HCC Cells Research Articles

Effect of Chitosan‐Silver Nanoparticles on DEN‐induced Hepatocellular Injury by Regulation of Wnt/β‐Catenin and Apoptosis Signaling Pathways in Rats

AbstractSilver nanoparticles (AgNPs) have gained remarkable attention as therapeutic agents since the appearance of nanotechnology. This study aims to assess the protective impact of chitosan‐silver nanoparticles (CS‐AgNPs) against DEN‐induced hepatocellular injury by regulation of Wnt/β‐Catenin and apoptosis signaling pathways in rats. CS‐AgNPs solutions were prepared by mixing different concentrations of chitosan and silver nitrate, and then subjected to gamma radiation at 20 kGy. The formation of CS‐AgNPs could be observed by naked eyes from the colour change from light yellow to reddish‐brown, and confirmed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Stability measurements confirmed that the prepared Cs‐AgNPs are highly stable for around 8 weeks. A crystal violet cytotoxicity assay of CS‐AgNPs has been detected in vitro on HepG2 hepatocellular carcinoma cell lines. The obtained results confirmed the selective influence of CS‐AgNPs on the HCC cell line in a time and dose dependent manner. Diethylnitrosamine (DEN) induced a marked increase in the expressions of Wnt, β‐Catenin, STAT3, MMP9, and Bcl‐2 with a significant decrease in the expression of BAX, p53, and P21. Additionally, DEN significantly decreased the levels of Cytc and TPTH while significantly increased the levels of COX‐2, PGE2, TGF‐1, AFP, and liver enzyme activity. The role of CS‐AgNPs is documented in diminishing the expressions of Wnt, β‐Catenin, STAT3, MMP9, and Bcl‐2. Also, CS‐AgNPs induced the decrease in the levels of COX‐2, PGE2, TGF‐1, AFP and liver enzyme activity accompanied by increased apoptosis. These results concluded that CS‐AgNPs might be a promising drug designed for treating liver diseases.

Histone Deacetylase 6 Inhibitor 5-Phenylcarbamoylpentyl Selenocyanide (SelSA) Suppresses Hepatocellular Carcinoma by Downregulating Phosphorylation of the Extracellular Signal-Regulated Kinase 1/2 Pathway.

Histone deacetylase 6 (HDAC6) enzyme plays a crucial role in a variety of cellular processes related to cancer, and inhibition of HDAC6 is emerging as an effective strategy for cancer treatment. Although several hydroxamate-based HDAC6 inhibitors showed promising anticancer activities, the intrinsic defects such as poor selectivity, stability, and pharmacokinetics limited their application. In this study, a potent selenocyanide-bearing HDAC6 inhibitor, 5-phenylcarbamoylpentyl selenocyanide (SelSA), was evaluated for its antihepatocellular carcinoma (HCC) activity and further explored for its antitumor mechanisms. In vitro studies demonstrated that SelSA exhibited excellent antiproliferative activity against three HCC cells HepG2 (2.3 ± 0.29 μM), Huh7 (0.83 ± 0.48 μM), and LM3 (2.6 ± 0.24 μM). Further studies indicated that SelSA could downregulate the expression of extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, inhibit the growth, invasion, and migration of Huh7 cells, and promote their apoptosis. Moreover, SelSA significantly suppressed tumor growth in Huh7 xenograft mouse models. Our findings suggest that SelSA could be a potential therapeutic agent for HCC.

Plant-derived extracellular nanovesicles: a promising biomedical approach for effective targeting of triple negative breast cancer cells.

Introduction: Triple negative breast cancer (TNBC), a highly aggressive subtype accounting for 15-20% of all breast cancer cases, faces limited treatment options often accompanied by severe side effects. In recent years, natural extracellular nanovesicles derived from plants have emerged as promising candidates for cancer therapy, given their safety profile marked by non-immunogenicity and absence of inflammatory responses. Nevertheless, the potential anti-cancer effects of Citrus limon L.-derived extracellular nanovesicles (CLENs) for breast cancer treatment is still unexplored. Methods: In this study, we investigated the anti-cancer effects of CLENs on two TNBC cell lines (4T1 and HCC-1806 cells) under growth conditions in 2D and 3D culture environments. The cellular uptake efficiency of CLENs and their internalization mechanism were evaluated in both cells using confocal microscopy. Thereafter, we assessed the effect of different concentrations of CLENs on cell viability over time using a dual approach of Calcein-AM PI live-dead assay and CellTiter-Glo bioluminescence assay. We also examined the influence of CLENs on the migratory and evasion abilities of TNBC cells through wound healing and 3D Matrigel drop evasion assays. Furthermore, Western blot analysis was employed to investigate the effects of CLENs on the phosphorylation levels of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and extracellular signal- regulated kinase (ERK) expression. Results: We found that CLENs were internalized by the cells via endocytosis, leading to decreased cell viability, in a dose- and time-dependent manner. Additionally, the migration and evasion abilities of TNBC cells were significantly inhibited under exposed to 40 and 80μg/mL CLENs. Furthermore, down-regulated expression levels of phosphorylated phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and extracellular signal-regulated kinase (ERK), suggesting that the inhibition of cancer cell proliferation, migration, and evasion is driven by the inhibition of the PI3K/AKT and MAPK/ERK signaling pathways. Discussion: Overall, our results demonstrate the anti-tumor efficiency of CLENs against TNBC cells, highlighting their potential as promising natural anti-cancer agents for clinical applications in cancer treatment.

T cell-related ubiquitination genes as prognostic indicators in hepatocellular carcinoma.

T lymphocytes, integral to the adaptive immune system, wield pivotal influence in bolstering anti-tumor responses, and are strictly regulated by ubiquitination modification. The objective of this investigation was to devise a novel prognostic and immunotherapeutic efficacy predictor for hepatocellular carcinoma patients utilizing T cell-related ubiquitination genes (TCRUG). The single-cell RNA sequencing (scRNA-seq) data and bulk RNA data of HCC patients are derived from the GEO database and TCGA database. Based on the processing of scRNA-seq, T cell marker genes are obtained and TCRUG is obtained. Further combined with WGCNA, differential analysis, univariate Cox regression analysis, LASSO analysis, and multivariate Cox regression analysis to filter and screen TCRUG. Finally construct a riskscore for predicting the prognosis of HCC patients, the predictive effect of which is validated in the GEO dataset. In addition, we also studied the correlation between riskscore and immunotherapy efficacy. Finally, the oncogenic role of UBE2E1 in HCC was explored through various in vitro experiments. Based on patients' scRNA-seq data, we finally obtained 3050 T cell marker genes. Combined with bulk RNA data and clinical data from the TCGA database, we constructed a riskscore that accurately predicts the prognosis of HCC patients. This riskscore is an independent prognostic factor for HCC and is used to construct a convenient column chart. In addition, we found that the high-risk group is more suitable for immunotherapy. Finally, the proliferation, migration, and invasion abilities of HCC cells significantly decreased after UBE2E1 expression reduction. This study developed a riskscore based on TCRUG that can accurately and stably predict the prognosis of HCC patients. This riskscore is also effective in predicting the immune therapy response of HCC patients.

WEE Family Kinase Inhibitors Combined with Sorafenib Can Selectively Inhibit HCC Cell Proliferation

Background: Sorafenib is currently the first choice for the treatment of patients with advanced hepatocellular carcinoma, but its therapeutic effect is still limited. Objectives: This study aims to examine whether WEE family kinase inhibitors can enhance the anticancer effect of sorafenib. Methods: We analyzed the expression levels of PKMYT1 kinase and WEE1 kinase in HCC, studied the inhibitory effect of PKMYT1 kinase inhibitor RP-6306, WEE1 kinase inhibitor adavosertib combined with sorafenib on the proliferation of HCC cells, and detected the effect of drug combination on CDK1 phosphorylation. Results: We found that PKMYT1 and WEE1 were upregulated in HCC and were detrimental to patient survival. Cell experiments showed that both RP-6306 and adavosertib (1-100 μM) inhibited the proliferation of HCC cell lines in a dose-dependent manner alone, and the combination of the two drugs had a synergistic effect. In HCC cell lines, sorafenib combined with RP-6306 or adavosertib showed a synergistic antiproliferation effect and less toxicity to normal cells. Sorafenib combined with RP-6306 and adavosertib further inhibited the proliferation of HCC cells and caused complete dephosphorylation of CDK1. Conclusion: Taken together, our findings provide experimental evidence for the future use of sorafenib in combination with RP-6306 or adavosertib for the treatment of HCC.

A novel single-cell model reveals ferroptosis-associated biomarkers for individualized therapy and prognostic prediction in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is a prevalent malignancy with a pressing need for improved therapeutic response and prognosis prediction. This study delves into a novel predictive model related to ferroptosis, a regulated cell death mechanism disrupting metabolic processes.ResultsSingle-cell sequencing data analysis identified subpopulations of HCC cells exhibiting activated ferroptosis and distinct gene expression patterns compared to normal tissues. Utilizing the LASSO-Cox algorithm, we constructed a model with 10 single-cell biomarkers associated with ferroptosis, namely STMN1, S100A10, FABP5, CAPG, RGCC, ENO1, ANXA5, UTRN, CXCR3, and ITM2A. Comprehensive analyses using these biomarkers revealed variations in immune infiltration, tumor mutation burden, drug sensitivity, and biological functional profiles between risk groups. Specific associations were established between particular immune cell subtypes and certain gene expression patterns. Treatment response analyses indicated potential benefits from anti-tumor immune therapy for the low-risk group and chemotherapy advantages for the high-risk group.ConclusionsThe integration of this single-cell level model with clinicopathological features enabled accurate overall survival prediction and effective risk stratification in HCC patients. Our findings illuminate the potential of ferroptosis-related genes in tailoring therapy and prognosis prediction for HCC, offering novel insights into the intricate interplay among ferroptosis, immune response, and HCC progression.

Kaempferol from Alpinia officinarum hance induces G2/M cell cycle arrest in hepatocellular carcinoma cells by regulating the ATM/CHEK2/KNL1 pathway

Ethnopharmacological relevanceAlpinia officinarum Hance (A. officinarum), a perennial herb known for its medicinal properties, has been used to treat various ailments, such as stomach pain, abdominal pain, emesis, and digestive system cancers. A. officinarum is extensively cultivated in the Qiongzhong and Baisha regions of Hainan, and it holds substantial therapeutic value for the local Li people of Hainan. Kaempferol, a flavonoid derived from A. officinarum, has demonstrated anticancer properties in various experimental and biological studies. Nevertheless, the precise mechanisms through which it exerts its anti-hepatocellular carcinoma (HCC) effects remain to be comprehensively delineated. Aim of the studyThis investigation aims to elucidate the anti-HCC effects of kaempferol derived from A. officinarum and to delve into its underlying mechanistic pathways. Materials and methodsUsing ultra-high performance liquid chromatography-mass spectrometry/mass spectrometry (UPLC-MS/MS) to identify active compounds in A. officinarum. HCCLM3 and Huh7 cells were used to study the anti-HCC effect of kaempferol from A. officinarum. The cytotoxicity and proliferation of kaempferol and A. officinarum were measured using CCK-8 and EDU staining. Wound-healing assays and three-dimensional tumor spheroid models were further used to evaluate migration and the anti-HCC activity of kaempferol. The cell cycle and apoptosis were evaluated by flow cytometry. Western blot and qRT-PCR were used to detect the expression of proteins and genes associated with the cell cycle checkpoints. Finally, bioinformatics was used to analyze the relationship between the differential expression of core targets in the ATM/CHEK2/KNL1 pathway and a poor prognosis in clinical HCC samples. ResultsUPLC-MS/MS was employed to detect five active compounds in A. officinarum, such as kaempferol. The CCK-8 and EDU assays showed that kaempferol and A. officinarum significantly inhibited the proliferation of HCC cells. A wound-healing assay revealed that kaempferol remarkably inhibited the migration of HCC cells. Kaempferol significantly suppressed the growth of tumor spheroids. In addition, kaempferol markedly induced G2/M arrest and promoted apoptosis of HCC cells. Mechanically, kaempferol significantly reduced the protein and mRNA expression levels of ATM, CHEK2, CDC25C, CDK1, CCNB1, MPS1, KNL1, and Bub1. Additionally, the combination of kaempferol and the ATM inhibitor KU55933 had a more significant anti-HCC effect. The results of bioinformatics showed that ATM, CHEK2, CDC25C, CDK1, and KNL1 were highly expressed in patients with HCC and cancer tissues, indicating that these genes have certain value in the clinical diagnosis of HCC. ConclusionsCollectively, our results revealed that kaempferol from A. officinarum inhibits the cell cycle by regulating the ATM/CHEK2/KNL1 pathway in HCC cells. In summary, our research presents an innovative supplementary strategy for HCC treatment.

Elevated SLC1A5 associated with poor prognosis and therapeutic resistance to transarterial chemoembolization in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is a common malignant tumor, and glutamine is vital for tumor cells. The role of glutamine transporter SLC1A5 in tumor progression and transarterial chemoembolization (TACE) efficacy is under study. This research seeks to determine the impact of SLC1A5 expression on the prognosis and TACE efficacy of HCC and elucidate its mechanisms.MethodsSLC1A5 expression in HCC, correlation with patient outcomes, and response to TACE were studied in an open access liver cancer dataset and confirmed in our cohort. Additionally, the correlation between SLC1A5 expression and hypoxia, angiogenesis and immune infiltration was analyzed and verified by immunohistochemistry, immunofluorescence and transcriptome sequencing. Liver cancer cell lines with SLC1A5 expression knockdown or overexpression were constructed, and cell proliferation, colony formation, apoptosis, migration and drug sensitivity as well as in vivo xenograft tumor were measured. A gene set enrichment analysis was conducted to determine the signaling pathway influenced by SLC1A5, and a western blot analysis was performed to detect protein expression alterations.ResultsSLC1A5 expression was higher in HCC tissue and associated with poor survival and TACE resistance. Hypoxia could stimulate the upregulation of glutamine transport, angiogenesis and SLC1A5 expression. The SLC1A5 expression was positively correlated with hypoxia and angiogenesis-related genes, immune checkpoint pathways, macrophage, Tregs, and other immunosuppressive cells infiltration. Knockdown of SLC1A5 decreased proliferation, colony formation, and migration, but increased apoptosis and increased sensitivity to chemotherapy drugs. Downregulation of SLC1A5 resulted in a decrease in Vimentin and N-cadherin expression, yet an increase in E-cadherin expression. Upregulation of SLC1A5 increased Vimentin and N-cadherin expression, while decreasing E-cadherin. Overexpression of β-catenin in SLC1A5-knockdown HCC cell lines could augment Vimentin and N-cadherin expression, suppress E-cadherin expression, and increase the migration and drug resistance.ConclusionsElevated SLC1A5 was linked to TACE resistance and survival shortening in HCC patients. SLC1A5 was positively correlated with hypoxia, angiogenesis, and immunosuppression. SLC1A5 may mediate HCC cell migration and drug resistance via Epithelial-mesenchymal transition (EMT) pathway.

Identifying disulfidptosis subtypes in hepatocellular carcinoma through machine learning and preliminary exploration of its connection with immunotherapy

BackgroundHepatocellular carcinoma (HCC) is a highly prevalent and deadly cancer, with limited treatment options for advanced-stage patients. Disulfidptosis is a recently identified mechanism of programmed cell death that occurs in SLC7A11 high-expressing cells due to glucose starvation-induced disintegration of the cellular disulfide skeleton. We aimed to explore the potential of disulfidptosis, as a prognostic and therapeutic marker in HCC.MethodsWe classified HCC patients into two disulfidptosis subtypes (C1 and C2) based on the transcriptional profiles of 31 disulfrgs using a non-negative matrix factorization (NMF) algorithm. Further, five genes (NEIL3, MMP1, STC2, ADH4 and CFHR3) were screened by Cox regression analysis and machine learning algorithm to construct a disulfidptosis scoring system (disulfS). Cell proliferation assay, F-actin staining and PBMC co-culture model were used to validate that disulfidptosis occurs in HCC and correlates with immunotherapy response.ResultsOur results suggests that the low disulfidptosis subtype (C2) demonstrated better overall survival (OS) and progression-free survival (PFS) prognosis, along with lower levels of immunosuppressive cell infiltration and activation of the glycine/serine/threonine metabolic pathway. Additionally, the low disulfidptosis group showed better responses to immunotherapy and potential antagonism with sorafenib treatment. As a total survival risk factor, disulfS demonstrated high predictive efficacy in multiple validation cohorts. We demonstrated the presence of disulfidptosis in HCC cells and its possible relevance to immunotherapeutic sensitization.ConclusionThe present study indicates that novel biomarkers related to disulfidptosis may serve as useful clinical diagnostic indicators for liver cancer, enabling the prediction of prognosis and identification of potential treatment targets.Graphical

The petroleum ether extracts of Chloranthus fortunei(A. Gray) Solms-Laub.with bioactivities: A rising source in HCC drug treatment

Ethnopharmacological relevanceHepatocellular Carcinoma (HCC) is an aggressive killer worldwide with high incidence and mortality. The herb Chloranthus fortunei (A. Gray) Solms-Laub is known as “Si Ji Feng” and is classified as a Feng-type medicine in classic Yao medicines. According to Yao's medical beliefs, Chloranthus fortunei has the functions of dispelling Feng, regulating qi, detoxifying, promoting blood circulation, etc. Folk uses its decoctions to treat stagnant liver conditions, such as liver abscesses, cirrhosis, hepatitis, and liver cancer. However, the bioactivity and mechanisms of Chloranthus fortunei extract against HCC have not been reported. Aim of the studyTo investigate the anti-HCC bioactivity and potential mechanism of the extract of Chloranthus fortunei (CFS). Materials and methodsUsing 70% ethanol for reflux extraction of CFS resulted in the CFS ethanol extract, followed by sequential extractions with petroleum ether, chloroform, ethyl acetate, and n-butanol, yielding four fractions. The CCK-8 assay was utilized to examine the cytotoxic effects of 4 fractions on MHCC97-H and HepG2 cells, exploring the most effective component, namely petroleum ether extracts of CFS (PECFS). The major active ingredients of PECFS were identified using LC/MS technology, and the impact on cell proliferation and apoptosis in HCC cells was studied. The key genes and proteins in the pathway were validated using RT-PCR and Western blotting. BALB/c nude mice were chosen for tumor xenotransplantation and PECFS therapy. hinders the proliferation of HCC cells and promotes apoptosis. ResultsAmong the four fractions, it was found that PECFS have the highest antiproliferative activity against MHCC97-H and HepG2 cells (IC50 = 13.86, 10.55 μg/mL), with sesquiterpene compounds being the primary active constituents. The antiproliferative activity of PECFS on HCC cells was linked to the inhibition of cell cloning, invasion, and metastasis abilities, as well as the arrest of the cell cycle at the G2/M phase. Additionally, exerts pro-apoptotic effects on HCC cells by upregulating the pro-apoptotic protein Bax, downregulating the anti-apoptotic protein Bcl-2, and activating the expression of the Caspase family. Moreover, protein and m-RNA expression data showed that PECFS inhibits HCC cell proliferation and promotes apoptosis by regulating the PI3K/AKT/mTOR pathway. Besides, after PECFS treatment, tumor growth in nude mice was suppressed. ConclusionPECFS can inhibit the viability of HCC cells by acting on the PI3K/AKT/mTOR pathway, demonstrating anti-tumor potential. This study's findings suggest that PECFS may represent a promising source of novel agents for liver cancer treatment, providing scientific evidence for the traditional application of CFS in treating HCC.

CircGNAO1 strengthens its host gene GNAO1 expression for suppression of hepatocarcinogenesis

BackgroundHepatocellular carcinoma (HCC) is one of the most prevalent primary liver carcinoma. Guanine nucleotide-binding protein, α-activating activity polypeptide O (GNAO1) was reported to be under-expressed in HCC tissues. This study aimed to investigate the GNAO1-derived circular RNA (circRNA) and its molecular mechanisms in HCC. MethodsReal-time quantitative polymerase chain reaction (RT-qPCR) and Western blot were applied to examine RNA and protein levels. Functional experiments were performed to study HCC cell proliferation, cell cycle and cellular senescence. The interactions among circGNAO1, GNAO1 and DNA methyltransferase 1 (DNMT1) were examined by mechanism assays. The methylation level was analyzed by bisulfite sequencing PCR (BSP). ResultsCircGNAO1 is down-regulated and positively associated with GNAO1 in HCC tissues. Overexpression of circGNAO1 inhibits cell proliferation, induces cell cycle arrest and facilitates cell senescence in HCC cells. CircGNAO1 facilitates the progression of HCC via modulating GNAO1. Mechanistically, circGNAO1 enhances the transcription of GNAO1 by sequestering DNMT1, thereby up-regulating GNAO1 expression in HCC cells. ConclusionsCircGNAO1 up-regulates its host gene GNAO1 expression for suppression of hepatocarcinogenesis.

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.

Effect of transcription factor WT1 on triple-negative breast cancer metastasis through PFKFB4-mediated glycolysis.

e13145 Background: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with a high probability of metastasis as well as a lack of specific targets and targeted therapeutics. Preliminary study suggested that Wilms' tumor gene 1 (WT1) is highly expressed in breast cancer patients with poor prognosis and may promote TNBC metastasis, but the underlying mechanism remains poorly defined. Methods: WT1 expression was evaluated by immunohistochemistry (IHC) in breast cancer patients. Kaplan–Meier survival analysis was performed to assess the prognostic significance of WT1 expression. WT1 was silenced in MDA-MB-231 and BT549 cells or overexpressed in HCC1806 cells. qRT-PCR and Western blot were used to detect the WT1 expression in tissues and cells. Wound healing assays, transwell assays and 3D spheroid assays were used to examine the migration and invasion abilities in TNBC cells. The lung metastasis model of mice was used to evaluate metastasis of TNBC in vivo. Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) and transcriptome sequencing (RNA-seq) were performed to find PFKFB4, a downstream target gene for WT1. ChIP-PCR and dual-luciferase reporter assays were used to explore the transcriptional regulation of PFKFB4 by WT1. Seahorse XF glycolysis stress assay, glucose uptake assay, and lactate production assay were used to investigate the role of WT1 in regulating glycolysis metabolites. Results: WT1 was highly expressed in TNBC and correlated to poor prognosis in TNBC patients. Functional assays revealed that WT1 promoted TNBC cell metastasis in vitro and in vivo. Our mechanistic investigations demonstrated that WT1 promoted TNBC cells migration and invasion by transcriptionally activating the expression of PFKFB4.This action leaded to increased glycolytic capacity, glucose uptake, and lactate production in cancer cells, therefore promoting metastasis of TNBC. Clinically, the combined expression of WT1 and PFKFB4 provides a reliable predictive biomarker for the prognosis of TNBC patients. Conclusions: Our findings reveal a molecular mechanism of WT1 promoting TNBC metastasis, which provides new targets for the precision treatment of TNBC and new perspectives for the development of targeted metabolic anticancer drugs.

Low protein expression of LZTR1 in hepatocellular carcinoma triggers tumorigenesis via activating the RAS/RAF/MEK/ERK signaling

LZTR1 is a substrate specific adaptor for E3 ligase involved in the ubiquitination and degradation of RAS GTPases, which inhibits the RAS/RAF/MEK/ERK signaling to suppress the pathogenesis of Noonan syndrome and glioblastoma. However, it's still unknown whether LZTR1 destabilizes RAS GTPases to suppress HCC progression by inhibiting these signaling pathway. Lenvatinib is the first-line drug for the treatment of advanced HCC, however, it has high drug resistance. To explore the roles of LZTR1 in HCC progression and the underlying mechanisms of lenvatinib resistance, techniques such as bioinformatics analysis, immunohistochemical staining, RT-qPCR, Western blot, cell functional experiments, small interfering RNA transfection and cycloheximide chase assay were applied in our study. Among these, bioinformatics analysis and immunohistochemical staining results indicated that LZTR1 protein was aberrantly expressed at low levels in HCC tissues, and low protein expression of LZTR1 was associated with poor prognosis of HCC patients. In vitro functional experiments confirmed that low expression of LZTR1 promoted HCC cell proliferation and migration via the aberrant activation of the RAS/RAF/MEK/ERK signaling due to the dysregulation of LZTR1-induced KRAS ubiquitination and degradation. Transwell assays revealed that blocking of LZTR1-mediated KRAS degradation could induce lenvatinib resistance in HCC cells. In conclusion, our study revealed that LZTR1 knockdown promoted HCC cell proliferation and migration, and induced lenvatinib resistance via activating the RAS/RAF/MEK/ERK signaling, which may provide new ideas for HCC treatment.

Anticancer Activity of HER2-targeting CPP-PTEN-THP Chimeric Proteins.

Protein phosphatase and tensin homolog (PTEN) is a tumor suppressor protein with potential to be a new biotechnological drug for PTEN-deficient cancer treatment. This study aimed to develop PTEN-based chimeric proteins (CPP-PTEN-THP) for human epidermal growth factor receptor 2 (HER2)-positive breast cancer treatment, addressing current limitations like inadequate delivery, poor tumor penetration, and low selectivity, while assessing their potential HER2-specific anticancer effects. pCEFL-EGFP vector was used for both TAT-PTEN-LTV and KLA-PTEN-LTV construction. Non-contact co-cultures were employed using HEK-293T cells for protein expression, and HCC-1954 and MCF-7 cell lines for cytotoxicity testing. Protein detection was analyzed by western blotting and a docking prediction analysis was performed to infer the interactions. Endogenous and recombinant PTEN protein expression was confirmed in cell lysates. A 54-kDa signal matching the theoretical size of PTEN was detected, showing a greater level in TAT-PTEN-LTV (215.1±26.45%) and KLA-PTEN-LTV (129.2±1.44%) compared to endogenous PTEN. After the noncontact co-culture method, cytotoxic studies showed HCC-1954 preferential cell inhibition growth, with 25.95±0.9% and 12.25±1.29% inhibition by KLA-PTEN-LTV and TAT-PTEN-LTV respectively, compared to MCF-7 cells. An LTV-HER2 interaction model was proposed, inferring that LTV interactions are mainly due to the Pro, Trp, and Tyr residues that target HER2. The developed PTEN-based chimeric proteins have HER2-specific anticancer activity against HCC-1954 cells.

Molecular characterization of breast cancer cell lines in correlation with clinical markers

Cell lines are indispensable for biomedical research, offering a consistent platform for investigating cellular processes and disease mechanisms. Despite their value, cell lines possess limitations such as genetic drift and phenotypic alterations over successive passages, potentially compromising their relevance. Monoclonal derivation may not represent in vivo heterogeneity, particularly in cancer. Therefore, characterizing them becomes imperative. We aimed to identify breast cancer subtype-specific cell lines by assessing ER, PR, and HER-2 expression via immunohistochemistry and their concordance with molecular expression. We selected cell lines with consistent phenotypes that mimic primary cells and confirmed concordance in ER, PR, and HER-2 expression at protein and mRNA levels. Evaluation of ER, PR, and HER-2 was conducted using Immunocytochemistry (ICC), Immunofluorescence (IF), and real-time PCR across commonly used breast cancer cell lines. Results revealed differential expression patterns, with MCF-7 and T-47D cells showing positive ER and PR staining but negative HER-2 staining, while SK-BR-3 and HCC1954 cells exhibited positive HER-2 staining and negative ER and PR staining. Conversely, MDA-MB-231 and MDA-MB-468 cells displayed negative staining for all three markers. Real-time PCR analysis highlighted variations in HER-2 expression among the cell lines. Our investigation underscores MCF-7, SK-BR-3, MDA-MB-231, and MDA-MB-468 cell lines as pertinent models for studying major molecular subtypes of breast cancer.

EDI3 knockdown in ER-HER2+ breast cancer cells reduces tumor burden and improves survival in two mouse models of experimental metastasis

BackgroundDespite progress understanding the mechanisms underlying tumor spread, metastasis remains a clinical challenge. We identified the choline-producing glycerophosphodiesterase, EDI3 and reported its association with metastasis-free survival in endometrial cancer. We also observed that silencing EDI3 slowed cell migration and other cancer-relevant phenotypes in vitro. Recent work demonstrated high EDI3 expression in ER-HER2+ breast cancer compared to the other molecular subtypes. Silencing EDI3 in ER-HER2+ cells significantly reduced cell survival in vitro and decreased tumor growth in vivo. However, a role for EDI3 in tumor metastasis in this breast cancer subtype was not explored. Therefore, in the present work we investigate whether silencing EDI3 in ER-HER2+ breast cancer cell lines alters phenotypes linked to metastasis in vitro, and metastasis formation in vivo using mouse models of experimental metastasis.MethodsTo inducibly silence EDI3, luciferase-expressing HCC1954 cells were transduced with lentiviral particles containing shRNA oligos targeting EDI3 under the control of doxycycline. The effect on cell migration, adhesion, colony formation and anoikis was determined in vitro, and significant findings were confirmed in a second ER-HER2+ cell line, SUM190PT. Doxycycline-induced HCC1954-luc shEDI3 cells were injected into the tail vein or peritoneum of immunodeficient mice to generate lung and peritoneal metastases, respectively and monitored using non-invasive bioluminescence imaging. Metabolite levels in cells and tumor tissue were analyzed using targeted mass spectrometry and MALDI mass spectrometry imaging (MALDI-MSI), respectively.ResultsInducibly silencing EDI3 reduced cell adhesion and colony formation, as well as increased susceptibility to anoikis in HCC1954-luc cells, which was confirmed in SUM190PT cells. No influence on cell migration was observed. Reduced luminescence was seen in lungs and peritoneum of mice injected with cells expressing less EDI3 after tail vein and intraperitoneal injection, respectively, indicative of reduced metastasis. Importantly, mice injected with EDI3-silenced cells survived longer. Closer analysis of the peritoneal organs revealed that silencing EDI3 had no effect on metastatic organotropism but instead reduced metastatic burden. Finally, metabolic analyses revealed significant changes in choline and glycerophospholipid metabolites in cells and in pancreatic metastases in vivo.ConclusionsReduced metastasis upon silencing supports EDI3’s potential as a treatment target in metastasizing ER-HER2+ breast cancer.

Overexpression of PLK1 Molecule Following Incomplete Thermal Ablation Promotes the Proliferation and Invasion of Residual Hepatocellular Carcinoma.

TAT, a widely used treatment for HCC, can exacerbate the progression of residual HCC. The present study investigated the mechanism of action of PLK1 following ITA of HCC. The PLK1 levels in HCC were determined using qRT-PCR from clinical patient samples, IHC from tissue microarray, and data from globally high-throughput data and microarrays. The PLK1 levels and their effect on the biological phenotype of heat-stress HCC cells were evaluated through in vitro experiments. We detected PLK1 abnormal expression in HCC models of nude mice subjected to ITA. We detected the effects of different PLK1 expression levels on EMT pathway proteins. PLK1 exhibited an overexpression in HCC tissues with an SMD of 1.19 (3414 HCC and 3036 non-HCC tissues were included), distinguishing HCC from non-HCC effectively (AUC = 0.9). The qRT-PCR data from clinical HCC patient samples and IHC from HCC tissue microarray results also indicated an overexpressed level. In the incomplete ablation models, an increased PLK1 expression was found in both heat-stress cells and subcutaneous tumors. The upregulation of PLK1 following ITA was found to enhance the malignancy of HCC and exacerbate the proliferation, migration, and invasion of residual HCC cells, whereas PLK1 knockdown suppressed the biological malignancy of HCC cells. Meanwhile, PLK1 has different regulatory effects on various EMT pathway proteins. PLK1 promotes the progression of residual HCC by activating EMT pathway after ITA, which might provide a novel idea for the treatment and prognosis of residual HCC.

Evaluation of drug resistance for EGFR-TKIs in lung cancer via multicellular lung-on-a-chip

Drug resistance to irreversible epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is a primary factor affecting their therapeutic efficacy in human non-small cell lung cancer (NSCLC). NSCLC cells can undergo epithelial-mesenchymal transition (EMT) induced by many factors in the tumour microenvironment (TME), which plays a crucial role in tumour drug resistance. In this study, a multicellular lung-on-a-chip that can realise the cell co-culture of the human non-small cell lung cancer cell line HCC827, human foetal lung fibroblasts (HFL-1), and human umbilical vein endothelial cells (HUVECs) is prepared. The TME was simulated on the chip combined with perfusion and other factors, and the drug evaluation of osimertinib was performed to explore the drug resistance mechanism of EGFR-TKIs. In the early stages, a two-dimensional static cell co-culture was achieved by microchip, and the results showed that HFL-1 cells could be transformed into cancer-associated fibroblasts (CAFs), and HCC827 cells could undergo EMT, both of which were mediated by Interleukin-6 (IL-6). Vimentin (VIM) and Alpha Skeletal Muscle Actin (a-SMA) expression of HFL-1 was upregulated, whereas E-cadherin (E-cad) expression of HCC827 was down-regulated. Further, N-cadherin (N-cad) expression of HCC827 was upregulated. In both the static cell co-culture and multicellular lung-on-a-chip, HCC827 cells with CAFs co-culture or IL-6 treatment developed resistance to osimertinib. Further use of the IL-6 antibody inhibitor tocilizumab could reverse EGFR-TKI resistance to a certain extent. Combination therapy with tocilizumab and EGFR-TKIs may provide a novel therapeutic strategy for overcoming EGFR-TKI resistance caused by EMT in NSCLC. Furthermore, the lung-on-a-chip can simulate complex TME and can be used for evaluating tumour resistance and exploring mechanisms, with the potential to become an important tool for personalised diagnosis, treatment, and biomedical research.

Discovery of novel EGFR-PROTACs capable of degradation of multiple EGFR-mutated proteins

Although three generations of Epidermal growth factor receptor (EGFR) - TK inhibitors have been approved for the treatment of Non-small-cell lung cancers (NSCLC), their clinical application is still largely hindered by acquired drug resistance mediated new EGFR mutations and side effects. The Proteolysis targeting chimera (PROTAC) technology has the potential to overcome acquired resistance from mutant EGFR through a novel mechanism of action. In this study, we developed the candidate degrader IV-3 by structural modifications of the lead compound 13, which exhibited limited antiproliferative activity against HCC-827 cells. Compared to compound 13, IV-3 exhibited remarkable anti-proliferative activity against HCC-827 cells, NCI–H1975 cells, and NCI–H1975-TM cells (IC50 = 0.009 μM, 0.49 μM and 3.24 μM, respectively), as well as significantly inducing degradation of EGFR protein in these cell lines (DC50 = 17.93 nM, 0.25 μM and 0.63 μM, respectively). Further investigations confirmed that IV-3 exhibited superior anti-tumor activity in all xenograft tumor models through the degradation of mutant EGFR protein. Moreover, IV-3 showed no inhibitory activity against A431 and A549 cells expressing wild-type EGFR, thereby eliminating potential toxic side effects emerging from wild-type EGFR inhibition. Overall, our study provides promising insights into EGFR-PROTACs as a potential therapeutic strategy against EGFR-acquired mutation.