Chaga Medicinal Mushroom, Inonotus obliquus (Agaricomycetes), Polysaccharides Alleviate Photoaging by Regulating Nrf2 Pathway and Autophagy.

Inonotus obliquus is a medicinal mushroom that contains the valuable I. obliquus polysaccharides (IOP), which is known for its bioactive properties. Studies have shown that IOP could inhibit oxidative stress induced premature aging and DNA damage, and delay body aging. However, the molecular mechanism of IOP in improving skin photoaging remains unclear, which prevents the development and utilization of I. obliquus in the field of skin care. In this study, ultraviolet B (UVB) induced human immortalized keratinocyte (HaCaT) cell photoaging model was used to explore the mechanism of IOP in relieving skin photoaging. Results showed that IOP inhibited cell senescence and apoptosis by reducing the protein expressions of p16, p21, and p53. IOP increased HO-1, SOD, and CAT expressions to achieve Nrf2/HO-1 pathway, thus improving antioxidant effects and preventing ROS generation. Furthermore, IOP enhanced the expression levels of p-AMPK, LC3B, and Beclin-1 to alleviate the autophagy inhibition in UVB-induced HaCaT cells. Based on these findings, our data suggested that IOP may be used to develop effective natural anti-photoaging ingredients to promote skin health.

Centella asiatica alleviates psoriasis through JAK/STAT3-mediated inflammation: An in vitro and in vivo study.

ETHNOPHARMACOLOGICAL RELEVANCE: Centella asiatica (L.) Urban (CA) is a dry herb of the Umbelliferae family, first mentioned in Shennong's Herbal Classic. It is known for its ability to clear heat and dampness, detoxify, and reduce swelling, making it a popular treatment for dermatitis, wound healing, and lupus erythematosus. Psoriasis is a chronic inflammatory skin disease that is characterized by clearly delineated erythema and squamous skin lesions. However, the effect of CA on regulating inflammation and its mechanism in the pathogenesis of psoriasis is still not fully understood. AIM OF THE STUDY: This study evaluated the effects of CA on inflammatory dermatosis by in vitro and in vivo studies. And clarified the important role of the JAK/STAT3 signaling pathway in the treatment of psoriasis with CA. METHODS AND MATERIALS: Different components of CA were extracted and analyzed for their total flavonoid and polyphenol contents. The antioxidant capacity of the CA extracts was determined using DPPH, ABTS, and FRAP methods. In vitro, HaCaT cells were induced by lipopolysaccharide (LPS, 20 µg·mL-1) to establish an inflammatory injury model, and the effects of CA extracts on oxidative stress, inflammation and skin barrier function were evaluated systematically. Annexin V-FITC/PI staining was utilized for detecting cell apoptosis, while the expression of NF-κB and JAK/STAT3 pathways were detected by RT-PCR and western blot. Combined with an in vivo mice model of Imiquimod (IMQ) induced psoriasis-like skin inflammation, the most effective CA extract for alleviating psoriasis was identified and its potential mechanism was investigated. RESULTS: CA extracts showed high antioxidant capacity and were able to increase the content of GSH and SOD while reducing intracellular ROS generation. Notably, CA ethyl acetate extract (CAE) was found to be the most effective. Furthermore, CA extracts effectively downregulate inflammatory factors (IFN-γ, CCL20, IL-6 and TNF-α) mRNA levels and improved the gene expressions of barrier protective factors AQP3 and FLG, among them CAE and n-hexane extract of CA (CAH) had better effects. Western blot analysis indicated that CAE and CAH had anti-inflammatory effects by inhibiting the activation of NF-κB and JAK/STAT3 pathways, and CAE exhibited the best regulatory effect at the dose of 25 µg·mL-1. In vivo experiment, the psoriasis-like skin inflammation mice model was established by 5% IMQ and treated CAE solution (10, 20, 40 mg·mL-1) for 7 days, the results showed that CAE intervention reduced the skin scale and blood scab, and significantly inhibited the secretion of inflammatory factors in both serum and skin lesions at the dose of 40 mg·mL-1. CONCLUSION: Centella asiatica extracts were effective in improving skin inflammation and skin barrier dysfunction, and also alleviated psoriasis through JAK/STAT3 pathway. The results provided experimental support for the potential use of Centella asiatica in functional food and skin care products.

SCYL3, as a novel binding partner and regulator of ROCK2, promotes hepatocellular carcinoma progression.

Background & Aims: SCY1-like pseudokinase 3 (SCYL3) was identified as a binding partner of ezrin, implicating it in metastasis. However, the clinical relevance and functional role of SCYL3 in cancer remain uncharacterized. In this study, we aimed to elucidate the role of SCYL3 in the progression of hepatocellular carcinoma (HCC). Methods: The clinical significance of SCYL3 in HCC was evaluated in publicly available datasets and by qPCR analysis of an in-house HCC cohort. The functional significance and mechanistic consequences of SCYL3 were examined in SCYL3-knockdown/overexpressing HCC cells. In vivo tumor progression was evaluated in Tp53 KO/c-Myc OE mice using the sleeping beauty transposon system. Potential downstream pathways were investigated by co-immunoprecipitation, western blotting analysis and immunofluorescence staining. Results: SCYL3 is often overexpressed in HCC; it is preferentially expressed in metastatic human HCC tumors and is associated with worse patient survival. Suppression of SCYL3 in HCC cells attenuated cell proliferation and migration as well as in vivo metastasis. Intriguingly, endogenous SCYL3 overexpression increased tumor development and metastasis in Tp53 KO/c-Myc OE mice. Mechanistic investigations revealed that SCYL3 physically binds and regulates the stability and transactivating activity of ROCK2 (Rho kinase 2) via its C-terminal domain, leading to the increased formation of actin stress fibers and focal adhesions. Conclusions: These findings reveal that SCYL3 plays a critical role in promoting the progression of HCC and have implications for developing new therapeutic strategies to tackle metastatic HCC. Impact and implications: SCYL3 was first reported to be a binding partner of a metastasis-related gene, ezrin. To date, the clinical relevance and functional role of SCYL3 in cancer remain uncharacterized. Herein, we uncover its crucial role in liver cancer progression. We show that it physically binds and regulates the stability and transactivating activity of ROCK2 leading to HCC tumor progression. Our data provide mechanistic insight that SCYL3-mediated ROCK2 protein stability plays a pivotal role in growth and metastasis of HCC cells. Targeting SCYL3/ROCK2 signaling cascade may be a novel therapeutic strategy for treatment of HCC patients.

Feasibility of machine learning-based modeling and prediction using multiple centers data to assess intrahepatic cholangiocarcinoma outcomes.

BACKGROUND AND AIMS: Currently, there are still no definitive consensus in the treatment of intrahepatic cholangiocarcinoma (iCCA). This study aimed to build a clinical decision support tool based on machine learning using the Surveillance, Epidemiology, and End Results (SEER) database and the data from the Fifth Medical Center of the PLA General Hospital in China. METHODS: 4,398 eligible patients from the SEER database and 504 eligible patients from the hospital data, who presented with histologically proven iCCA, were enrolled for modeling by cross-validation based on machine learning. All the models were trained using the open-source Python library scikit-survival version 0.16.0. Shapley additive explanations method was used to help clinicians better understand the obtained results. Permutation importance was calculated using library ELI5. RESULTS: All involved treatment modalities could contribute to a better prognosis. Three models were derived and tested using different data sources, with concordance indices of 0.67, 0.69, and 0.73, respectively. The prediction results were consistent with those under actual situations involving randomly selected patients. Model 2, trained using the hospital data, was selected to develop an online tool, due to its advantage in predicting short-term prognosis. CONCLUSION: The prediction model and tool established in this study can be applied to predict the prognosis of iCCA after treatment by inputting the patient's clinical parameters or TNM stages and treatment options, thus contributing to optimal clinical decisions.KEY MESSAGESA prognostic model related to disease staging and treatment mode was conducted using the method of machine learning, based on the big data of multi centers.The online calculator can predict the short-term survival prognosis of intrahepatic cholangiocarcinoma, thus, help to make the best clinical decision.The online calculator built to calculate the mortality risk and overall survival can be easily obtained and applied.

Prognostic analysis of patients with combined hepatocellular-cholangiocarcinoma after radical resection: A retrospective multicenter cohort study.

BACKGROUND: Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is a form of rare primary liver cancer that combines intrahepatic cholangiocarcinoma (ICC) and hepatocellular carcinoma. AIM: To investigate overall survival (OS) and recurrence-free survival (RFS) after radical resection in patients with cHCC-CCA, and the clinicopathological factors affecting prognosis in two center hospitals of China. METHODS: We reviewed consecutive patients with cHCC-CCA who received radical resection between January 2005 and September 2021 at Peking Union Medical College and the 5th Medical Center of the PLA General Hospital retrospectively. Regular follow-up and clinicopathological characteristics were systematic collected for baseline and prognostic analysis. RESULTS: Our study included 95 patients who received radical resection. The majority of these patients were male and 82.7% of these patients were infected with HBV. The mean tumor size was 4.5 cm, and approximately 40% of patients had more than one lesion. The median OS was 26.8 (95%CI: 18.5-43.0) mo, and the median RFS was 7.27 (95%CI: 5.83-10.3) mo. Independent predictors of OS were CA19-9 ≥ 37 U/mL (HR = 8.68, P = 0.002), Child-Pugh score > 5 (HR = 5.52, P = 0.027), tumor number > 1 (HR = 30.85, P = 0.002), tumor size and transarterial chemoembolization (TACE) after surgery (HR = 0.2, P = 0.005). CONCLUSION: The overall postoperative survival of cHCC-CCA patients is poor, and most patients experience relapse within a short period of time after surgery. Preoperative tumor biomarker (CA19-9, alpha-fetoprotein) levels, tumor size, and Child-Pugh score can significantly affect OS. Adjuvant TACE after surgery prolongs RFS, suggesting that TACE is a possible option for postoperative adjuvant therapy in patients with cHCC-CCA.

A New Prognostic Model Covering All Stages of Intrahepatic Cholangiocarcinoma.

Background and Aims: Intrahepatic cholangiocarcinoma (ICC) is the second most common primary hepatic malignancy that causes a poor survival. We aimed to identify its prognostic factors and to develop a nomogram that will predict survival of ICC patients among all stages. Methods: A total of 442 patients with pathology-proven ICC registered at the Fifth Medical Center of PLA General Hospital between July 2007 and December 2019 were enrolled. Subjects were followed for survival status until June 30, 2020. A prognostic model visualized as a nomogram was constructed in the training cohort using multivariate cox model, and was then validated in the validation cohort. Results: The median age was 55 years. With a median follow-up of 50.4 months, 337 patients died. The median survival was 11.6 months, with 1-, 3- and 5-year survival rates of 48.3%, 22.7% and 16.2%, respectively. Factors associated with overall survival were multiple tumors, lymph node involvement, vascular invasion, distant metastasis, decreased albumin, elevated lactate dehydrogenase (LDH), decreased iron, elevated fibrinogen, elevated CA125 and elevated CA19-9. A nomogram predicting survival of ICC patients at the time of diagnosis achieved a Harrel's c-statistic of 0.758, significantly higher than the 0.582 of the TNM stage alone. Predicted median survivals of those within the low, mid and high-risk subgroups were 35.6, 12.1 and 6.2 months, respectively. Conclusions: A nomogram based on imaging data and serum biomarkers at diagnosis showed good ability to predict survival in patients with all stages of ICC. Further studies are needed to validate the prognostic capability of our new model.

ISOC1 is a novel potential tumor suppressor in hepatocellular carcinoma.

Isochorismatase domain-containing 1 (ISOC1) plays a carcinogenic role in various tumors. However, its expression and role in hepatocellular carcinoma (HCC) have not been elucidated. This is the first study to investigate the involvement of ISOC1 in HCC growth and migration. ISOC1 expression was analyzed using public databases and clinical samples, and clinical specimens were analyzed by real-time quantitative polymerase chain reaction, western blotting, and immunohistochemistry. ISOC1 was also overexpressed in two HCC cell lines (Huh7 and HepG2) to explore how ISOC1 affects HCC cells. Finally, a nude mouse xenograft tumor model was used to investigate the role of ISOC1 in HCC cell tumorigenicity. ISOC1 was downregulated in HCC tissues compared to that in matched paracancerous tissues, and low ISOC1 expression was associated with a poor prognosis. The proliferation and single-cell colony-forming ability of the ISOC1-overexpressing cell lines Huh7 and HepG2 were significantly inhibited. Moreover, ISOC1 overexpression suppressed the migration and invasion abilities of HCC cells in vitro, and ISOC1 upregulation hindered tumor growth in the xenograft tumor model in vivo. Therefore, ISOC1 is a potential HCC suppressor protein.

Virtual Screening and Optimization of Novel mTOR Inhibitors for Radiosensitization of Hepatocellular Carcinoma.

BACKGROUND: Radiotherapy has an ameliorative effect on a wide variety of tumors, but hepatocellular carcinoma (HCC) is insensitive to this treatment. Overactivated mammalian target of rapamycin (mTOR) plays an important part in the resistance of HCC to radiotherapy; thus, mTOR inhibitors have potential as novel radiosensitizers to enhance the efficacy of radiotherapy for HCC. METHODS: A lead compound was found based on pharmacophore modeling and molecular docking, and optimized according to the differences between the ATP-binding pockets of mTOR and PI3K. The radiosensitizing effect of the optimized compound (2a) was confirmed by colony formation assays and DNA double-strand break assays in vitro. The discovery and preclinical characteristics of this compound are described. RESULTS: The key amino acid residues in mTOR were identified, and a precise virtual screening model was constructed. Compound 2a, with a 4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine scaffold, exhibited promising potency against mTOR (mTOR IC50=7.1 nmol/L (nM)) with 126-fold selectivity over PI3Kα. Moreover, 2a significantly enhanced the sensitivity of HCC to radiotherapy in vitro in a dose-dependent manner. CONCLUSION: A new class of selective mTOR inhibitors was developed and their radiosensitization effects were confirmed. This study also provides a basis for developing mTOR-specific inhibitors for use as radiosensitizers for HCC radiotherapy.

Bone morphogenetic protein-7 represses hepatic stellate cell activation and liver fibrosis via regulation of TGF-ß/Smad signaling pathway.

BACKGROUND: Liver fibrosis is a refractory disease whose persistence can eventually induce cirrhosis or even liver cancer. Early liver fibrosis is reversible by intervention. As a member of the transforming growth factor-beta (TGF-ß) superfamily, bone morphogenetic protein 7 (BMP7) has anti-liver fibrosis functions. However, little is known about BMP7 expression changes and its potential regulatory mechanism as well as the relationship between BMP7 and TGF-ß during liver fibrosis. In addition, the mechanism underlying the anti-liver fibrosis function of BMP7 needs to be further explored. AIM: To investigate changes in the dynamic expression of BMP7 during liver fibrosis, interactions between BMP7 and TGF-ß1, and possible mechanisms underlying the anti-liver fibrosis function of BMP7. METHODS: Changes in BMP7 expression during liver fibrosis and the interaction between BMP7 and TGF-ß1 in mice were observed. Exogenous BMP7 was used to treat mouse primary hepatic stellate cells (HSCs) to observe its effect on activation, migration, and proliferation of HSCs and explore the possible mechanism underlying the anti-liver fibrosis function of BMP7. Mice with liver fibrosis received exogenous BMP7 intervention to observe improvement of liver fibrosis by using Masson's trichrome staining and detecting the expression of the HSC activation indicator alpha-smooth muscle actin (α-SMA) and the collagen formation associated protein type I collagen (Col I). Changes in the dynamic expression of BMP7 during liver fibrosis in the human body were further observed. RESULTS: In the process of liver fibrosis induced by carbon tetrachloride (CCl4) in mice, BMP7 protein expression first increased, followed by a decrease; there was a similar trend in the human body. This process was accompanied by a sustained increase in TGF-ß1 protein expression. In vitro experiment results showed that TGF-ß1 inhibited BMP7 expression in a time- and dose-dependent manner. In contrast, high doses of exogenous BMP7 inhibited TGF-ß1-induced activation, migration, and proliferation of HSCs; this inhibitory effect was associated with upregulation of pSmad1/5/8 and downregulation of phosphorylation of Smad3 and p38 by BMP7. In vivo experiment results showed that exogenous BMP7 improved liver fibrosis in mice. CONCLUSION: During liver fibrosis, BMP7 protein expression first increases and then decreases. This changing trend is associated with inhibition of BMP7 expression by sustained upregulation of TGF-ß1 in a time- and dose-dependent manner. Exogenous BMP7 could selectively regulate TGF-ß/Smad pathway-associated factors to inhibit activation, migration, and proliferation of HSCs and exert anti-liver fibrosis functions. Exogenous BMP7 has the potential to be used as an anti-liver fibrosis drug.

Focal Adhesion Kinase Regulates Hepatic Stellate Cell Activation and Liver Fibrosis.

Understanding the underlying molecular mechanisms of liver fibrosis is important to develop effective therapy. Herein, we show that focal-adhesion-kinse (FAK) plays a key role in promoting hepatic stellate cells (HSCs) activation in vitro and liver fibrosis progression in vivo. FAK activation is associated with increased expression of α-smooth muscle actin (α-SMA) and collagen in fibrotic live tissues. Transforming growth factor beta-1 (TGF-ß1) induces FAK activation in a time and dose dependent manner. FAK activation precedes the α-SMA expression in HSCs. Inhibition of FAK activation blocks the α-SMA and collagen expression, and inhibits the formation of stress fibers in TGF-ß1 treated HSCs. Furthermore, inhibition of FAK activation significantly reduces HSC migration and small GTPase activation, and induces apoptotic signaling in TGF-ß1 treated HSCs. Importantly, FAK inhibitor attenuates liver fibrosis in vivo and significantly reduces collagen and α-SMA expression in an animal model of liver fibrosis. These data demonstrate that FAK plays an essential role in HSC activation and liver fibrosis progression, and FAK signaling pathway could be a potential target for liver fibrosis.

Interaction of Src and Alpha-V Integrin Regulates Fibroblast Migration and Modulates Lung Fibrosis in A Preclinical Model of Lung Fibrosis.

Src kinase is known to regulate fibroblast migration. However, the contribution of integrin and Src kinase interaction to lung fibrosis has not been mechanistically investigated. Our data demonstrate that integrin alpha v (αV) recruited Src kinase and that leads to subsequent Src activation in fibroblasts plated on fibrotic matrix, osteopontin. Src interaction with integrin αV is required for integrin αV-mediated Src activation, and the subsequent fibroblast migration. The study identified that ß5 and ß3 are the major integrins for this effect on osteopontin. In contrast, integrins ß1, ß6, and ß8 did not have a critical role in this phenomenon. Importantly, Src inhibitor significantly reduces fibroblast migration stimulated by PDGF-BB and reduced in vivo lung fibrosis in mice. Src inhibitor reduced Src activation and blocked the signaling transduction by integrin αV, inhibited migration signaling pathways and reduced extracellular matrix protein production, and blocked myofibroblast differentiation in vivo in mouse lung tissues. The present study supports that the interaction of Src Kinase and integrins plays a critical role in the development of lung fibrosis and the signaling involved may present a novel opportunity to target deadly fibrotic diseases.

Age and Ebola viral load correlate with mortality and survival time in 288 Ebola virus disease patients.

BACKGROUND: A Chinese medical team managed Ebola virus disease (EVD) patients in Sierra Leone from October 2014 to March 2015 and attended to 693 suspected patients, of whom 288 had confirmed disease. METHODS: A retrospective study was conducted of the 288 patients with confirmed disease. Clinical symptoms, manifestations, and serum viral load were analyzed and compared among the different groups for mortality and survival time. RESULTS: Among the 288 confirmed EVD patients (149 male and 139 female, median age 28 years, and median log viral load 6.68), 98 died, 36 recovered, and 154 were lost to follow-up. Common symptoms were fever (77.78%), fatigue (64.93%), abdominal pain (64.58%), headache (62.85%), and diarrhea (61.81%). Compared to patients aged<18 years, those who were older than 40 years had a higher probability of death (odds ratio 2.855, p=0.044). Patients with a viral load of >10(6) copies/ml had a higher case fatality rate than those with <10(6) copies/ml (odds ratio 3.095, p=0.004). Cox regression showed that age, viral load, and the presence of diarrhea correlated with mortality. CONCLUSION: Patients with a high viral load, of older age, and with diarrhea had a higher mortality and shorter survival time.

S100A4 promotes pancreatic cancer progression through a dual signaling pathway mediated by Src and focal adhesion kinase.

S100A4 expression is associated with poor clinical outcomes of patients with pancreatic cancer. The effects of loss or gain of S100A4 were examined in pancreatic cancer cell lines. S100A4 downregulation remarkably reduces cell migration and invasion, inhibits proliferation, and induces apoptosis in pancreatic tumor cells. S100A4 downregulation results in significant cell growth inhibition and apoptosis in response to TGF-ß1, supporting a non-canonical role of S100A4 in pancreatic cancer. The role of S100A4 in tumor progression was studied by using an orthotopic human pancreatic cancer xenograft mouse model. Tumor mass is remarkably decreased in animals injected with S100A4-deficient pancreatic tumor cells. P27(Kip1) expression and cleaved caspase-3 are increased, while cyclin E expression is decreased, in S100A4-deficient pancreatic tumors in vivo. S100A4-deficient tumors have lower expression of vascular endothelial growth factor, suggesting reduced angiogenesis. Biochemical assays revealed that S100A4 activates Src and focal adhesion kinase (FAK) signaling events, and inhibition of both kinases is required to maximally block the tumorigenic potential of pancreatic cancer cells. These findings support that S100A4 plays an important role in pancreatic cancer progression in vivo and S100A4 promotes tumorigenic phenotypes of pancreatic cancer cells through the Src-FAK mediated dual signaling pathway.

Hepatic angiomyolipoma mimicking hepatocellular carcinoma: magnetic resonance imaging and clinical pathological characteristics in 9 cases.

Hepatic angiomyolipoma (HAML) is a rare mesenchymal tumor of the liver with marked histological diversity. The present study was to review the magnetic resonance imaging (MRI) and clinical pathological features of HAML resembling hepatocellular carcinoma (HCC). Nine patients who underwent surgical resection and had pathological diagnosis of HAML were retrospectively analyzed. All of 9 patients (5 males and 4 females) had a solitary hepatic mass with a median size of 4 cm (from 1.4 cm to 15.3 cm). Seven cases were identified as incidental liver tumors during health screening and 2 patients were diagnosed for hepatic mass when visited hospitals with unspecific abdominal discomfort. Before resection, 6 cases were diagnosed as HCC on MRI. MRI on chemical shift imagings showed a large amount of lipids in 5 cases. The enhancement pattern of MRI was classified into 2 types: in 2 cases, lesions with small or no vessels that demonstrated prolonged enhancement (1 mixed subtype and 1 myomatous subtype) and in 7 cases, lesions with abundant central vessels that show rapid washout (3 mixed subtypes and 4 myomatous subtypes) in the portal venous/delayed phase. All patients underwent resection of hepatic tumor and no recurrence was observed during follow-up (range: 2-24 months) of median 10 months. By immunohistochemistry, the tumor cells demonstrated positive immunostaining for human melanoma black-45, smooth muscle actin, and CD34. In conclusion, all of 9 patients with HAML presented with none or nonspecific clinical manifestations. The diagnosis of HAML relies on disease and immunohistochemistry, but not MRI due to its resemblance to HCC.

Hypomethylation of long interspersed nuclear element-1 promoter is associated with poor outcomes for curative resected hepatocellular carcinoma.

BACKGROUND: Epigenetic alterations are well documented in hepatocarcinogenesis. However, hypomethylation of long interspersed nuclear element 1(LINE-1) promoter and its relationship with clinicopathological features in hepatocellular carcinoma(HCC) remain unknown. METHODS: The bisulfite-specific PCR and DNA sequencing analysis was performed to assess the methylation status of LINE-1 promoter in a pilot cohort of 71 patients with HCC. Additionally,methylation levels of two hot CpG sites of LINE-1 promoter, site 7 and 18 were measured by real-time PCR and compared with clinicopathological parameters in a cohort of 172 HCC. All the patients included were in BCLC stage A or B. RESULTS: Most patients with HCC (87.3%) showed hypomethylation of LINE-1 promoter compared with HBV-related cirrhosis and normal controls (P < 0.001). The HCC patients with LINE-1 promoter hypomethylation had a median tumour-free survival (TFS) and overall survival (OS)post-resection of 22.0 (95% CI: 13.3­30.7) months and 35.0 (95% CI: 24.0­46.1) months, respectively, compared with 40 months and ~60 months for those with LINE-1 promoter hypermethylation (P < 0.05). Multivariate analyses showed that the hypomethylation level at CpG site 7 and 18 of LINE-1 promoter, along with tumour size and tumour differentiation, was independently associated with both TFS and OS for patients with HCC after resection. CONCLUSION: Promoter hypomethylation of LINE-1, especially at the CpG site 7 and 18, was associated with a poor prognosis in HCC.

Long interspersed nuclear element ORF-1 protein promotes proliferation and resistance to chemotherapy in hepatocellular carcinoma.

AIM: To clarify the specific roles and mechanisms of long interspersed nuclear element-1 ORF-1 protein [human long interspersed nuclear element-1 (LINE-1), ORF-1p] in chemotherapeutic drug resistance and cell proliferation regulation in hepatocellular carcinoma (HCC) cells. METHODS: MTT assays were performed to identify the effect of the chemotherapeutic drug toxicity on HepG2 cells. Cell proliferation inhibition and the IC50 were calculated by the Origin 8.0 software. Western blotting assays were performed to investigate whether LINE-1 ORF-1p modulates the expression of some important genes, including p53, p27, p15, Bcl-2, mdr, and p-gp. To corroborate the proliferation and anchor-independent growth results, the HepG2 cells were analyzed by flow cytometry to investigate the effect of LINE-1 ORF-1p on the apoptosis regulation. RESULTS: LINE-1 ORF-1p contributed to the resistance to several chemotherapeutic drugs (cisplatin and epirubicin) in HepG2 cells. The IC50 of the epirubicin and cisplatin increased from 36.04 nmol/L to 59.11 nmol/L or from 37.94 nmol/L to 119.32 nmol/L. Repression of LINE-1 ORF-1p expression by the siRNA could markedly enhance the response of HepG2 cells to the epirubicin and cisplatin. The IC50 correspondingly decreased from 28.06 nmol/L to 3.83 nmol/L or from 32.04 nmol/L to 2.89 nmol/L. Interestingly, down-regulation of LINE-1 ORF-1p level by siRNA could promote the response of HepG2 cells to the paclitaxel. The IC50 decreased from 35.90 nmol/L to 7.36 nmol/L. However, overexpression of LINE-1 ORF-1p did not modulate the paclitaxel toxicity in HepG2 cells. Further Western blotting revealed that LINE-1 ORF-1p enhanced mdr and p-gp gene expression. As a protein arrested in the nucleus, LINE-1 ORF-1p may function through modulating transcriptional activity of some important transcription factors. Indeed, LINE-1 ORF-1p promoted HepG2 cell proliferation, anchor-independent growth and protected the cells against apoptosis through modulating the expression of p15, p21, p53, and Bcl-2 genes. CONCLUSION: LINE-1 ORF-1p promotes HepG2 cell proliferation and plays an important role in the resistance of chemotherapeutic drugs. By establishing novel roles and defining the mechanisms of LINE-1 ORF-1p in HCC chemotherapeutic drug resistance and cell proliferation regulation, this study indicates that LINE-1 ORF-1p is a potential target for overcoming HCC chemotherapeutic resistance.

High intratumoral metastasis-associated in colon cancer-1 expression predicts poor outcomes of cryoablation therapy for advanced hepatocellular carcinoma.

BACKGROUND: Cryoablation is one of the local therapies for hepatocellular carcinoma (HCC), but its safety and effect has not been studied in patients with Child class A or B and Barcelona Clinic Liver Cancer (BCLC) stage C HCC. Metastasis-associated in colon cancer-1 (MACC1) overexpression has been associated with poor prognosis of HCC, but its predictive value to post-cryoablation outcomes remains unknown in patients with BCLC stage C HCC. METHODS: This study assessed the safety and outcomes of cryoablation measured by time to progression (TTP) and overall survival (OS), and predictive value of MACC1 mRNA and protein overexpression in tumorous tissue to post-cryoablation outcomes in 120 advanced HCC patients with child-pugh class A or B by quantitative polymerase chain reaction and immunohistochemical staining. The potenial correlation of MACC1 and c-Met expression to tumor cell proliferation and apoptosis was also analyzed. RESULTS: The cryoablation in patients with advanced unresectable HCC resulted in a median TTP and OS of 5.5 (4.2- 6.7) months and 10.5 (9.0-12.0) months, respectively and no significant complications, comparable to the historical report for RFA therapy. The MACC1 mRNA and nuclear protein expression was significantly increased in tumorous tissues in these patients than that in normal liver tissue controls. Higher expression of MACC1 mRNA and nuclear protein in tumorous tissues in these patients was associated with shorter post cryoablation median TTP and OS than that with lower MACC1 expression. CONCLUSIONS: Cryoablation is a safe and effective therapeutic option for patients with advanced HCC and Child-pugh class A or B cirrhosis; and a higher intratumoral expression of MACC1 or nuclear translocation predicts poor outcomes of cryotherapy in these patients.

Tumour seeding after percutaneous cryoablation for hepatocellular carcinoma.

AIM: To assess the rate and risk factors for tumour seeding in a large cohort of patients. METHODS: Over an 8-year period, 1436 hepatocellular carcinoma (HCC) patients with 2423 tumour nodules underwent 3015 image-guided percutaneous cryoablation sessions [1215 guided by ultrasonography and 221 by spiral computed tomography (CT)]. Follow-up CT or magnetic resonance imaging was performed every 3 mo. The detailed clinical data were recorded to analyse the risk factors for seeding. RESULTS: The median follow-up time was 18 (range 1-90) mo. Seeding was detected in 11 patients (0.76%) at 1-24 (median 6.0) mo after cryoablation. Seeding occurred along the needle tract in 10 patients and at a distant location in 1 patient. Seeded tumours usually showed similar imaging and histopathological features to the primary HCCs. Univariate analyses identified subcapsular tumour location and direct subcapsular needle insertion as risk factors for seeding. Multivariate analysis showed that only direct subcapsular needle insertion was an independent risk factor for seeding (P = 0.017; odds ratio 2.57; 95%CI: 1.47-3.65). Seeding after cryoablation occurred earlier in patients with poorly differentiated HCC than those with well or moderately differentiated HCC [1.33 ± 0.577 mo vs 11.12 ± 6.896 mo; P = 0.042; 95%CI: (-19.115)-(-0.468)]. CONCLUSION: The risk of seeding after cryoablation for HCC is small. Direct puncture of subcapsular tumours should be avoided to minimise seeding.

Overexpression of metastasis-associated in colon cancer 1 predicts a poor outcome of hepatitis B virus-related hepatocellular carcinoma.

AIM: To investigate the intratumoral expression of metastasis-associated in colon cancer 1 (MACC1) and c-Met and determine their clinical values associated with hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). METHODS: A retrospective study admitted three hundred fifty-four patients with HBV-related HCC. The expression and distribution of MACC1 and c-Met were assessed by quantitative real-time polymerase chain reaction and immunohistochemistry staining. Prognostic factors influencing survival, metastasis and recurrence were assessed. RESULTS: Intratumoral MACC1 level was found to be associated with HCC disease progression. Both median tumor-free survival (TFS) and overall survival (OS) were significantly shorter in the postoperative HCC patients with high intratumoral MACC1 expression, as compared to those with low intratumoral MACC1 levels (TFS: 34 mo vs 48.0 mo, P < 0.001; OS: 40 mo vs 48 mo, P < 0.01). Multivariable analysis indicated that high MACC1 expression or co-expression with c-Met were independent predictors for HCC clinic outcome (P < 0.001). CONCLUSION: High intratumoral MACC1 expression can be associated with enhanced tumor progression and poor outcome of HBV-related HCC. MACC1 may serve as a prognostic biomarker for postoperative HCC.

Decrease in size of non-treated lesions after cryoablation for hepatocellular carcinoma.

We present the case of one 58-year-old man with advancd hepatocellular carcinoma and hepatitis-B virus-related liver cirrhosis who received hepatic cryoablation. Magnetic resonance imaging (MRI) showed multiple liver tumors and the diameter of the largest tumor was more than 10cm. The patient received 2 percutaneous cryoablations in December 2009 and January 2010. Ten months later, MRI showed that not only the treated areas underwent necrosis but also the non-treated area decreased. The a-fetoprotein (AFP) level and the frequency of circulated regulatory T cell (Treg) before treatment were 13,800ng/mL and 15.6%, respectively. Following the cryoablations they dropped to 436ng/mL and 7.6%, respectively, 10 months later. The patient remains in good condition until now.