Amentoflavone reverses epithelial-mesenchymal transition in hepatocellular carcinoma cells by targeting p53 signalling pathway axis.

Epithelial-mesenchymal transition (EMT) and its reversal process are important potential mechanisms in the development of HCC. Selaginella doederleinii Hieron is widely used in Traditional Chinese Medicine for the treatment of various tumours and Amentoflavone is its main active ingredient. This study investigates the mechanism of action of Amentoflavone on EMT in hepatocellular carcinoma from the perspective of bioinformatics and network pharmacology. Bioinformatics was used to screen Amentoflavone-regulated EMT genes that are closely related to the prognosis of HCC, and a molecular prediction model was established to assess the prognosis of HCC. The network pharmacology was used to predict the pathway axis regulated by Amentoflavone. Molecular docking of Amentoflavone with corresponding targets was performed. Detection and evaluation of the effects of Amentoflavone on cell proliferation, migration, invasion and apoptosis by CCK-8 kit, wound healing assay, Transwell assay and annexin V-FITC/propidium iodide staining. Eventually three core genes were screened, inculding NR1I2, CDK1 and CHEK1. A total of 590 GO enrichment entries were obtained, and five enrichment results were obtained by KEGG pathway analysis. Genes were mainly enriched in the p53 signalling pathway. The outcomes derived from both the wound healing assay and Transwell assay demonstrated significant inhibition of migration and invasion in HCC cells upon exposure to different concentrations of Amentoflavone. The results of Annexin V-FITC/PI staining assay showed that different concentrations of Amentoflavone induces apoptosis in HCC cells. This study revealed that the mechanism of Amentoflavone reverses EMT in hepatocellular carcinoma, possibly by inhibiting the expression of core genes and blocking the p53 signalling pathway axis to inhibit the migration and invasion of HCC cells.

Construction and validation of a nomogram for cancer specific survival of postoperative pancreatic cancer based on the SEER and China database.

BACKGROUND: The recurrence rate and mortality rate among postoperative pancreatic cancer patients remain elevated. This study aims to develop and validate the cancer-specific survival period for individuals who have undergone pancreatic cancer surgery. METHODS: We extracted eligible data from the Surveillance, Epidemiology, and End Results database and randomly divided all patients into a training cohort and an internal validation cohort. External validation was performed using a separate Chinese cohort. The nomogram was developed using significant risk factors identified through univariate and multivariate Cox proportional hazards regression. The effectiveness of the nomogram was assessed using the area under the time-dependent curve, calibration plots, and decision curve analysis. Kaplan-Meier survival curves were utilized to visualize the risk stratification of nomogram and AJCC stage. RESULTS: Seven variables were identified through univariate and multivariate analysis to construct the nomogram. The consistency index of the nomogram for predicting overall survival was 0.683 (95% CI: 0.675-0.690), 0.689 (95% CI: 0.677-0.701), and 0.823 (95% CI: 0.786-0.860). The AUC values for the 1- and 2-year time-ROC curves were 0.751 and 0.721 for the training cohort, 0.731 and 0.7554 for the internal validation cohort, and 0.901 and 0.830 for the external validation cohorts, respectively. Calibration plots demonstrated favorable consistency between the predictions of the nomogram and actual observations. Moreover, the decision curve analysis indicated the clinical utility of the nomogram, and the risk stratification of the nomogram effectively identified high-risk patients. CONCLUSION: The nomogram guides clinicians in assessing the survival period of postoperative pancreatic cancer patients, identifying high-risk groups, and devising tailored follow-up strategies.

[Study on mechanism of icariin-induced ferroptosis in HepG2 hepatoma carcinoma cells through PPARG/FABP4/GPX4 pathway].

The aim of this study was to explore the mechanism of icaritin-induced ferroptosis in hepatoma HepG2 cells. By bioinformatics screening, the target of icariin's intervention in liver cancer ferroptosis was selected, the protein-protein interaction(PPI) network was constructed, the related pathways were focused, the binding ability of icariin and target protein was evaluated by molecular docking, and the impact on patients' survival prognosis was predicted and the clinical prediction model was built. CCK-8, EdU, and clonal formation assays were used to detect cell viability and cell proliferation; colorimetric method and BODIPY 581/591 C1 fluorescent probe were used to detect the levels of Fe~(2+), MDA and GSH in cells, and the ability of icariin to induce HCC cell ferroptosis was evaluated; RT-qPCR and Western blot detection were used to verify the mRNA and protein levels of GPX4, xCT, PPARG, and FABP4 to determine the expression changes of these ferroptosis-related genes in response to icariin. Six intervention targets(AR, AURKA, PPARG, AKR1C3, ALB, NQO1) identified through bioinformatic analysis were used to establish a risk scoring system that aids in estimating the survival prognosis of HCC patients. In conjunction with patient age and TNM staging, a comprehensive Nomogram clinical prediction model was developed to forecast the 1-, 3-, and 5-year survival of HCC patients. Experimental results revealed that icariin effectively inhibited the activity and proliferation of HCC cells HepG2, significantly modulating levels of Fe~(2+), MDA, and lipid peroxidation ROS while reducing GSH levels, hence revealing its potential to induce ferroptosis in HCC cells. Icariin was found to diminish the expression of GPX4 and xCT(P<0.01), inducing ferroptosis in HCC cells, potentially in relation to inhibition of PPARG and FABP4(P<0.01). In summary, icariin induces ferroptosis in HCC cells via the PPARG/FABP4/GPX4 pathway, providing an experimental foundation for utilizing the traditional Chinese medicine icariin in the prevention or treatment of HCC.

Construction and verification of a novel circadian clock related long non-coding RNA model and prediction of treatment for survival prognosis in patients with hepatocellular carcinoma.

Circadian clock genes are significant in the occurrence and development of HCC and long-non coding RNAs (lncRNAs) are closely related to HCC progression. In this study, we aimed to establish a prognostic risk model for HCC. Circadian clock-related lncRNAs expressed in HCC were extracted from The Cancer Genome Atlas. A nomogram was established to predict individual survival rate. Biological processes enriched for risk model transcripts were investigated by Gene Set Enrichment Analysis. Further, we evaluated the relationship between risk score and immune-checkpoint inhibitor-related gene expression level. The Genomics of Drug Sensitivity in Cancer (GDSC) database was used to assess the sensitivity of tumors in high- and low-risk score groups to different drugs. A total of 11 circadian clock-related lncRNAs were included in multi-Cox proportional hazards model analysis to establish a risk model. Univariate and multivariate Cox regression analysis showed that the risk model was an independent risk factor in HCC. The risk model was a significantly associated with the immune signature. Further GDSC analysis indicated that patients in each risk score group may be sensitive to different anti-cancer drugs. QRT-PCR analysis results showed that C012073.1, PRRT3-AS1, TMCC1-AS1, LINC01138, MKLN1-AS, KDM4A-AS1, AL031985.3, POLH-AS1, LINC01224, and AC099850.3 were more highly expressed in Huh-7 and HepG2, compared to LO2, while AC008549.1 were lower expressed. Our work established a prognostic model for HCC. Risk score analysis indicated that the model is significantly associated with modulation tumor immunity and could be used to guide more effective therapeutic strategies in the future.

Comprehensive analysis of m6A regulators associated with immune infiltration in Hepatitis B virus-related hepatocellular carcinoma.

BACKGROUND: N6A methylation (m6A) is a significant epigenetic modification that critically impacts post-transcriptional regulation and tumor occurrence and development. While previous studies have identified a role for epigenetic regulation in hepatocellular carcinoma (HCC), the potential function of the m6A cluster in Hepatitis B virus (HBV)-related HCC remains unclear. METHODS: The related information was downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). Based on the expression of 20 m6A regulators, we comprehensively evaluated the m6A clusters and systematically explored the correlation between these clusters and immune cell infiltration characteristics of the tumor microenvironment (TME). The patients were divided into low- and high-m6A score groups. Then, the immune cell infiltration, chemokines, and cytokines levels, and drug sensitivity were further explored between the two groups. RESULTS: The m6A cluster predicted a better prognosis that was accompanied by increased immune cell infiltration. Using these results, an m6A score was established that could predict overall survival, immune checkpoints, and clinical treatments for patients with HBV-related HCC. This study demonstrated that m6A modifications affected tumorigenesis, TME, and the prognosis of patients with HBV-related HCC. CONCLUSION: A comprehensive assessment of m6A patterns could improve the current understanding of immune cell infiltration patterns and inform the development of individualized cancer treatments.

Brasesquilignan A-E, Five New Furofurans Lignans from Selaginella braunii Baker.

Five new furofurans lignans, Brasesquilignan A-E (1-5), were isolated from the aqueous ethanol extract of Selaginella braunii Baker. Their structures were elucidated by extensive analysis of NMR and HRESIMS data. Their absolute configurations were determined by CD spectra, enzymatic hydrolysis, and GCMS analysis. Furthermore, all compounds were evaluated for anti-proliferative activities against various human cancer cellsin vitro. Compounds 2 and 3 exhibited weak inhibitorypotency against five human cancer cells.

Circadian clock: a regulator of the immunity in cancer.

The circadian clock is an endogenous timekeeper system that controls and optimizes biological processes, which are consistent with a master circadian clock and peripheral clocks and are controlled by various genes. Notably, the disruption of circadian clock genes has been identified to affect a wide range of ailments, including cancers. The cancer-immunity cycle is composed of seven major steps, namely cancer cell antigen release and presentation, priming and activation of effector immunity cells, trafficking, and infiltration of immunity to tumors, and elimination of cancer cells. Existing evidence indicates that the circadian clock functions as a gate that govern many aspects of the cancer-immunity cycle. In this review, we highlight the importance of the circadian clock during tumorigenesis, and discuss the potential role of the circadian clock in the cancer-immunity cycle. A comprehensive understanding of the regulatory function of the circadian clock in the cancer-immunity cycle holds promise in developing new strategies for the treatment of cancer. Video Abstract.

[Clinical features and epidemiological trend of prostate cancer in the Changsha area of Hunan Province: Analysis of 2 877 cases].

OBJECTIVE: To analyze the epidemiological characteristics of PCa in the Changsha area of Hunan Province and provide some reference for the formulation of the strategies for the prevention and control of the malignancy. METHODS: We collected the data on the age, pathological type and TCM syndrome type of 2 877 PCa patients diagnosed and treated in Xiangya Hospital of Central South University, the First Affiliated Hospital of Hunan University of Chinese Medicine and the Affiliated Hospital of Hunan Research Institute of Chinese Medicine from January 1, 2010 to December 31, 2019. We analyzed the data obtained and the current prevalence and epidemiological trend of PCa. RESULTS: Of the total number of cases of PCa diagnosed and treated, there were 291 in 2010, 315 in 2011, 213 in 2012, 220 in 2013, 159 in 2014, 226 in 2015, 199 in 2016, 180 in 2017, 577 in 2018 and 497 in 2019. The age-related incidence rate was the lowest in the <40-year-olds (1.77%) and the highest in the 65- to 79-year-olds (18.4%). The incidence rate was increased year by year in the 65- to 79-year-olds, elevated to 63.9% in the 10 years, and most significantly in the ≥80-year-olds, soaring to 97.9% in the 10 years. As for the pathological types, prostatic adenocarcinoma (PAC) accounted for 50.1% (n = 1 441), acinar cell PAC 7.0% (n = 201), follicular PAC 1.29% (n = 37), ductal PCa 0.94% (n = 27), non-specific PCa 9.49% (n = 273), and other PACs 5.77% (n = 166). TCM syndrome differentiation was performed for 157 cases, which revealed kidney-yin deficiency in 40 cases (25.5%) and kidney-yang deficiency in 69 cases (43.9%). CONCLUSIONS: The incidence of PCa from 2010 to 2019 showed an aging-related trend in the Changsha area of Hunan Province, the highest among 65- to 69-year-olds. Males aged 65－79 years are a high-risk population for PCa, which calls for strengthened health education, early diagnosis and early treatment.

Development of a Nomogram Using the Inflammatory Risk Score for Prognostication in Moderate and Advanced Hepatocellular Carcinoma Associated with Hepatitis B Virus.

Background: The changes in risk scores of inflammatory markers among patients diagnosed with hepatocellular carcinoma (HCC) remain unknown. Aims: To investigate the relationship between the inflammation risk score and other contributing factors and the prognostic outcomes in patients with moderate and advanced hepatitis B virus (HBV)-related HCC. Study Design: A retrospective cohort study. Methods: A total of 174 patients with moderate and advanced HBV related HCC were recruited to investigate the impact of stratified inflammatory risk scores and other associated risk factors on disease prognosis. Based on the optimal cut-off values calculated by the Youden index, the patients were divided into high-risk and low-risk groups based on their inflammation risk scores. Results: The study found a significant difference in median survival time between the low-risk and high-risk groups based on the inflammation risk score. Furthermore, the inflammation risk score, alpha-fetoprotein levels, transarterial chemoembolization treatment, and Barcelona Clinic Liver Cancer stage were identified as independent prognostic factors. The four variables were used to construct a prognostic nomogram for HCC. Subsequent evaluations using time-dependent receiver operating characteristic analysis and calibration curve tests revealed the nomogram's commendable discriminatory ability. As a result, the nomogram proved to be an effective tool for predicting survival at 2- to 4-years. Conclusion: The inflammation risk score has been identified as a significant prognostic factor for HBV-related HCC. The development of nomogram models has provided a practical and effective tool for determining the prognosis of patients affected by HBV-related HCC.

Brazilin Actuates Ferroptosis in Breast Cancer Cells via p53/SLC7A11/GPX4 Signaling Pathway.

OBJECTIVE: To investigate the mechanism of induction of ferroptosis by brazilin in breast cancer cells. METHODS: Breast cancer 4T1 cells were divided into 6 groups: control, brazilin 1/2 half maximal inhibitory concentration (IC50), IC50, 2×IC50, erastin (10 µg/mL) and capecitabine (10 µg/mL) groups. The effect of brazilin on the proliferation of 4T1 cells was detected by cell counting kit-8 assay, and the treatment dose of brazilin was screened. The effect of brazilin on the mitochondrial morphology of 4T1 cells, and the mitochondrial damage was evaluated under electron microscopy. The levels of Fe2+, reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH) and glutathione peroxidase 4 (GPX4) were estimated using various detection kits. The invasion and migration abilities of 4T1 cells were detected by scratch assay and transwell assay. The expressions levels of tumor protein p53, solute carrier family 7 member 11 (SLC7A11), GPX4 and acyl-CoA synthetase long-chain family member 4 (ACSL4) proteins were quantified by Western blot assay. RESULTS: Compared to the control group, the 10 (1/2 IC50), 20 (IC50) and 40 (2×IC50) µg/mL brazilin, erastin, and capecitabine groups showed a significant decrease in the cell survival rate, invasion and migration abilities, GSH, SLC7A11 and GPX4 protein expression levels, and mitochondrial volume and ridge (P<0.05), and a significant increase in the mitochondria membrane density, Fe2+, ROS and MDA levels, and p53 and ACSL4 protein expression levels (P<0.05). CONCLUSIONS: Brazilin actuated ferroptosis in breast cancer cells, and the underlying mechanism is mainly associated with the p53/SLC7A11/GPX4 signaling pathway.

Polyphyllin II (PPII) Enhances the Sensitivity of Multidrug-resistant A549/DDP Cells to Cisplatin by Modulating Mitochondrial Energy Metabolism.

BACKGROUND/AIM: Cisplatin resistance often leads to treatment futility and elevated mortality rates in patients with lung cancer. One promising strategy to address this challenge involves the integration of traditional Chinese medicine (TCM) with chemotherapeutic drugs. Currently, the potential synergistic effect and underlying mechanism of polyphyllin II (PPII) and cisplatin combination in combating cisplatin (DDP) resistance in lung cancer remain unexplored. MATERIALS AND METHODS: In this study, we established a cisplatin resistance model using A549 cells and explored the underlying mechanisms of PPII in combination with cisplatin in A549/DDP resistant cells. Specifically, we assessed the impact of PPII combined with cisplatin on A549/DDP cell proliferation, viability, and the expression of apoptosis-related proteins. To gain deeper insights into the underlying mechanism, we examined the effects of PPII and cisplatin on mitochondrial function in A549/DDP cells. RESULTS: This combination induced cell cycle arrest at both the S phase and G2/M phase in A549/DDP cells, thereby promoting apoptosis. Western blotting confirmed that DDP acted synergistically with PPII to enhance the expression of apoptotic proteins, diminish the expression of anti-apoptotic proteins, and promote the expression of anti-proliferation proteins in the mitochondrial pathway of A549/DDP cells. CONCLUSION: The combination of PPII and cisplatin effectively modulated the mitochondrial function, thereby reversing drug resistance in A549/DDP cells. This innovative combination therapy shows significant promise as a novel strategy for overcoming cisplatin resistance in lung cancer.

Construction and validation of a SEER-based prognostic nomogram for young and middle-aged males patients with hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common digestive tumor, and we aimed to develop and validate nomogram models, predicting the overall survival (OS) of young and middle-aged male patients with HCC. METHODS: We extracted eligible data from relevant patients between 2000 and 2017 from the Surveillance, Epidemiology, and End Results (SEER) database. In addition, randomly divided all patients into two groups (training and validation = 7:3). The nomogram was established using effective risk factors based on univariate and multivariate analysis. The area under the time-dependent curve, calibration plots, and decision curve analysis (DCA) were used to evaluate the effective performance of the nomogram. The risk stratifications of the nomogram and the AJCC criteria-based tumor stage were compared. RESULTS: 11 variables were selected by univariate and multivariate analysis to establish the nomogram of HCC. The AUC values of 3, 4, and 5 years of the time-ROC curve are 0.858, 0.862 and 0.859 for the training cohort, and 0.858, 0.877 and 0.869 for the validation cohort, respectively, indicating that the nomogram has a good ability of discrimination. The calibration plots showed favorable consistency between the prediction of the nomogram and actual observations in both the training and validation cohorts. In addition, the decision curve DCA showed that the nomogram was clinically useful and had better discriminative ability to recognize patients at high risk than the AJCC criteria-based tumor stage. CONCLUSION: Prognostic nomogram of young and middle-aged male patients with HCC was developed and validated to help clinicians evaluate the prognosis of patients.

Construction and validation of a nomogram for hepatocellular carcinoma patients treated by traditional Chinese medicine based on inflammation, nutrition, and blood lipid indicators.

PURPOSE: To establish a nomogram for hepatocellular carcinoma (HCC) patients treated by traditional Chinese medicine (TCM). METHODS: Clinical cases of HCC patients treated by TCM at Hunan Integrated Traditional Chinese and Western Medicine Hospital, and it was randomly divided into the training cohort (n = 222) and the validation cohort (n = 95). In the training cohort, independent risk factors were determined by Cox regression analysis and a nomogram was constructed. The efficiency and clinical applicability of nomograms were evaluated using time-dependent curves, calibration, and the decision curve (DCA), and the patients were divided into high-risk, middle-risk and low-risk groups using X-tile software. RESULTS: Multivariate Cox regression analysis screened 6 independent risk factors to construct a nomogram of HCC patients, including TNM stage, treatment methods, high-density lipoprotein (HDL), neutrophil-to-lymphocyte ratio (NLR), albumin-to-globulin ratio (AGR), and prognostic nutritional index (PNI). The consistency index (C-index) of the nomogram of the training was 0.811 (0.794-0.829) and the validation cohort was 0.825 (0.800-0.849). The time dependency showed the AUC values of the nomogram for 3 and 5 years in training cohort were 0.894 (95% CI 0.840-0.948) and 0.952 (95% CI 0.914-0.990), and the validation cohort were 0.928 (95% CI 0.865-0.990) and 0.96 3(95% CI 0.916-1.010). The calibration plot showed the nomogram fits well onto perfect curves, and the DCA curve showed the net benefit of the nomogram at a certain probability threshold is significantly higher than the net benefit of the TNM stage at the same threshold probability. Finally, all the patients were divided into high-risk, middle-risk and low-risk groups based on the total score of nomogram, and it showed effectively how to identify to high-risk patients. CONCLUSION: The nomogram established by the independent risk factors of TNM stage, treatment methods, HDL, AGR, NLR and PNI can predict the prognosis of HCC patients treated by TCM, providing an effective tool to clinical workers to evaluate the prognosis and survival time of HCC patients.

Construction of an anoikis-associated lncRNA-miRNA-mRNA network reveals the prognostic role of ß-elemene in non-small cell lung cancer.

ß-Elemene is the main active ingredient in Curcumae Rhizoma that exerts antitumour effects. Anoikis affects tumour development through various biological pathways in non-small cell lung cancer (NSCLC), but the regulation between ß-elemene and anoikis remains to be explored. First, we explored the molecular expression patterns of anoikis-associated genes (AAGs) using consensus clustering and characterized the impact of AAGs on patient prognosis, clinical characteristics, and genomic instability. In addition, we revealed that AAG regulatory genes have rich interactions with ß-elemene targets, and established a lncRNA-miRNA-mRNA network to explain the effect of ß-elemene on anoikis. Finally, to reveal the prognostic effect of their correlation, the prognostic scoring model and clinical nomogram of ß-elemene and anoikis were successfully established by least absolute shrinkage and selection operator (LASSO) and random forest algorithms. This prognostic scoring model containing noncoding RNA (ncRNA) can indicate the immunotherapy and mutational landscape, providing a novel theoretical basis and direction for the study of the antitumour mechanism of ß-elemene in NSCLC patients.

Lipids, cholesterols, statins and liver cancer: a Mendelian randomization study.

Aim: To investigate the causal relationship of serum lipid indicators and lipid-lowering drugs with the risk of liver cancer using Mendelian randomization study. Methods: A two-sample Mendelian randomization (TSMR) study was performed to investigate the causal relationship between serum levels of lipid indicators and liver cancer, including low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c), triglycerides (TG), total cholesterol (TC), Apolipoprotein B (ApoB), and Apolipoprotein A1 (ApoA1).Furthermore, instrumental variable weighted regression (IVW) and summary data-based MR (SMR) analyses were performed to investigate the causal effects of lipid-lowering drugs, including statins and PCSK9 inhibitors, on the risk of liver cancer. Results: Serum LDL-c and serum TC levels showed negatively associated with liver cancer (n = 22 SNPs, OR = 0.363, 95% CI = 0.231 - 0.570; p = 1.070E-5) (n = 83 SNPs; OR = 0.627, 95% CI = 0.413-0.952; p = 0.028). However, serum levels of TG, HDL-c, and ApoA1 did not show any significant correlation with liver cancer. In the drug target MR (DMR) analyses, HMGCR-mediated level of LDL-c showed an inverse relationship with the risk of liver cancer in the IVW-MR analysis (n = 5 SNPs, OR = 0.201, 95% CI = 0.064 - 0.631; p = 5.95E-03) and SMR analysis (n = 20 SNPs, OR = 0.245, 95% CI = 0.065 - 0.926; p = 0.038) However, PCSK9 did not show any significant association with liver cancer based on both the IVW-MR and SMR analyses. Conclusion: Our results demonstrated that reduced levels of LDL-c and TC were associated with an increased risk of liver cancer. Furthermore, lipid-lowering drugs targeting HMGCR such as statins were associated with increased risk of liver cancer.

Construction and validation of a nomogram for hepatocellular carcinoma patients based on HCC-GRIm score.

PURPOSE: To construct a nomogram for hepatocellular carcinoma (HCC) patients base on HCC-GRIm score. METHODS: Clinical cases of HCC patients diagnosed at Hunan Integrated Traditional Chinese and Western Medicine Hospital were included, and these were randomly divided into the training cohort (n = 219) and the validation cohort (n = 94), and those patients were divided into low GRIm-Score group (scores 0, 1, and 2) and high GRIm-Score group (scores 3, 4, and 5). In the training cohort, independent risk factors were determined by Cox regression analysis, and a nomogram was constructed by independent risk factors. The efficiency and the clinical applicability of nomograms were evaluated using ROC curves, calibration plot, and the decision curve (DCA), and the patients were divided into high-risk, middle-risk, and low-risk groups according to total score of nomogram. RESULTS: Compared to low HCC-GRIm score group, high HCC-GRIm score group with BCLC stage is more advanced (P < 0.001), and fewer patients received TACE (P = 0.005) and surgical treatment (P = 0.001). There was higher rate of the presence of vascular invasion (P < 0.001) and distant metastasis (P < 0.001). Multivariate Cox regression analysis screened 4 independent risk factors to construct a nomogram of HCC patients, including HCC-GRIm score, BCLC stage, albumin-to-globulin ratio (AGR), and glutamyl trans-peptidase (GGT). The consistency index (C-index) of the nomogram of the training was 0.843 (0.832-0.854) and the validation was 0.870 (0.856-0.885). The time-dependent parameter showed the AUC values of the training cohort at 1, 3, and 5 years were 0.954 (95% CI 0.929-0.980), 0.952 (95% CI 0.919-0.985), and 925 (95% CI 0.871-0.979), while the AUC values of validation cohort at 1, 3, and 5 years were 0.974 (95% CI 0.950-0.998), 0.965 (95% CI 0.931-0.999), and 0.959 (95% CI 0.898-1.021). The calibration plot showed the nomogram fits well onto perfect curves, and the DCA curve showed the net benefit of the nomogram at a certain probability threshold is significantly higher than the net benefit of the BCLC stage at the same threshold probability. Finally, all patients were divided into high-risk, middle-risk, and low-risk groups based on the total score of nomogram, and it showed effectively to identify high-risk patients. CONCLUSION: The nomogram constructed by the independent risk factors can predict the prognosis of HCC patients, providing an effective tool with clinical workers to evaluate the prognosis and survival time of HCC patients.

Hederagenin Induces Apoptosis of Human Hepatoma HepG2 Cells via the Mitochondrial Pathway.

OBJECTIVE: The objective of this study is to assess the antitumor effects of hederagenin (HDG) in liver cancer (LC) cells and explore the related mechanisms. METHODS AND MATERIALS: HepG2 cells were treated with HDG and cisplatin, respectively. The CCK8 assay was used to detect cell activity, DAPI staining was used to detect the proportion of living cells, TUNEL assay to detect the proportion of apoptotic cells, flow cytometry to detect the membrane potential, fluoroscopic electron microscopy to detect microstructural changes to the mitochondrial, and western blot analysis and high-content screening to detect apoptosisrelated proteins. RESULTS: Treatment with HDG inhibited the growth of HepG2 cells, decreased the proportion of viable cells, increased the proportion of apoptotic cells, and significantly increased the proportion of cells in the G1 phase. Fluorescence staining showed that HDG damaged the mitochondria of HepG2 cells and significantly decreased the number of mitochondria. Flow cytometry showed that HDG decreased the mitochondrial membrane potential of HepG2 cells. Observations by electron microscopy showed that HDG caused swelling and vacuole formation of the mitochondria of HepG2 cells. HDG significantly reduced the average fluorescence intensity of Bcl-2 in HepG2 cells and significantly increased that of the pro-apoptosis proteins Bax, Cytochrome-c, and Caspase-3. CONCLUSION: HDG induced apoptosis of HepG2 cells via the mitochondrial pathway.

Identification and validation of a novel cuproptosis-related genes signature associated with prognosis, clinical implications and immunotherapy of hepatocellular carcinoma.

Background: Cuproptosis is a novel type of regulated cell death and is reported to promote tumor occurrence and progression. However, whether a cuproptosis-related signature has an impact on hepatocellular carcinoma (HCC) is still unclear. Materials and methods: We analyzed the transcriptome data of HCC from The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) database, and searched for tumor types with different cuproptosis patterns through consistent clustering of cuproptosis genes. We then constructed a Cuproptosis-Related Genes (CRGs)-based risk signature through LASSO COX regression, and further analyzed its impact on the prognosis, clinical characteristics, immune cell infiltration, and drug sensitivity of HCC. Results: We identified the expression changes of 10 cuproptosis-related genes in HCC, and all the patients can be divided into two subtypes with different prognosis by applying the consensus clustering algorithm. We then constructed a cuproptosis-related risk signature and identified five CRGs, which were highly correlated with prognosis and representative of this gene set, namely G6PD, PRR11, KIF20A, EZH2, and CDCA8. Patients in the low CRGs signature group had a favorable prognosis. We further validated the CRGs signature in ICGC cohorts and got consistent results. Besides, we also discovered that the CRGs signature was significantly associated with a variety of clinical characteristics, different immune landscapes and drug sensitivity. Moreover, we explored that the high CRGs signature group was more sensitive to immunotherapy. Conclusion: Our integrative analysis demonstrated the potential molecular signature and clinical applications of CRGs in HCC. The model based on CRGs can precisely predict the survival outcomes of HCC, and help better guide risk stratification and treatment strategy for HCC patients.

m6A-mediated upregulation of lncRNA-AC026356.1 promotes cancer stem cell maintenance in lung adenocarcinoma via activating Wnt signaling pathway.

LncRNA plays a pivotal role in the stemness and drug resistance of lung cancer. Here, we found that lncRNA-AC026356.1 was upregulated in stem spheres and chemo-resistant lung cancer cells. Our fish assay also shows that AC026356.1 was predominantly located in the cytoplasm of lung cancer cells and does not have protein-coding potential. Silencing AC026356.1 significantly inhibited proliferation and migration but increased apoptosis in A549-cisplatin (DDP) cells. Additionally, IGF2BP2 and the lncRNA-AC026356.1 positively regulated the proliferation and stemness of stem-like lung cancer cells. Further mechanistic investigation revealed that METTL14/IGF2BP2-mediated m6A modification and stabilization of the AC026356.1 RNA. Functional analysis corroborated that AC026356.1 acted as a downstream target of METTL14/IGF2BP2 and AC026356.1 silencing could block the oncogenicity of lung cancer stem-like cells. AC026356.1 expression was correlated with immune cell infiltration and T cell exhaustion. Compared with paired adjacent normal tissues, lung cancer specimens exhibited consistently upregulated METTL14/IGF2BP2/AC026356.1. M6A-modified METTL14/IGF2BP2/AC026356.1 loop may serve as a potential therapeutic target and prognostic predictor for lung cancer therapy and diagnosis in the clinic.

Antihepatoma peptide, scolopentide, derived from the centipede scolopendra subspinipes mutilans.

BACKGROUND: Centipedes have been used to treat tumors for hundreds of years in China. However, current studies focus on antimicrobial and anticoagulation agents rather than tumors. The molecular identities of antihepatoma bioactive components in centipedes have not yet been extensively investigated. It is a challenge to isolate and characterize the effective components of centipedes due to limited peptide purification technologies for animal-derived medicines. AIM: To purify, characterize, and synthesize the bioactive components with the strongest antihepatoma activity from centipedes and determine the antihepatoma mechanism. METHODS: An antihepatoma peptide (scolopentide) was isolated and identified from the centipede scolopendra subspinipes mutilans using a combination of enzymatic hydrolysis, a Sephadex G-25 column, and two steps of high-performance liquid chromatography (HPLC). Additionally, the CCK8 assay was used to select the extracted fraction with the strongest antihepatoma activity. The molecular weight of the extracted scolopentide was characterized by quadrupole time of flight mass spectrometry (QTOF MS), and the sequence was matched by using the Mascot search engine. Based on the sequence and molecular weight, scolopentide was synthesized using solid-phase peptide synthesis methods. The synthetic scolopentide was confirmed by MS and HPLC. The antineoplastic effect of extracted scolopentide was confirmed by CCK8 assay and morphological changes again in vitro. The antihepatoma effect of synthetic scolopentide was assessed by the CCK8 assay and Hoechst staining in vitro and tumor volume and tumor weight in vivo. In the tumor xenograft experiments, qualified model mice (male 5-week-old BALB/c nude mice) were randomly divided into 2 groups (n = 6): The scolopentide group (0.15 mL/d, via intraperitoneal injection of synthetic scolopentide, 500 mg/kg/d) and the vehicle group (0.15 mL/d, via intraperitoneal injection of normal saline). The mice were euthanized by cervical dislocation after 14 d of continuous treatment. Mechanistically, flow cytometry was conducted to evaluate the apoptosis rate of HepG2 cells after treatment with extracted scolopentide in vitro. A Hoechst staining assay was also used to observe apoptosis in HepG2 cells after treatment with synthetic scolopentide in vitro. CCK8 assays and morphological changes were used to compare the cytotoxicity of synthetic scolopentide to liver cancer cells and normal liver cells in vitro. Molecular docking was performed to clarify whether scolopentide tightly bound to death receptor 4 (DR4) and DR5. qRT-PCR was used to measure the mRNA expression of DR4, DR5, fas-associated death domain protein (FADD), Caspase-8, Caspase-3, cytochrome c (Cyto-C), B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), x-chromosome linked inhibitor-of-apoptosis protein and Cellular fas-associated death domain-like interleukin-1ß converting enzyme inhibitory protein in hepatocarcinoma subcutaneous xenograft tumors from mice. Western blot assays were used to measure the protein expression of DR4, DR5, FADD, Caspase-8, Caspase-3, and Cyto-C in the tumor tissues. The reactive oxygen species (ROS) of tumor tissues were tested. RESULTS: In the process of purification, characterization and synthesis of scolopentide, the optimal enzymatic hydrolysis conditions (extract ratio: 5.86%, IC50: 0.310 mg/mL) were as follows: Trypsin at 0.1 g (300 U/g, centipede-trypsin ratio of 20:1), enzymolysis temperature of 46 °C, and enzymolysis time of 4 h, which was superior to freeze-thawing with liquid nitrogen (IC50: 3.07 mg/mL). A peptide with the strongest antihepatoma activity (scolopentide) was further purified through a Sephadex G-25 column (obtained A2) and two steps of HPLC (obtained B5 and C3). The molecular weight of the extracted scolopentide was 1018.997 Da, and the peptide sequence was RAQNHYCK, as characterized by QTOF MS and Mascot. Scolopentide was synthesized in vitro with a qualified molecular weight (1018.8 Da) and purity (98.014%), which was characterized by MS and HPLC. Extracted scolopentide still had an antineoplastic effect in vitro, which inhibited the proliferation of Eca-109 (IC50: 76.27 µg/mL), HepG2 (IC50: 22.06 µg/mL), and A549 (IC50: 35.13 µg/mL) cells, especially HepG2 cells. Synthetic scolopentide inhibited the proliferation of HepG2 cells (treated 6, 12, and 24 h) in a concentration-dependent manner in vitro, and the inhibitory effects were the strongest at 12 h (IC50: 208.11 µg/mL). Synthetic scolopentide also inhibited the tumor volume (Vehicle vs Scolopentide, P = 0.0003) and weight (Vehicle vs Scolopentide, P = 0.0022) in the tumor xenograft experiment. Mechanistically, flow cytometry suggested that the apoptosis ratios of HepG2 cells after treatment with extracted scolopentide were 5.01% (0 µg/mL), 12.13% (10 µg/mL), 16.52% (20 µg/mL), and 23.20% (40 µg/mL). Hoechst staining revealed apoptosis in HepG2 cells after treatment with synthetic scolopentide in vitro. The CCK8 assay and morphological changes indicated that synthetic scolopentide was cytotoxic and was significantly stronger in HepG2 cells than in L02 cells. Molecular docking suggested that scolopentide tightly bound to DR4 and DR5, and the binding free energies were-10.4 kcal/mol and-7.1 kcal/mol, respectively. In subcutaneous xenograft tumors from mice, quantitative real-time polymerase chain reaction and western blotting suggested that scolopentide activated DR4 and DR5 and induced apoptosis in SMMC-7721 Liver cancer cells by promoting the expression of FADD, caspase-8 and caspase-3 through a mitochondria-independent pathway. CONCLUSION: Scolopentide, an antihepatoma peptide purified from centipedes, may inspire new antihepatoma agents. Scolopentide activates DR4 and DR5 and induces apoptosis in liver cancer cells through a mitochondria-independent pathway.