (98 day old) male hSOD1G93A animal model at the Zusanli (ST36) acupoint, which is known to mediate an anti-inflammatory effect. For measurement of motor activity, rotarod test was performed and survival statistics were analyzed by Kaplan-Meier survival curves. The effects of BV treatment on anti-neuroinflammation of hSOD1G93A mice were assessed via immunoreactions using Iba 1 as a microglia marker and TNF-α antibody. Activation of ERK, Akt, p38 MAP Kinase (MAPK), and caspase 3 proteins was evaluated by western blotting. RESULTS: BV-treated mutant hSOD1 transgenic mice showed a decrease in the expression levels of microglia marker and phospho-p38 MAPK in the spinal cord and brainstem. Interestingly, treatment of BV in symptomatic ALS animals improved motor activity and the median survival of the BV-treated group (139 ± 3.5 days) was 18% greater than control group (117 ± 3.1 days). Furthermore, we found that BV suppressed caspase-3 activity and blocked the defects of mitochondrial structure and cristae morphology in the lumbar spinal cord of hSOD1G93A mice at the symptomatic stage. CONCLUSION: From these findings, our research suggests BV could be a potential therapeutic agent for anti-neuroinflammatory effects in an animal model of ALS.

Electroacupuncture reduces neuroinflammatory responses in symptomatic amyotrophic lateral sclerosis model.

Amyotrophic lateral sclerosis (ALS) is a paralyzing disorder that is characterized by the progressive degeneration and death of motor neurons. Acupuncture or electroacupuncture (EA) has been used for the treatment of various conditions including osteoarthritis, asthma, and other types of chronic pain conditions. It has been hypothesized that acupuncture exerts anti-inflammatory and anti-nociceptive effects on inflammatory reactions processes. The purpose of this study was to determine whether acupuncture at a specific acupoint could produce anti-inflammatory responses and suppress motor neuron loss in the hG93ASOD1 mouse, commonly used as a model for inherited ALS. We delivered EA at the Zusanli (ST36) acupuncture point in the symptomatic hSOD1G93A animal model. The EA-treated mutant hSOD1 transgenic mice showed decreases in microglial cell activity and TNF-alpha expression in the spinal cord and brain stem. Furthermore, EA significantly improved motor activity compared to the control group and reduced neuronal cell loss in hSOD1G93A mice. Our research suggests a potential functional link between EA therapy and anti-neuroinflammatory response in an ALS animal model.

Anti-toxoplasmosis effects of oleuropein isolated from Fraxinus rhychophylla.

Toxoplasmosis is a parasitic disease caused by Toxoplasma gondii, with very few therapeutic treatment options. Typically, the choices for treatment are pyrimethamine and sulfadiazine, however their utility is limited because of drug toxicity and serious side effects. For these reasons, new drugs with lower toxicity are urgently needed. In this study, the compound oleuropein isolated from Fraxinus rhynchophylla showed anti-T. gondii effects in vitro and in vivo. In Madin-Darby bovine kidney cells, the selectivity of oleuropein was 8.9, which was higher than sulfadiazine and pyrimethamine (3.8 and 2.5, respectively). In infected mice, the inhibition ratio of T. gondii in the peritoneal cavity was 55.4% compared to the negative control group after treatment with 300 mg/kg oleuropein. In addition, inhibitory effects on granuloma, apoptosis, necrosis and cyst-formation were shown in sections of spleen and liver. Oleuropein is therefore a potentially useful anti-T. gondii candidate for clinical application.

Prodomain processing of recombinant plasmepsin II and IV, the aspartic proteases of Plasmodium falciparum, is auto- and trans-catalytic.

Prodomain processing of the four food vacuole plasmepsins (PMs), the malarial aspartic proteases, is prerequisite for their activity on hemoglobin degradation of the parasite Plasmodium falciparum. Although previous studies have suggested the involvement of a calpain-like PM convertase in the processing of PMs, the underlying mechanism of their processing remains to be clarified. Here, to investigate the mechanism by which food vacuole PM II and IV are processed, we used their wild-type and mutant proteins in which the catalytic Asp residue in two active-site motifs was mutated, as well as protease inhibitors. Autocatalytic processing of wild-type PM II and IV was inhibited only by an aspartic protease inhibitor pepstatin A. Unexpectedly, their proteolytic activities were inhibited not only by pepstatin A but also by calpain inhibitor ALLN. The active-site mutants of both PM II and IV showed neither autocatalytic processing nor proteolytic activities. However, the mutants of both PMs were efficiently processed upon incubation with their respective wild type proteins. Furthermore, the mutants of both PMs were processed upon incubation with each other's wild-type PM in both pepstatin A- and ALLN-sensitive manners. These results suggest that the processing of PM II and IV occurs via an intra- and inter-molecular autocatalytic event as well as via a transcatalytic event between them.