Optimizing CT and MRI criteria for differentiating intrahepatic mass-forming cholangiocarcinoma and hepatocellular carcinoma.

BACKGROUND: Accurate diagnosis of intrahepatic mass-forming cholangiocarcinoma (IMCC) is crucial with regard to the choice of patient management and treatment options. PURPOSE: To evaluate the feasibility and diagnostic performance of the LI-RADS M (LR-M) targetoid criteria on computed tomography (CT) and gadoxetic acid-enhanced magnetic resonance imaging (EOB-MRI) in differentiating IMCC from hepatocellular carcinoma (HCC). MATERIAL AND METHODS: A total of 118 patients with IMCC and HCC were included who underwent CT and EOB-MRI examinations. Multivariate analysis was used to determine the strongest predictors differentiating IMCC from HCC. Using these predictors, a predictive model for differentiating IMCC from HCC was constructed and the performance of the model was confirmed using the receiver operating characteristic curve. RESULTS: Multivariate analyses revealed rim-like arterial phase hyperenhancement (rim APHE) on CT and rim APHE, delayed central enhancement (DCE), and targetoid hepatobiliary phase (HBP) on MRI as independent variables significantly differentiating IMCC from HCC. The multivariate logistic regression model incorporating the three variables on EOB-MRI was constructed with an area under the curve (AUC) of 0.946, sensitivity of 87.80%, specificity of 92.21%, and accuracy of 94.60%. Per the DeLong test, the multivariate logistic regression model showed significantly higher AUC than rim APHE on CT (0.946 vs. 0.871; P = 0.008) and MRI (0.946 vs. 0.876; P = 0.003), whereas rim APHE on CT and MRI did not differ significantly (P = 0.809). CONCLUSION: The multivariate logistic regression model based on rim APHE, DCE, and targetoid HBP on EOB-MRI can effectively distinguish IMCC from HCC and is superior to any other targetoid appearance criterion of LI-RADS on CT and EOB-MRI.

A gadoxetic acid-enhanced MRI-based multivariable model using LI-RADS v2018 and other imaging features for preoperative prediction of macrotrabecular-massive hepatocellular carcinoma.

PURPOSE: To identify imaging features of macrotrabecular-massive hepatocellular carcinoma (MTM-HCC) using LI-RADS v2018 and other imaging features and to develop a gadoxetic acid-enhanced MRI (EOB-MRI)-based model for pretreatment prediction of MTM-HCC. MATERIALS AND METHODS: A total of 93 patients with pathologically proven HCC (39 MTM-HCC and 54 non-MTM-HCC) were retrospectively evaluated with EOB-MRI at 3 T. Imaging analysis according to LI-RADS v2018 was evaluated by two readers. Univariate and multivariate analyses were performed to determine independent predictors for MTM-HCC. Different logistic regression models were built based on MRI features, including model A (enhancing capsule, blood products in mass and ascites), model B (enhancing capsule and ascites), model C (blood products in mass and ascites), and model D (blood products in mass and enhancing capsule). Diagnostic performance was assessed by receiver operating characteristic (ROC) curves. RESULTS: After multivariate analysis, absence of enhancing capsule (odds ratio = 0.102, p = 0.010), absence of blood products in mass (odds ratio = 0.073, p = 0.030), and with ascites (odds ratio = 55.677, p = 0.028) were identified as independent differential factors for the presence of MTM-HCC. Model A yielded a sensitivity, specificity, and AUC of 35.90% (21.20,52.80), 94.44% (84.60, 98.80), and 0.731 (0.629, 0.818). Model A achieved a comparable AUC than model D (0.731 vs. 0.699, p = 0.333), but a higher AUC than model B (0.731 vs. 0.644, p = 0.048) and model C (0.731 vs. 0.650, p = 0.005). CONCLUSION: The EOB-MRI-based model is promising for noninvasively predicting MTM-HCC and may assist clinicians in pretreatment decisions.

A Computed Tomography Nomogram for Assessing the Malignancy Risk of Focal Liver Lesions in Patients With Cirrhosis: A Preliminary Study.

PURPOSE: The detection and characterization of focal liver lesions (FLLs) in patients with cirrhosis is challenging. Accurate information about FLLs is key to their management, which can range from conservative methods to surgical excision. We sought to develop a nomogram that incorporates clinical risk factors, blood indicators, and enhanced computed tomography (CT) imaging findings to predict the nature of FLLs in cirrhotic livers. METHOD: A total of 348 surgically confirmed FLLs were included. CT findings and clinical data were assessed. All factors with P < 0.05 in univariate analysis were included in multivariate analysis. ROC analysis was performed, and a nomogram was constructed based on the multivariate logistic regression analysis results. RESULTS: The FLLs were either benign (n = 79) or malignant (n = 269). Logistic regression evaluated independent factors that positively affected malignancy. AFP (OR = 10.547), arterial phase hyperenhancement (APHE) (OR = 740.876), washout (OR = 0.028), satellite lesions (OR = 15.164), ascites (OR = 156.241), and nodule-in-nodule architecture (OR =27.401) were independent predictors of malignancy. The combined predictors had excellent performance in differentiating benign and malignant lesions, with an AUC of 0.959, a sensitivity of 95.24%, and a specificity of 87.5% in the training cohort and AUC of 0.981, sensitivity of 94.74%, and specificity of 93.33% in the test cohort. The C-index was 96.80%, and calibration curves showed good agreement between the nomogram predictions and the actual data. CONCLUSIONS: The nomogram showed excellent discrimination and calibration for malignancy risk prediction, and it may aid in making FLLs treatment decisions.

Corrigendum: USP5 Promotes Metastasis in Non-Small Cell Lung Cancer by Inducing Epithelial-Mesenchymal Transition via Wnt/ß-Catenin Pathway.

[This corrects the article DOI: 10.3389/fphar.2020.00668.].

The deubiquitinating enzyme UCHL1 promotes resistance to pemetrexed in non-small cell lung cancer by upregulating thymidylate synthase.

Rationale: Resistance to pemetrexed (PEM)-based chemotherapy is a major cause of progression in non-small cell lung cancer (NSCLC) patients. The deubiquitinating enzyme UCHL1 was recently found to play important roles in chemoresistance and tumor progression. However, the potential roles and mechanisms of UCHL1 in PEM resistance remain unclear. Methods: Bioinformatics analyses and immunohistochemistry were used to evaluate UCHL1 expression in NSCLC specimens. Kaplan-Meier analysis with the log-rank test was used for survival analyses. We established PEM-resistant NSCLC cell lines by exposing them to step-wise increases in PEM concentrations, and in vitro and in vivo assays were used to explore the roles and mechanisms of UCHL1 in PEM resistance using the NSCLC cells. Results: In chemoresistant tumors from NSCLC patients, UCHL1 was highly expressed and elevated UCHL1 expression was strongly associated with poor outcomes. Furthermore, UCHL1 expression was significantly upregulated in PEM-resistant NSCLC cells, while genetic silencing or inhibiting UCHL1 suppressed resistance to PEM and other drugs in NSCLC cells. Mechanistically, UCHL1 promoted PEM resistance in NSCLC by upregulating the expression of thymidylate synthase (TS), based on reduced TS expression after UCHL1 inhibition and re-emergence of PEM resistance upon TS restoration. Furthermore, UCHL1 upregulated TS expression, which mitigated PEM-induced DNA damage and cell cycle arrest in NSCLC cells, and also conferred resistance to PEM and other drugs. Conclusions: It appears that UCHL1 promotes PEM resistance by upregulating TS in NSCLC cells, which mitigated DNA damage and cell cycle arrest. Thus, UCHL1 may be a therapeutic target for overcoming PEM resistance in NSCLC patients.

USP5 Promotes Metastasis in Non-Small Cell Lung Cancer by Inducing Epithelial-Mesenchymal Transition via Wnt/ß-Catenin Pathway.

Ubiquitin-specific protease 5 (USP5) is a deubiquitinating enzyme that functions as an oncoprotein in a variety of human cancers. However, the expression and role of USP5 in the metastasis of non-small cell lung cancer (NSCLC) have not been addressed. In this study, we examined the expression and prognostic significance of USP5 in NSCLC. The results revealed that USP5 was overexpressed and correlated with metastasis and overall survival in NSCLC tissues. A further in vitro study revealed that the levels of USP5 protein in NSCLC cells were associated with epithelial-mesenchymal transition (EMT) markers. Furthermore, USP5 overexpression significantly enhanced, whereas USP5 silencing significantly decreased the expression of EMT proteins and migration and invasion of NSCLC cells. In addition, the results from western blotting demonstrated that USP5 regulated EMT via the Wnt/ß-catenin signaling pathway. Further immunohistochemical analysis revealed that USP5 was significantly associated with the expression of ß-catenin and EMT markers in NSCLC tissues. Overall, USP5 upregulation is associated with tumor metastasis and poor prognosis in patients with NSCLC. USP5 promotes EMT and the invasion and migration of NSCLC cells. Therefore, USP5 may serve as a novel prognostic biomarker and provide a potential target for the treatment of metastasis in NSCLC.

Cathepsin L activated by mutant p53 and Egr-1 promotes ionizing radiation-induced EMT in human NSCLC.

BACKGROUND: Ionizing radiation (IR) is one of the major clinical therapies of cancer, although it increases the epithelial-mesenchymal transition (EMT) of non-small cell lung cancer (NSCLC), unexpectedly. The cellular and molecular mechanisms underlying this role are not completely understood. METHODS: We used NSCLC cell lines as well as tumor specimens from 78 patients with NSCLC to evaluate p53, Cathepsin L (CTSL) and EMT phenotypic changes. Xenograft models was also utilized to examine the roles of mutant p53 (mut-p53) and CTSL in regulating IR-induced EMT of NSCLC. RESULTS: Expression of CTSL was markedly increased in human NSCLC tissues with mutant p53 (mut-p53), and p53 mutation positively correlated with metastasis of NSCLC patients. In human non-small cell lung cancer cell line, H1299 cells transfected with various p53 lentivirus vectors, mut-p53 could promote the invasion and motility of cells under IR, mainly through the EMT. This EMT process was induced by elevating intranuclear CTSL which was regulated by mut-p53 depending on Early growth response protein-1 (Egr-1) activation. In the subcutaneous tumor xenograft model, IR promoted the EMT of the cancer cells in the presence of mut-p53, owing to increase expression and nuclear transition of its downstream protein CTSL. CONCLUSION: Taken together, these data reveal the role of the mut-p53/Egr-1/CTSL axis in regulating the signaling pathway responsible for IR-induced EMT.

Paclitaxel­resistant gastric cancer MGC­803 cells promote epithelial­to­mesenchymal transition and chemoresistance in paclitaxel­sensitive cells via exosomal delivery of miR­155­5p.

Paclitaxel is a first­line chemotherapeutic agent for gastric cancer; however, resistance limits its effectiveness. Investigation into the underlying mechanisms of paclitaxel resistance is urgently required. In the present study, a paclitaxel­resistant gastric cancer cell line (MGC­803R) was generated with a morphological phenotype of epithelial­to­mesenchymal transition (EMT) and increased expression levels of microRNA (miR)­155­5p. MGC­803R cell­derived exosomes were effectively taken up by paclitaxel­sensitive MGC­803S cells, which exhibited EMT and chemoresistance phenotypes. miR­155­5p was enriched in MGC­803R­exosomes and could be delivered into MGC­803S cells. miR­155­5p overexpression in MGC­803S cells via transfection with mimics resulted in similar phenotypic effects as treatment with MGC­803R exosome and increased miR­155­5p content in MGC­803S exosomes, which then capable of inducing the malignant phenotype in the sensitive cells. GATA binding protein 3 (GATA3) and tumor protein p53­inducible nuclear protein 1 (TP53INP1) were identified as targets of miR­155­5p. Exosomal miR­155­5p inhibited these targets by directly targeting their 3' untranslated regions. Knockdown of miR­155­5p was observed to reverse the EMT and chemoresistant phenotypes of MGC­803R cells, potentially via GATA3 and TP53INP1 upregulation, which inhibited MGC­803R­exosomes from inducing the malignant phenotype. These results demonstrated that exosomal delivery of miR­155­5p may induce EMT and chemoresistant phenotypes from paclitaxel­resistant gastric cancer cells to the sensitive cells, which may be mediated by GATA3 and TP53INP1 suppression. Targeting miR­155­5p may thus be a promising strategy to overcome paclitaxel resistance in gastric cancer.

K-ras mutation promotes ionizing radiation-induced invasion and migration of lung cancer in part via the Cathepsin L/CUX1 pathway.

K-ras mutation is involved in cancer progression including invasion and migration, but the underlying mechanism is not yet clear. Cathepsin L is a lysosomal cysteine protease and has recently been associated with invasion and migration in human cancers when it is overexpressed. Our recent studies have shown that ionizing radiation (IR) enhanced expression of cathepsin L and increased invasion and migration of tumor cells, but the molecular mechanism is still unclear. In the present study, the effects of K-ras mutation and IR induced invasion and migration of lung cancer as well as the underlying mechanisms were investigated both in vitro and in vivo. Firstly, the levels of cathepsin L and epithelial mesenchymal transition (EMT) marker proteins remarkably changed in A549 (K-ras mutant) after irradiation compared with H1299 (K-ras wild), thereby promoting invasion and migration. Additionally, cathepsin L and its downstream transcription factor CUX1/p110 were increased after irradiation in A549 transfected with CUX1/p200, and the proteolytic processing of CUX1 by cathepsin L was remarkably increased after co-transfection of CUX1/p200 and cathepsin L-lentivirus in H1299. In addition, delivery of a mutant K-ras (V12) into HEK 293 cells stimulated EMT after irradiation due to the accumulation of cathepsin L. Moreover, mutated K-ras was associated with IR-induced cathepsin L and EMT in BALB/c nude mice. Finally, the level of cathepsin L expression was higher in samples carrying a K-ras mutation than in wild-type K-ras samples and the mesenchymal markers were upregulated in the samples of mutant K-ras, whereas the epithelial marker E-cadherin was downregulated in non-small cell lung cancers tissues. In conclusion, the findings demonstrated that mutated K-ras promotes cathepsin L expression and plays a pivotal role in EMT of human lung cancer. The regulatory effect of IR-induced cathepsin L on lung cancer invasion and migration was partially attributed to the Cathepsin L /CUX1-mediated EMT signaling pathway. This study will provide cathepsin L as a potential target for tumor therapy.

Cathepsin L is involved in X-ray-induced invasion and migration of human glioma U251 cells.

An important therapeutic method of glioblastoma, the most common primary brain tumor, is radiotherapy. However, several studies reported recently that radiation could also promote the invasion and migration of malignant tumor. Herein, we have identified that a significant increase of migration and invasiveness of human glioma U251 cells undergoing X-ray was observed compared to controls, accompanied by the increase of cathepsin L (CTSL), which is a lysosomal cysteine protease overexpressed and secreted by tumor cells. To verify if there was a relationship between CTSL and the X-ray-induced glioma invasion, a CTSL specific inhibitor Z-FY-CHO or a short hairpin RNA interference was used to pretreat U251 cells. As a result, the cell invasion and migration was impaired via down-regulation of CTSL. Additionally, a marked reduction of the cell-signaling molecules Rho kinase was also detected compared with controls. We also found that CTSL is involved in EMT progress: both in vitro and in clinical specimens. Overall, our findings show that CTSL is an important protein which mediates cell invasion and migration of human glioma U251 cells induced by X-ray, and the inhibition of CTSL expression might diminish the invasion of U251 cells by reducing the activity of RhoA and CDC42 as well as EMT positive markers.

Cathepsin L upregulation-induced EMT phenotype is associated with the acquisition of cisplatin or paclitaxel resistance in A549 cells.

AIM: Cathepsin L (CTSL), a lysosomal acid cysteine protease, is known to play important roles in tumor metastasis and chemotherapy resistance. In this study we investigated the molecular mechanisms underlying the regulation of chemoresistance by CTSL in human lung cancer cells. METHODS: Human lung cancer A549 cells, A549/PTX (paclitaxel-resistant) cells and A549/DDP (cisplatin-resistant) cells were tested. The resistance to cisplatin or paclitaxel was detected using MTT and the colony-formation assays. Actin remodeling was observed with FITC-Phalloidin fluorescent staining or immunofluorescence. A wound-healing assay or Transwell assay was used to assess the migration or invasion ability. The expression of CTSL and epithelial and mesenchymal markers was analyzed with Western blotting and immunofluorescence. The expression of EMT-associated transcription factors was measured with Western blotting or q-PCR. BALB/c nude mice were implanted subcutaneously with A549 cells overexpressing CTSL, and the mice were administered paclitaxel (10, 15 mg/kg, ip) every 3 d for 5 times. RESULTS: Cisplatin or paclitaxel treatment (10-80 ng/mL) induced CTSL expression in A549 cells. CTSL levels were much higher in A549/PTX and A549/DDP cells than in A549 cells. Silencing of CTSL reversed the chemoresistance in A549/DDP and A549/TAX cells, whereas overexpression of CTSL attenuated the sensitivity of A549 cells to cisplatin or paclitaxel. Furthermore, A549/DDP and A549/TAX cells underwent morphological and cytoskeletal changes with increased cell invasion and migration abilities, accompanied by decreased expression of epithelial markers (E-cadherin and cytokeratin-18) and increased expression of mesenchymal markers (N-cadherin and vimentin), as well as upregulation of EMT-associated transcription factors Snail, Slug, ZEB1 and ZEB2. Silencing of CTSL reversed EMT in A549/DDP and A549/TAX cells; In contrast, overexpression of CTSL induced EMT in A549 cells. In xenograft nude mouse model, the mice implanted with A549 cells overexpressing CTSL exhibited significantly reduced sensitivity to paclitaxel treatment, and increased expression of EMT-associated proteins and transcription factors in tumor tissues. CONCLUSION: Cisplatin and paclitaxel resistance is associated with CTSL upregulation-induced EMT in A549 cells. Thus, CTSL-mediated EMT may be exploited as a target to enhance the efficacy of cisplatin or paclitaxel against lung cancer and other types of malignancies.

Cathepsin L knockdown enhances curcumin-mediated inhibition of growth, migration, and invasion of glioma cells.

Curcumin can be used to prevent and treat cancer. However, its exact underlying molecular mechanisms remain poorly understood. Cathepsin L, a lysosomal cysteine protease, is overexpressed in several cancer types. This study aimed to determine the role of cathepsin L in curcumin-mediated inhibition of growth, migration, and invasion of glioma cells. Results revealed that the activity of cathepsin L was enhanced in curcumin-treated glioma cells. Cathepsin L knockdown induced by RNA interference significantly promoted curcumin-induced cytotoxicity, apoptosis, and cell cycle arrest. The knockdown also inhibited the migration and invasion of glioma cells. Our results suggested that the inhibition of cathepsin L can enhance the sensitivity of glioma cells to curcumin. Therefore, cathepsin L may be a new target to enhance the efficacy of curcumin against cancers.

Knockdown of Cathepsin L promotes radiosensitivity of glioma stem cells both in vivo and in vitro.

The presence of glioma stem cells (GSCs) in tumor is relevant for glioma treatment resistance. This study assessed whether knockdown of Cathepsin L can influence GSC growth, tumor radiosensitivity, and clinical outcome. Protein levels of Cathepsin L and stem cell markers (CD133 and Nestin) were analyzed in samples from 90 gliomas of different WHO grades and 6 normal brain tissues by immunohistochemistry. Two glioma stem cell lines with overexpressed Cathepsin L were stably transfected with Cathepsin L short hairpin RNA expression vectors. The effects of Cathepsin L inhibition on radiosensitivity, self-renewal, stemness, DNA damage, and apoptosis were evaluated. In addition, an intracranial animal model and subcutaneous tumor xenografts in nude mice were used to assess tumor response to Cathepsin L inhibition in vivo. Our results proved that expressions of Cathepsin L and CD133, but not of Nestin, correlated with malignant grades of glioma tissues. GSCs with high Cathepsin L and CD133 co-expression were extraordinarily radioresistant. Cathepsin L inhibition with radiotherapy significantly reduced GSC growth, promoted apoptosis, and improved radiosensitivity. Knockdown of Cathepsin L resulted in a dramatic reduction of CD133 expression, as well as the decreased phosphorylation of DNA repair checkpoint proteins (ATM and DNA-PKcs). Furthermore, combination of Cathepsin L inhibition and radiotherapy potently blocked tumor growth and decreased blood vessel formation in vivo. Taken together, these findings suggest Cathepsin L as a promising therapeutic target for clinical therapy in GBM patients.

A novel approach for the efficient extraction of silybin from milk thistle fruits.

BACKGROUND: Milk Thistle fruit is an important herb popularly consumed worldwide for a very long time. Silybin is the main bioactive constituent of the herb, and it has been approved by US Food and Drug Administration (FDA) as a medicine to treat liver diseases. Presently, using conventional technology, the meal of Milk Thistle fruit is used as the raw material to extract silybin. OBJECTIVE: To investigate the necessity of detaching husk from kernel of the herb and also to propose a novel approach to enhance the extraction technology in pharmaceutical practices. MATERIALS AND METHODS: The husk of Milk Thistle fruit was detached from the kernel of the herb using an automatic huller specially designed for this application. The husk and the meal of Milk Thistle fruit was subsequently refluxed, separately, with production rate of silybin as index for comparison of their extraction effect. RESULTS: The highest production rate was achieved under optimized condition. The husk was extracted 2 times (3 hrs each) using ethyl acetate, and the ratio of solvent to raw material was 8:1. The extract was allowed to be crystallized out. CONCLUSION: The separation of kernel from the husk of Milk Thistle fruit and using only the husk as raw material can largely enhance the extraction of silybin.

Matrine induction of reactive oxygen species activates p38 leading to caspase-dependent cell apoptosis in non-small cell lung cancer cells.

Non-small cell lung carcinoma (NSCLC) is one of the most refractory cancers in the clinic; it is insensitive to chemotherapy and is usually excised. However, screening natural compounds from herbs is also considered a possible method for its therapy. In the present study, we investigated whether matrine, a natural compound isolated from Sophora flavescens Ait. and exerting an inhibitory effect on lung cancer cells, also indicates inhibition on NSCLC cells and elucidated its molecular mechanism. Firstly, it is confirmed that matrine induces apoptosis of human NSCLC cells with anti-apoptotic factors inhibited and dependent on caspase activity. In addition, we found that matrine increases the phosphorylation of p38 but not its total protein, and inhibition of the p38 pathway with SB202190 partially prevents matrine-induced apoptosis. Furthermore, matrine generates reactive oxygen species (ROS) in a dose- and time-dependent manner, which is reversed by pretreatment with N-acetyl-L-cysteine (NAC). Additionally, inhibition of cell proliferation and increase of phosphorylation of p38 was also partially reversed by NAC. Collectively, matrine activates p38 pathway leading to a caspase-dependent apoptosis by inducing generation of ROS in NSCLC cells and may be a potential chemical for NSCLC.