Hepatocellular Carcinoma Cell Lines Hep3B Research Articles

Circular RNA circ_0006168 accelerates the development of hepatocellular carcinoma through sponging microRNA-125b.

This study explored the role of circular RNA circ_0006168 in the progression of hepatocellular carcinoma (HCC) and its interaction with microRNA-125b. The expression of circ_0006168 was examined in 42 pairs of HCC tumor and adjacent tissue specimens using quantitative polymerase chain reaction (qPCR). Elevated circ_0006168 expression in HCC tissues was significantly associated with advanced pathological staging and lower overall survival rates. Lentivirus-mediated circ_0006168 knockdown in HCC cell lines (Hep3B and Huh7) demonstrated a notable reduction in cell proliferation and an increase in apoptosis. MicroRNA-125b expression exhibited a marked reduction in HCC tissues, negatively correlating with circ_0006168 levels. Luciferase reporting assays indicated that circ_0006168 was a direct target of microRNA-125b. Additionally, cell recovery experiments suggested a reciprocal regulation between circ_0006168 and microRNA-125b, contributing to the accelerated malignant progression of HCC. The study underscored the significantly increased expression of circ_0006168 in both HCC tissues and cell lines, highlighting its association with advanced pathological stages and poor prognosis in HCC patients. Furthermore, circ_0006168 appeared to play a pivotal role in elevating the proliferation rate of HCC cells through its modulation of microRNA-125b. These findings contribute to a deeper understanding of the molecular mechanisms underlying HCC development and may offer potential therapeutic targets for intervention.

Chalcomoracin promotes apoptosis and endoplasmic reticulum stress in hepatocellular carcinoma cells.

Chalcomoracin (CMR), a Diels-Alder adduct obtained from mulberry leaves, demonstrated wide-spectrum anti-cancer activity. Herein, we aimed to explore the function of CMR and how it works in hepatocellular carcinoma (HCC). Human HCC cell lines Hep3B and SNU-387 were cultured and treated with various concentrations of CMR (1.5, 3, and 6 µM). Subsequently, the effects of CMR on cell viability, colony formation, apoptosis, migration, and invasion abilities were studied in vitro. Furthermore, the levels of endoplasmic reticulum (ER) stress-related proteins and mitogen-activated protein kinase (MAPK) pathway-related proteins in cells under CMR exposure were detected using western blot. Experiments in vivo were conducted to examine the effects of CMR on tumor growth in HCC. CMR administration inhibited the viability and clonogenic, migration, and invasion abilities, as well as promoted cell apoptosis and ER stress in Hep3B and SNU-387 cells. In addition, CMR treatment reduced the phosphorylation levels of ERK, P38, and JNK in the MAPK pathway. Moreover, an in vivo study showed that CMR administration could inhibit tumorigenesis and MAPK pathway activity in HCC. Our data indicate that CMR has the potential to inhibit the development of HCC, potentially through the inhibition of the MAPK pathway. These findings suggest that CMR may have promising applications as an anticancer agent in future therapeutics for HCC.

Psilostachyin C reduces malignant properties of hepatocellular carcinoma cells by blocking CREBBP-mediated transcription of GATAD2B.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality globally. Many herbal medicines and their bioactive compounds have shown anti-tumor properties. This study was conducted to examine the effect of psilostachyin C (PSC), a sesquiterpenoid lactone isolated from Artemisia vulgaris L., in the malignant properties of HCC cells. CCK-8, flow cytometry, wound healing, and Transwell assays revealed that 25 μM PSC treatment significantly suppressed proliferation, cell cycle progression, migration, and invasion of two HCC cell lines (Hep 3B and Huh7) while promoting cell apoptosis. Bioinformatics prediction suggests CREB binding protein (CREBBP) as a promising target of PSC. CREBBP activated transcription of GATA zinc finger domain containing 2B (GATAD2B) by binding to its promoter. CREBBP and GATAD2B were highly expressed in clinical HCC tissues and the acquired HCC cell lines, but their expression was reduced by PSC. Either upregulation of CREBBP or GATAD2B restored the malignant properties of HCC cells blocked by PSC. Collectively, this evidence demonstrates that PSC pocessess anti-tumor functions in HCC cells by blocking CREBBP-mediated transcription of GATAD2B.

All Three AKT Isoforms Can Upregulate Oxygen Metabolism and Lactate Production in Human Hepatocellular Carcinoma Cell Lines.

Hepatocellular carcinoma (HCC), the main pathological type of liver cancer, is related to risk factors such as viral hepatitis, alcohol intake, and non-alcoholic fatty liver disease (NAFLD). The constitutive activation of the PI3K/AKT signaling pathway is common in HCC and has essential involvement in tumor progression. The serine/threonine kinase AKT has several downstream substrates, which have been implicated in the regulation of cellular metabolism. However, the contribution of each of the three AKT isoforms, i.e., AKT1, AKT2 and AKT3, to HCC metabolism has not been comprehensively investigated. In this study, we analyzed the functional role of AKT1, AKT2 and AKT3 in HCC metabolism. The overexpression of activated AKT1, AKT2 and AKT3 isoforms in the human HCC cell lines Hep3B and Huh7 resulted in higher oxygen consumption rate (OCR), ATP production, maximal respiration and spare respiratory capacity in comparison to vector-transduced cells. Vice versa, lentiviral vector-mediated knockdowns of each AKT isoform reduced OCR in both cell lines. Reduced OCR rates observed in the three AKT isoform knockdowns were associated with reduced extracellular acidification rates (ECAR) and reduced lactate production in both analyzed cell lines. Mechanistically, the downregulation of OCR by AKT isoform knockdowns correlated with an increased phosphorylation of the pyruvate dehydrogenase on Ser232, which negatively regulates the activity of this crucial gatekeeper of mitochondrial respiration. In summary, our data indicate that each of the three AKT isoforms is able to upregulate OCR, ECAR and lactate production independently of each other in human HCC cells through the regulation of the pyruvate dehydrogenase.

Methanol Extract of Petasites japonicas Promotes Apoptosis via the MAPK and ROS-dependent Signaling Pathways

Background: Petasites japonicus (PJ), also known as Butterbur, has a rich history as a traditional healing remedy across numerous countries. Objectives: This study was designed to evaluate the potential anti-cancer properties of the methanol extract derived from PJ (PJE). Materials and Methods: Cell viability was measured with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. Cell cycle analysis, caspase activity assays, western blotting, and reactive oxygen species (ROS) assays were also used to investigate the anticancer effects of PJE on cancer cells. Results: It was shown that PJE inhibited the cell viability of the colon carcinoma cell line Caco-2 (half-maximal inhibitory concentration [IC50]: 268.4 µg/mL), of the hepatocellular carcinoma cell line Hep3B (IC50: 420.2 µg/mL), and of the bladder carcinoma cell line 5637 (IC50: 99.43 µg/mL). Analysis of DNA content indicated an increase in the sub-G1 population of 5637 cells as a result of PJE treatment. Furthermore, PJE caused a reduction in mitochondrial membrane potential and the ratio of Bcl-2 to Bax. Moreover, PJE enhanced the levels of various components involved in the proapoptotic cascade, such as caspase-3, caspase-9, and poly-adenosine diphosphate-ribose polymerase. Moreover, it was observed that PJE modulated mitogen-activated protein kinases (MAPKs) activation and induced an elevation in intracellular production of ROS. Conclusion: These combined results strongly suggest that PJE possesses significant proapoptotic properties as an herbal medicine, acting through ROS-dependent MAPK signaling pathways in bladder cancer cells.

SKI-349, a Sphingosine Kinases 1/2 Inhibitor, Suppresses Cell Viability, Invasion, and AKT/mTOR Signaling Pathway, and Shows Synergistic Cytotoxic Effects with Sorafenib in Hepatocellular Carcinoma.

SKI-349 is a novel sphingosine kinases (SPHK) inhibitor with anti-tumor effects. This study aimed to assess the effect of SKI-349 on cell biological behaviors, downstream pathways, and its synergistic effect with sorafenib in hepatocellular carcinoma (HCC). HCC cell lines (Huh7 and Hep3B) were treated with SKI-349 at concentrations of 1, 2, 4, or 8 μM. Then, SPHK1/2 activity, cell viability, proliferation, apoptosis, invasion, and protein expressions of phosphorylated-protein kinase B (p-AKT), AKT, phosphorylated-mammalian target of rapamycin (p-mTOR) and mTOR were detected. Combination index values of SKI-349 (0, 1, 2, 4, or 8 μM) and sorafenib (0, 2.5, 5, 10, or 20 μM) were calculated. SKI-349 decreased the relative SPHK1 and SPHK2 activity compared with blank control in a dose-dependent manner in the Huh7 and Hep3B cell lines. Meanwhile, SKI-349 reduced cell viability, 5-ethynyl-2'-deoxyuridine (EdU) positive cells, and invasive cells, while it increased apoptotic cells compared to blank control in a dose-dependent manner in Huh7 and Hep3B cell lines. Based on the western blot assay, SKI-349 decreased the ratio of p-AKT to AKT and that of p-mTOR to mTOR compared with blank control in a dose-dependent manner in the Huh7 and Hep3B cell lines. Additionally, SKI-349 combined with sorafenib declined cell viability with concentration gradient effects compared to SKI-349 sole treatment, and they had synergistic cytotoxic effects in Huh7 and Hep3B cell lines. SKI-349 suppresses SPHK1 and SPHK2 activity, cell viability, invasion, and AKT/mTOR signaling pathway, as well as exhibits a synergistic cytotoxic effect with sorafenib in HCC.

A HIF-1α inhibitor combined with palmitic acid and L-carnitine treatment can prevent the fat metabolic reprogramming under hypoxia and induce apoptosis in hepatocellular carcinoma cells

BackgroundA hypoxic environment often persists within solid tumors, including hepatocellular carcinoma (HCC). Hypoxia-inducible factor-1α (HIF-1α) can accelerate cancer malignancy by inducing hypoxia-dependent expression of various genes. Tumor hypoxia can also induce metabolic reprogramming of fatty acid (FA) metabolism, through which HIF-1α plays an essential role in diminishing fatty acid β-oxidation (FAO) in hypoxic cancer cells.MethodsWe aimed to investigate potential new drug therapy options for targeting hypoxic cancer cells within HCC tumors, specifically through combining HIF-1α inhibition with palmitic acid (PA) + L-carnitine (LC) treatment to effectively induce apoptosis in hypoxic HCC cells. To test this hypothesis, in vitro and in vivo studies were performed.ResultsWe first demonstrated that hypoxia-dependent apoptosis was induced by an overload of PA in two HCC cell lines (HepG2 and Hep3B) via excessive production of reactive oxygen species (ROS). Moreover, this observed PA-induced apoptosis was enhanced by HIF-1α knockdown (KD) in these cells under hypoxia. In addition, the combination of PA with FAO activator LC increased FAO activity and led to stronger cell death than PA alone in hypoxic HIF-1α KD cells, specifically through further ROS generation. To clarify the mechanism of hypoxia-induced FA metabolism reprogramming, expression levels of the genes encoding FAO enzymes CPT1A, ACSL1, MCAD, and LCAD, FA transporter CD36, and FA esterification enzymes DGAT and APGAT were analyzed using HIF-1α KD and scramble control (SC) cells. The results suggested that HIF-1α could repress mRNA expression of the FAO-related enzymes and CD36, while it upregulated FA esterification gene expression. This suggested a central role for HIF-1α in hypoxia-induced reprogramming of FA metabolism in HCC cells. Using a nude mouse model, PA administration was found to induce apoptosis from ROS overproduction in HIF-1α KD tumors compared with SC tumors. Additional LC treatment synergistically enhanced the PA-induced apoptosis in HIF-1α KD tumors. Finally, in vivo therapy composed of HIF-1α inhibitor YC-1 with PA + LC could induce ROS-mediated apoptosis in HepG2 tumors without significant toxicity.ConclusionsA combination therapy of YC-1 with PA + LC may be a unique anti-tumor therapy for targeting hypoxic HCC cells, specifically by ROS overproduction leading to forced FAO activation.

Antitumor from Several Plant Compounds Against Hepatocellular Carcinoma Cell Line Hep3B: Mini Systematic Review

Liver cancer is a global health challenge, with more than one million cases expected by 2025. Hepatocellular carcinoma (HCC) is the most common form of liver cancer and accounts for approximately 90% of cases. Nature provides a large source of natural medicines and compounds, one of which is plants. So, this article is made to contain information on the potential of several plants as antitumor agents. The method of preparing articles through literature review studies that collect articles from various sources such as ScienceDirect, google scholar, PubMed and others in the range of 2013 to 2023. There are ten plant bioactive compounds that have been tested in vitro on Hep3B cell lines to see their toxicity. These plant bioactive compounds play an important role in the proliferation phase, cell cycle, and cell apoptosis in liver cancer and have potential as antitumor.

Upregulation of FAM134B inhibits endoplasmic reticulum stress-related degradation protein expression and promotes hepatocellular carcinogenesis.

Endoplasmic reticulum (ER) stress can stimulate the proliferation and metastasis of hepatocellular carcinoma (HCC) cells while hindering apoptosis and immune system function, but the molecular mechanism of ER stress in HCC has yet to be fully studied. We aim to investigate the molecular mechanism by which FAM134B inhibits autophagy of HCC cells by reducing the expression of ER stress-related degradation proteins. Clinical samples were collected for this study. Normal liver cell lines HL7702 and Hep3B and Huh7 HCC cell lines were cultured. Construction of FAM134B knockdown cell line. Cell proliferation was measured using the CCK-8 assay, while cell migration and invasion capabilities were detected using the plate colony formation assay. Flow cytometry was used to detect the apoptosis rate. Transmission electron microscopy was used to observe the formation of autophagosomes. qRT-PCR and WB detective expression changes related to autophagy proteins. Finally, the expression of the relevant proteins was observed by immunohistochemistry. The expression of FAM134B was significantly increased in human liver cancer tissue and HCC cell lines Hep3B and Huh7. After the lentiviral vector was transfected into Hep3B cells with sh-FAM134B, results showed that sh-FAM134B could effectively inhibit Hep3B cell proliferation and promote HCC cell apoptosis. Meanwhile, sh-FAM134B could effectively induce the autophagy of Hep3B liver cancer cells. Immunohistochemistry results showed that sh-FAM134B could effectively induce ER stress. FAM134B inhibits HCC cell autophagy and promotes the progression of liver cancer by inhibiting the expression of ER stress-related degradation factors such as DERL2, EDEM1, SEL1L and HRD1.

Arsenic Sulfide Inhibits Hepatocellular Carcinoma Metastasis by Suppressing the HIF-1α/VEGF Pathway.

Metastasis is one of the principal reasons of cancer mortality from hepatocellular carcinoma (HCC). The goal of our investigation was to examine the mechanism by which arsenic sulfide (As4S4) represses the metastasis of HCC. The cell counting kit-8 (CCK-8) assay was conducted to observe cell viability of HCC cell lines HepG2 and Hep3B following As4S4 treatment, and their metastasis was studied using the wound-healing and transwell assays. HCC-induced angiogenesis of human umbilical vein endothelial cells (HUVEC) was assessed by tube formation assay. Enzyme-linked immunosorbent assay (ELISA), western blot, quantitative polymerase chain reaction and immunofluorescence staining were utilized to evaluate key molecules involved in metastasis, including hypoxia-inducible factor 1α (HIF-1α), vascular endothelial growth factor (VEGF), Vimentin, N-cadherin and E-cadherin. As4S4 suppressed the proliferation, migration and invasion of HepG2 and Hep3B cell lines in a concentration-dependent pattern, and inhibited HCC cell-induced angiogenesis of HUVEC in the tube formation assay. Treatment with As4S4 also decreased the expression of crucial elements involved in the metastasis of HCC cells, including HIF-1α and VEGF, while reducing epithelial-mesenchymal transition, as shown by Western blot, ELISA and immunofluorescence staining. Overall, results above indicate that As4S4 suppresses the metastasis of HCC cells via the HIF-1α/VEGF pathway.

Yeni PDI tabanlı antiproliteratif kromoforlar

The primary objective of the work was to monitor the antiproliferative activity of two newly synthesized and described perylene diimides. The MTT test was used to investigate the antiproliferative effect against PC3 the prostat cancer cell line, Panc1 pancreatic cancer cell line, Saos2 osteosarcoma cell line, Hep3B hepatoma cell line and HUVEC non-cancerous human umbilical vein endothelial cells. To PC3, Panc1, and Saos 2 cells, both compounds were found to be considerably cytotoxic; however, a comparable impact was not seen in Hep3B cells. Compound 2 had the lowest IC50 value of 40,88 µg/ml and significantly inhibited PC3 cell proliferation when compared to other cell lines.

Creation of a Prognostic Model Using Cuproptosis-Associated Long Noncoding RNAs in Hepatocellular Carcinoma.

Cuproptosis is an unusual form of cell death caused by copper accumulation in mitochondria. Cuproptosis is associated with hepatocellular carcinoma (HCC). Long noncoding RNAs (LncRNAs) have been shown to be effective prognostic biomarkers, yet the link between lncRNAs and cuproptosis remains unclear. We aimed to build a prognostic model of lncRNA risk and explore potential biomarkers of cuproptosis in HCC. Pearson correlations were used to derive lncRNAs co-expressed in cuproptosis. The model was constructed using Cox, Lasso, and multivariate Cox regressions. Kaplan-Meier survival analysis, principal components analysis, receiver operating characteristic curve, and nomogram analyses were carried out for validation. Seven lncRNAs were identified as prognostic factors. A risk model was an independent prognostic predictor. Among these seven lncRNAs, prostate cancer associated transcript 6 (PCAT6) is highly expressed in different types of cancer, activating Wnt, PI3K/Akt/mTOR, and other pathways; therefore, we performed further functional validation of PCAT6 in HCC. Reverse transcription-polymerase chain reaction results showed that PCAT6 was aberrantly highly expressed in HCC cell lines (HepG2 and Hep3B) compared to LO2 (normal hepatocytes). When its expression was knocked down, cells proliferated and migrated less. PCAT6 might be a potential biomarker for predicting prognosis in HCC.

MESP1-knockdown inhibits the proliferation and epithelial mesenchymal transition of hepatocellular carcinoma and enhances the tumor-suppressive effect of 5-fluorouracil

Primary liver hepatocellular carcinoma (HCC) is the third most deadly malignancy worldwide,in part, because it is often diagnosed at an advanced stage. Thus, molecular markers are needed to aid in the early diagnosis and treatment of HCC. Expression of abnormal mesoderm posterior-1 (MESP1) promotes tumorigenesis; however,its role in the regulation of HCC proliferation, apoptosis,and invasion is unknown. Here,we analyzed data in The Cancer Genome Atlas (TCGA)and Genotype Tissue Expression (GTEx) databases on the pan-cancer expression of MESP1 and its relationship with clinical characteristics and prognosis of patients with HCC. The expression of MESP1 was measured in 48 HCC tissues using immunohistochemical staining,and the results were correlated with clinical stage, tumor differentiation, tumor size,and metastasis. MESP1 expression was downregulated using small interfering RNA (siRNA) in the HCC cell lines HepG2 and Hep3B,and cell viability, proliferation,cell cycle, apoptosis,and invasion were analyzed. Finally,we also evaluated the tumor suppression effect of MESP1 downregulation combined with 5-fluorouracil (5-FU) treatment. Our results showed that MESP1 is a pan-oncogene associated with poor prognosis in patients with HCC. siRNA-induced downregulation of MESP1 expression in HepG2 and Hep3B cells exhibited downregulation of β-catenin and GSK3β expression 48h after transfection, along with an increase in apoptosis rate, arrest in the G1-S phase,and a decrease in mitochondrial membrane potential. Moreover,the expression levels of c-Myc, PARP1, bcl2, Snail1, MMP9, and immune checkpoint genes (TIGIT, CTLA4,LAG3,CD274,and PDCD1) were downregulated, while those of caspase3 and E-cadherin were upregulated. Tumor cells also showed decreased migration ability. Furthermore, siRNA interference of MESP1 expression combined with 5-FU-treatment of HCC cells significantly enhanced the G1-S phase block and apoptosis. MESP1 showed an aberrant high expression in HCC and was associated with poor clinical outcomes; therefore, MESP1 may be a potential target for the diagnosis and treatment of HCC.

The regulation of APGAT4 on the growth and lenvatinib resistance of hepatocellular carcinoma

Objective: To investigate the effect of 1-acyl-sn-glycerol-3-phosphate acyltransferaseδ (APGAT4) on the growth and lenvatinib resistance of hepatocellular carcinoma (HCC), and provide novel targets for HCC treatment. Methods: Using the bioinformatics methods to screen out upregulated genes in lenvatinib resistant cell lines from GEO dataset and survival related genes from TCGA dataset. Immumohistochemical staining was used to detect the expression AGPAT4 in HCC tissues, and its correlation with patients' survival. CCK8, EdU, cell cycle, and cell apoptosis assays were used to investigate the impact of role AGPAT4 on the proliferation and lenvatinib reistance of HCC cells. AGPAT4 stable knockdown cell line and subcutaneous nude mouse model were established to test the therapeutic effects of Lenvatinib. Analysis of variance was used to compare the differences between data sets. Results: APGAT4 was the common factor that predicted poor survival and Lenvatinib resistance. The mRNA and protein levels of APGAT4 were significantly upregulated in HCC tissues compared to the para-tumor tissues (P < 0.05). Using siRNA could significantly knocked down the mRNA and protein expression of APGAT4 in HCC cell lines Hep3B and HCCLM3. Compared with the control group, the proliferation ability of HCC cell lines (Hep3B and HCCLM3) in APGAT4 knockdown group was significantly inhibited, and the cell cycle was arrested in G2/M phase (P < 0.05). In addition, compared to the control group, HCC cell lines (Hep3B and HCCLM3) in APGAT4 knockdown group showed significant decrease in the Lenvatinib half maximal inhibitory concentration, and were more sensitive to lenvatinib-induced apoptosis (P < 0.05). In HCC subcutaneous nude mouse model, compared to the control group, the growth of tumor in APGAT4 knockdown group was significantly suppressed, and more apoptosis cells were induced (P < 0.05). Conclusion: APGAT4 promotes the growth and Lenvatinib resistance of HCC, which is a potential target for HCC treatment. Targeting APGAT4 treatment is conducive to inhibit the growth and Lenvatinib resistance of HCC.

Abstract 5985: Cyclin dependent kinase 9 (CDK9) inhibition increased efficacy of programmed cell death protein 1 (PD-1) blockade for hepatocellular carcinoma (HCC) through decreasing programmed death-ligand 1 (PD-L1) expression

Abstract Background: PD-1 blockade has been approved as treatment for advanced HCC. We previously demonstrated the efficacy of CDK9 inhibition in reducing the expression of inducible proteins and as a treatment for HCC (Shao et al. Oncology 2022). We thus examined the influence of CDK9 inhibition on the expression of PD-L1 in HCC and the potential of improving the efficacy of PD-1 blockade with the combination of CDK9 inhibitors. Methods: We first examined the influence of specific CDK9 inhibitors, AZD4573 and atuveciclib, on interferon-γ (IFN-γ) induced PD-L1 expression of human HCC cell lines HuH7 and Hep3B. Overexpression of CDK9 and siRNA to CDK9 were used to confirm the findings. Similar experiments were repeated in the mouse HCC cell line BNL. To test the in vivo efficacy, we orthotopically implanted BNL cells in the subcapsular area of BALB/c mice, which were then treated with vehicle, CDK9 inhibitors, anti-PD-L1 antibodies, or their combination. To compare with tumor sizes, all mice were sacrificed after 14 days of treatment. To compare survival, the mice were only sacrificed if they met the criteria of animal euthanasia. Results: Both HuH7 and Hep3B cells had minimal PD-L1 expression, which however could be induced using IFN-γ. AZD4573 and atuveciclib decreased the IFN-γ induced PD-L1 expression of HuH7 and Hep3B cells in a dose dependent manner. The flow cytometry results confirmed that membrane PD-L1 expression was also decreased by CDK9 inhibition. Overexpression of CDK9 decreased the influence of these CDK9 inhibitors on PD-L1 expression. CDK9 knockdown using siRNA reduced the IFN-γ induced PD-L1 expression. AZD4573 and atuveciclib also reduce the PD-L1 expression in BNL cell lines. In the orthotopic animal model, the tumors of the sacrificed mice were significantly smaller in mice treated with atuveciclib and an anti-PD-L1 antibody than mice treatment with either alone. The survival of mice treated with the combination was also longer than mice treated with each therapy alone. Conclusion: We demonstrated that CDK9 inhibition could reduce the IFN-γ induced PD-L1 expression of HCC cells. Combination of CDK9 inhibitors and anti-PD-L1 antibodies was more effective than either therapy alone. Citation Format: Ching-Tso Chen, Yu-Yun Shao, Hung-Wei Hsu, Rita R. Wo, Han-Yu Wang, Ann-Lii Cheng, Chih-Hung Hsu. Cyclin dependent kinase 9 (CDK9) inhibition increased efficacy of programmed cell death protein 1 (PD-1) blockade for hepatocellular carcinoma (HCC) through decreasing programmed death-ligand 1 (PD-L1) expression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5985.

Green synthesis of fluorescent carbon nanodots from sage leaves for selective anticancer activity on 2D liver cancer cells and 3D multicellular tumor spheroids.

Carbon nanodots, a family of carbon-based nanomaterials, have been synthesized through different methods from various resources, affecting the properties of the resulting product and their application. Herein, carbon nanodots (CNDs) were synthesized with a green and simple hydrothermal method from sage leaves at 200 °C for 6 hours. The obtained CNDs are well dispersed in water with a negative surface charge (ζ-potential = -11 mV) and an average particle size of 3.6 nm. The synthesized CNDs showed concentration-dependent anticancer activity toward liver cancer (Hep3B) cell lines and decreased the viability of the cancer cells to 23% at the highest used concentration (250 μg ml-1 of CNDs). More interestingly, the cytotoxicity of the CNDs was tested in normal liver cell lines (LX2) revealed that the CNDs at all tested concentrations didn't affect their viability including at the highest concentration showing a viability of 86.7%. The cellular uptake mechanisms of CNDs were investigated and they are thought to be through energy-dependent endocytosis and also through passive diffusion. The main mechanisms of endocytosis were lipid and caveolae-mediated endocytosis. In addition, the CNDs have hindered the formation of 3D spheroids from the Hep3B hepatocellular carcinoma cell line. Hence, it would be concluded that the synthesized CNDs from sage are more highly selective to liver cancer cells than normal ones. The CNDs' cancer-killing ability would be referred to as the production of reactive oxygen species.

Nrf2 and Parkin-Hsc70 regulate the expression and protein stability of p62/SQSTM1 under hypoxia

Solid tumors often contain regions with very low oxygen concentrations or hypoxia resulting from altered metabolism, uncontrolled proliferation, and abnormal tumor blood vessels. Hypoxia leads to resistance to both radio- and chemotherapy and a predisposition to tumor metastases. Under hypoxia, sequestosome 1 (SQSTM1/p62), a multifunctional stress-inducible protein involved in various cellular processes, such as autophagy, is down-regulated. The hypoxic depletion of p62 is mediated by autophagic degradation. We herein demonstrated that hypoxia down-regulated p62 in the hepatoma cell line Hep3B at the transcriptional and post-translational levels. At the transcriptional level, hypoxia down-regulated p62 mRNA by inhibiting nuclear factor erythroid 2-related factor 2 (Nrf2). The overexpression of Nrf2 and knockdown of Siah2, a negative regulator of Nrf2 under hypoxia, diminished the effects of hypoxia on p62 mRNA. At the post-translational level, the proteasome inhibitor MG132, but not the lysosomal inhibitors ammonium chloride and bafilomycin, prevented the hypoxic depletion of p62, suggesting the involvement of the proteasome pathway. Under hypoxia, the expression of the E3 ubiquitin ligase Parkin was up-regulated in a hypoxia-inducible factor 1α-dependent manner. Parkin ubiquitinated p62 and led to its proteasomal degradation, ensuring low levels of p62 under hypoxia. We demonstrated that the effects of Parkin on p62 required heat shock cognate 71kDa protein (Hsc70). We also showed that the overexpression of Nrf2 and knockdown of Parkin or Hsc70 induced the accumulation of p62 and reduced the viability of cells under hypoxia. We concluded that a decrease in p62, which involves regulation at the transcriptional and post-translational levels, is critical for cell survival under hypoxia. The present results show the potential of targeting Nrf2/Parkin-Hsc70-p62 as a novel strategy to eradicate hypoxic solid tumors.

Hepatocellular carcinoma cells loss lenvatinib efficacy in vitro through autophagy and hypoxia response-derived neuropilin-1 degradation

Despite pharmacological advances such as lenvatinib approval, therapeutic failure of hepatocellular carcinoma (HCC) remains a big challenge due to the complexity of its underlying molecular mechanisms. Neuropilin-1 (NRP1) is a co-receptor involved in several cellular processes associated to chemoresistance development. Since both the double-edged process of autophagy and hypoxia-derived response play crucial roles in the loss of therapeutic effectiveness, herein we investigated the interplay among NRP1, autophagy and hypoxia in development of lenvatinib resistance in HCC cell lines. We first analyzed NRP1 expression levels in human HCC samples from public databases, found significantly increased NRP1 expression in human HCC samples as well as its correlation with advanced tumor and metastasis stages. Among 3 HCC cell lines (HepG2, Huh-7 and Hep3B), Hep3B and Huh-7 cells showed significantly increased NRP1 expression levels and cell migration ability together with higher susceptibility to lenvatinib. We demonstrated that NRP1 gene silencing significantly enhanced the anticancer effects of lenvatinib on Hep3B and Huh-7 cells. Furthermore, lenvatinib suppressed NRP1 expression through promoting autophagy in Hep3B and Huh-7 cells; co-treatment with bafilomycin A1 attenuated the antitumor effects of lenvatinib, and NRP1 silencing prevented this loss of in vitro effectiveness of lenvatinib even in the presence of bafilomycin A1. In addition, exposure to a hypoxic microenvironment significantly decreased NRP1 expression through autophagy in Hep3B and Huh-7 cells. Under hypoxia, HIF-1α directly modulated NRP1 expression; HIF-1α silencing not only enhanced the anticancer effects of combined lenvatinib and hypoxia, but also prevented the loss of effectiveness caused by bafilomycin A1, highlighting the potential role of HIF-1α-derived hypoxia response in the adaptive cellular response to lenvatinib and promoting resistance acquisition by autophagy modulation. Overall, NRP1 may constitute a potential therapeutic target to prevent lenvatinib failure derived from a hypoxia-associated modulation of autophagy in advanced HCC.

Gundelia tournefortii inhibits hepatocellular carcinoma progression by lowering gene expression of the cell cycle and hepatocyte proliferation in immunodeficient mice

Gundelia (G.) tournefortii has antibacterial, anti-inflammatory, and hypolipemic effects. We evaluated the anticancer effect of G. tournefortii in an hepatocellular carcinoma (HCC) mouse model of an HCC cell line (Hep3B) injected into NOD.CB17-Prkdc-SCID/NCrHsD male mice. Tumorigenicity was assessed by tumor size, histology, serum α-fetoprotein (αFP), and glypican 3 (GPC3). HCC-related gene expression of the cell cycle (Cyclin-dependent kinase inhibitor 2A (CDNK2A)), proliferation (MKI67), and platelet-derived growth factor receptor α (PDGFA) were measured. HCC cell cycle alterations, apoptosis, and antioxidant markers in serum and liver following treatment with G. tournefortii were determined. Signaling pathways of liver p53 and phosphorylated PI3K, AKT, and mTOR were also evaluated. Results indicate a significant increase in tumor size in HCC animals associated with elevated αFP, GPC3, and MKI67. Tumor markers of p53 and phosphorylated AKT/PI3K/mTOR signaling pathway were diminished, with less proliferating cells and reduced PDGFRA gene expression following G. tournefortii infection. H&E staining showed a remarkable reduction in inflammatory lesions in HCC mice treated with G. tournefortii. This result was in line with a significant delay in the G2/M phase of HCC-primary hepatocytes by 1.39- to 2.4-fold and reduced HCC necrosis associated with inhibited CDNK2A gene expression. Antioxidant activity was significantly lower in the HCC mice than in the control group. Moreover, G. tournefortii inhibited the HCC formation of 3D MCTS spheroids. G. tournefortii treatment markedly restored antioxidant levels and displayed anticancer and antiproliferative effects and could be a promising cancer therapy.

Highly Oxidized Germacranolides from Elephantopus tomentosus and the Configurational Revision of Some Previously Reported Analogues.

Highly oxidized germacranolides are mainly found in the genus Elephantopus, contain a characteristic ten-membered molecular core that is highly flexible, and exhibit potential cytotoxic properties. However, their configurations were assigned ambiguously in previous reports due to spectroscopic observation of macrocyclic systems. Herein, 17 highly oxidized germacranolides, including 12 new germacranolides (1-12), were isolated from Elephantopus tomentosus. Their structures were characterized by spectroscopic data analysis combined with X-ray crystallography and ECD calculations, and it was possible to propose configurational revisions of five previously reported analogues (13-17). Cytotoxic activities for 1-17 against two hepatocellular carcinoma cell lines (HepG2 and Hep3B) were tested, and compounds 1-10 and 13-16 generated IC50 values of 2.2-9.8 μM. Furthermore, the observed cytotoxic activity of 1 was determined as being mediated by inducing the apoptosis of HepG2 and Hep3B cells via mitochondrial dysfunction.