Icaritin promotes apoptosis and inhibits proliferation by down-regulating AFP gene expression in hepatocellular carcinoma

Abstract Background Icaritin, an active ingredient of the Chinese herb Epimedium, plays an anti-tumor role in liver cancer by inhibiting the proliferation of hepatocellular cells and promoting their apoptosis. In China, phase II and a large phase III clinical trial of icaritin reagent for the treatment of hepatocellular cancer is under-going, but the specific mechanism of icaritin action was unclear. Alpha-fetoprotein (AFP), an oncofetal protein, produced in the healthy fetal liver and yolk sac. Intracellular AFP promoted cellular proliferation and inhibited cellular apoptosis in hepatocellular carcinoma (HCC). The study was aimed to investigate the effect of icaritin on HCC through p53/AFP pathway. Methods Real-time RT PCR and western blot were used to detect p53 and AFP expression levels in HCC cells treated with icaritin. The mechanism of icaritin affecting p53 expression was verified by ubiquitination experiment, and the binding activity of icaritin on p53 in AFP promoter region was verified by luciferase experiment. EdU, MTT and flow cytometry were used to determine whether icaritin affected HCC cellular proliferation and apoptosis through p53/ AFP pathway. Expression levels of p53 and AFP in xenograft mouse model were determined by western blotting. Results Our results showed icaritin inhibited AFP expression at mRNA and protein level. AFP was also identified as the target gene of the p53 transcription factor. Icaritin abrogated murine double minute (Mdm) 2-mediated p53 ubiquitination degradation to improve the stability of p53. Up-regulated p53 protein levels then transcriptionally inhibited the AFP promoter. Icaritin-mediated decrease of AFP through Mdm2/p53 pathways inhibited HCC cellular proliferation and promoted HCC cellular apoptosis. Conclusion Our findings revealed the mechanism of icaritin in promoting apoptosis and inhibiting proliferation in liver cancer cells. The regulatory mechanism of icaritin in AFP protein down-regulation provides a theoretical and experimental basis for further research into new drugs for the treatment of liver cancer.

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Hypericin Exerts Detrimental Effect on Huh-7 As a Delegacy of Hepatocellular Carcinoma: A P53 Dependent Pathway

Galen Medical Journal ◽

10.31661/gmj.v9i0.1896 ◽

2020 ◽

Vol 9 ◽

pp. 1896

Author(s):

Maedeh Olya ◽

Hamid Zaferani Arani ◽

Amirhossein Shekarriz ◽

Amirhossein Zabolian ◽

Hadi Zare Marzouni ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Flow Cytometry ◽

Liver Cancer ◽

Cell Line ◽

Real Time ◽

Real Time Pcr ◽

Huh7 Cell ◽

P53 Expression ◽

Huh7 Cells ◽

Huh7 Cell Line

Background: Hepatocellular carcinoma is the most common type of liver cancer which arises from the main cells in the liver. We address many studies investigating anti-cancer role of hypericin, however the proposing corresponding molecular pathway seems to be still a debate. Therefore, the present study aimed to evaluate the apoptotic effect of hypericin on the Huh7 as the liver cancer cell line and its relation with the gate keeper gene P53. Materials and Methods: In this study, the Huh7 cell line and fibroblast cells (as control group) were treated with different concentrations of hypericin for 24 and 48 hours. Detection of cell death was performed by MTT assay and flow cytometry. The expression of bax, bcl2 and p53 mRNAs was evaluated by Real-time PCR. Also, Immunocytochemistry (ICC) analysis was used for further evaluation of P53expression. Results: The results showed that hypericin has a dose-dependent cytotoxic effect on the Huh7 cell line, with no or marginal effect on fibroblastic cells. According to flow cytometry results, about 53%cells underwent apoptosis after exposure to LD50 of hypericin for 24 hours. Real-time PCR data demonstrated that the pro-apoptotic genes Bax and P53 expression level increased. Expectedly ICC results confirmed the up-regulation of P53 proteins in treated samples. Conclusion: Our results indicate the cytotoxicity of hypericin on Huh7 cells by affecting the expression of the gate keeper gene P53; furthermore it is suggested that this herb can be utilized simultaneously with modalities targeting P53 up-regulation or related molecular pathways. [GMJ.2020;9:e1896]

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Thymoquinone exerts anti-tumor activities on human hepatocellular carcinoma cells: role of angiogenesis-related genes VCAN, Grb2 and EZH2

European Journal of Cell Science ◽

10.34154/2019-ejcs-0101-10-16/euraass ◽

2019 ◽

pp. 10-16

Author(s):

Mohammed Y. Alhassani ◽

Samir F. Zohny ◽

Ryan A. Sheikh ◽

Mohammed A. Hassan ◽

Abdulaziz A. Kalantan ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Liver Cancer ◽

Molecular Mechanisms ◽

Target Genes ◽

Nigella Sativa ◽

Human Hepatocellular Carcinoma ◽

Biologically Active ◽

New Drugs ◽

Pcr Analysis ◽

Dependent Manner

Human hepatocellular carcinoma (HCC) is the most prevalent and recurrent type of primary adult liver cancer without any effective therapy. Thus, there is an increase demands for finding new drugs and treatment strategies with selective and potent effects towards HCC. Plant-derived compounds acting as anti-cancer agents can induce apoptosis through targeting several signaling pathways. Thymoquinone (TQ), the major biologically active compound of the black seed oil (Nigella sativa) has demonstrated inhibitory activities on various cancers by targeting several pathways. In the present study, we have evaluated the molecular mechanisms that underlie the anti-proliferative, anti-metastatic, and pro-apoptotic activities exerted by TQ on liver cancer cell lineHepG2, a well-documented HCC in vitro model. Cell proliferation was determined by WST-1 assay, apoptosis rate was assessed by flow cytometry using annexin-V/7AAD staining, wound healing assay to investigate the metastasis, and the expression of target genes was assessed by Real-time RT–PCR analysis. We found that TQ significantly reduced HepG2 cell viability and induced apoptosis in a dose-dependent manner. Migration of HepG2 cells was suppressed in response to TQ. Moreover, TQ decreased the expression of several angiogenesis-related genes including versican (VCAN), growth factor receptor-bound protein 2 (Grb2), and the histone methyltransferase for lysine 27 of histone 3 (EZH2). The findings suggest that TQ exerts inhibitory effects on HCC most likely through targeting key genes involved in the invasiveness and

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Lupeol-Loaded Nanoparticles Enhance the Radiosensitivity of Hepatocellular Carcinoma by Inhibiting the Hyperactivation in Raf/Mitogen-Activated Protein Kinase and Phospatidylinositol-3 Kinase/mTOR Pathways

Journal of Biomedical Nanotechnology ◽

10.1166/jbn.2021.3194 ◽

2021 ◽

Vol 17 (11) ◽

pp. 2247-2258

Author(s):

Yinghai Xie ◽

Changwei Liu ◽

Shuping Zhou ◽

Qi Wang ◽

Xiaolong Tang

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Cycle Progression ◽

Cellular Proliferation ◽

Mitogen Activated Protein Kinase ◽

Proliferation Index ◽

Nude Mouse Model ◽

Ki 67 ◽

Mitogen Activated Protein ◽

Cellular Apoptosis

Radioresistance limits the effectiveness of radiotherapy for hepatocellular carcinoma. Raf and PI3K signaling cascades promote the formation of radioresistance in hepatocellular carcinoma (HCC). Lupeol has anticancer activity despite itspoor solubility in water and is toxic effect on normal tissue. In this study, nanoparticles (lupeol-NPs) were constructed using PEG-PLGA diblock copolymer vector, and results revealed that Lupeol-NPs reversed the radioresistance of hepatocellular carcinoma by inhibiting cellular proliferation and cell-cycle progression and promoting cellular apoptosis through blocking Raf/MAPK and PI3K/Akt signal transduction in radioresistant Huh-7R cells. In vivo, Lupeol-NPs combined with radiotherapy inhibited the growth of radioresistant hepatocellular carcinoma in a xenogenic nude mouse model. Ki-67 proliferation index decreased significantly (p < 0.05). We conclude that Lupeol-NPs can increase the sensitivity of radioresistant hepatocellular carcinoma to radiotherapy through inhibiting the Raf and PI3K signal cascades.

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Recent Advances in Systemic Therapy for Hepatocellular Carcinoma in an Aging Society: 2020 Update

Liver Cancer ◽

10.1159/000511001 ◽

2020 ◽

Vol 9 (6) ◽

pp. 640-662

Author(s):

Masatoshi Kudo

Keyword(s):

Hepatocellular Carcinoma ◽

Clinical Trials ◽

Systemic Therapy ◽

Checkpoint Inhibitors ◽

Treatment Options ◽

New Drugs ◽

Phase Iii ◽

Second Line ◽

First Line ◽

Recent Advances

Systemic therapy for hepatocellular carcinoma (HCC) has changed markedly since the introduction of the molecular targeted agent sorafenib in 2007. Sorafenib increased the available treatment options for patients with extrahepatic spread and vascular invasion and improved survival in patients with advanced HCC; however, various shortcomings such as low response rates and relatively high toxicity (e.g., hand-foot skin reaction) prompted concerted efforts aimed at developing new molecular targeted agents to provide more treatment options and second-line agents for patients with disease progression or intolerance to sorafenib. Despite many attempts to develop new drugs between 2007 and 2016, all first-line and second-line clinical trials conducted during this period failed. However, between 2017 and 2019, 4 drugs (lenvatinib as a first-line agent and regorafenib, cabozantinib, and ramucirumab as second-line agents) emerged in quick succession from clinical trials and became available for clinical use. In addition, nivolumab and pembrolizumab were approved as second-line agents after sorafenib. A recent phase III trial (IMbrave150) showed that combination immunotherapy with atezolizumab plus bevacizumab increases overall survival compared with sorafenib therapy; Food and Drug Agency already approved this combination therapy, and worldwide approval is expected soon. This review describes the recent advances in systemic therapy and the use of tyrosine kinase inhibitors (sorafenib, lenvatinib, regorafenib, and cabozantinib), monoclonal antibodies (ramucirumab and bevacizumab), and immune checkpoint inhibitors (nivolumab, pembrolizumab, and atezolizumab) in elderly patients and the similarity of their efficacy and safety profiles to those in the general population.

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SIX1 Down-Regulation Influence Drug Resistance, Epithelial-Mesenchymal Transcription Factors and Self-Renewal Capacity in Hepatocellular Carcinoma

10.21203/rs.3.rs-376584/v1 ◽

2021 ◽

Author(s):

Pelin Balcik Ercin ◽

Arzu Aysan ◽

Nazli Salik ◽

Esma Erciyas

Keyword(s):

Hepatocellular Carcinoma ◽

Drug Resistance ◽

Liver Cancer ◽

Expression Profile ◽

Epithelial Mesenchymal Transition ◽

Profile Analysis ◽

Sorafenib Treatment ◽

Mesenchymal Transition ◽

Expression Levels ◽

Sine Oculis

Abstract Sine oculis homeoprotein 1 (SIX1) was discovered to exert an essential role in embryonic development and it was also identified to be re-activated in various types of mammalian cancer. Immunohistochemical and SIX1 gene expression analyses were performed to determine the prognostic role of SIX1 expression. SIX1 expression was suppressed by short hairpin RNA transduction in the SNU398 HCC cell line. The effects of SIX1 on proliferation, epithelial-mesenchymal transition, apoptosis, drug resistance, and sphere formation were assessed in SIX1 knock-down cells. The upregulated expression levels of SIX1 were revealed to be correlated with the stage of the disease in breast, colon and liver cancer, with liver cancer exhibiting the highest expression profile. SIX1 knockdown significantly affected the cell morphology, proliferation, downregulated the protein expression levels of ZEB1, ZEB2 and SNAI1 and upregulated the expression levels of TWIST1 in hepatocellular carcinoma cells. Furthermore, SIX1 knockdown cells were more sensitive to sorafenib treatment; however, the expression profile analysis of the drug resistance genes ABCB1, ABCC1 and ABCG2 did not explain this sensitivity. Finally, SIX1 knockdown cells were identified to have decreased CD90 levels and lost their sphere-forming ability, which is essential for cancer stem cell properties. Overall, these results indicated that SIX1 expression may be useful as a diagnostic marker for patients with HCC.

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Long non-coding RNA HOMER3-AS1 drives hepatocellular carcinoma progression via modulating the behaviors of both tumor cells and macrophages

Cell Death and Disease ◽

10.1038/s41419-021-04309-z ◽

2021 ◽

Vol 12 (12) ◽

Author(s):

Jian Pu ◽

Wenchuan Li ◽

Anmin Wang ◽

Ya Zhang ◽

Zebang Qin ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Tumor Cells ◽

Cellular Proliferation ◽

Migration And Invasion ◽

Downstream Target ◽

Non Coding Rna ◽

Cellular Apoptosis ◽

Positive Correlations ◽

Long Non Coding Rna

AbstractThe crosstalk between cancer cells and tumor microenvironment plays critical roles in hepatocellular carcinoma (HCC). The identification of long non-coding RNAs (lncRNAs) mediating the crosstalk might promote the development of new therapeutic strategies against HCC. Here, we identified a lncRNA, HOMER3-AS1, which is over-expressed in HCC and correlated with poor survival of HCC patients. HOMER3-AS1 promoted HCC cellular proliferation, migration, and invasion, and reduced HCC cellular apoptosis. Furthermore, HOMER3-AS1 promoted macrophages recruitment and M2-like polarization. In vivo, HOMER3-AS1 significantly facilitated HCC progression. Mechanism investigations revealed that HOMER3-AS1 activated Wnt/β-catenin signaling via upregulating HOMER3. Functional rescue experiments revealed that HOMER3/Wnt/β-catenin axis mediated the roles of HOMER3-AS1 in promoting HCC cellular malignant phenotypes. Furthermore, colony stimulating factor-1 (CSF-1) was also identified as a critical downstream target of HOMER3-AS1. HOMER3-AS1 increased CSF-1 expression and secretion. Blocking CSF-1 reversed the roles of HOMER3-AS1 in inducing macrophages recruitment and M2 polarization. Furthermore, positive correlations between HOMER3-AS1 and HOMER3 expression, HOMER3-AS1 and CSF-1 expression, and HOMER3-AS1 expression and M2-like macrophages infiltration were found in human HCC tissues. In summary, our findings demonstrated that HOMER3-AS1 drives HCC progression via modulating the behaviors of both tumor cells and macrophages, which are dependent on the activation of HOMER3/Wnt/β-catenin axis and CSF-1, respectively. HOMER3-AS1 might be a promising prognostic and therapeutic target for HCC.

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Translational Considerations to Improve Response and Overcome Therapy Resistance in Immunotherapy for Hepatocellular Carcinoma

Cancers ◽

10.3390/cancers12092495 ◽

2020 ◽

Vol 12 (9) ◽

pp. 2495

Author(s):

Sophia Heinrich ◽

Darko Castven ◽

Peter R. Galle ◽

Jens U. Marquardt

Keyword(s):

Hepatocellular Carcinoma ◽

Liver Cancer ◽

Current Knowledge ◽

Primary Liver Cancer ◽

Objective Response ◽

Response Rates ◽

Therapy Resistance ◽

Phase Iii ◽

Treatment Modalities ◽

Molecular Characteristics

Over the last decade, progress in systemic therapies significantly improved the outcome of primary liver cancer. More recently, precision oncological and immunotherapeutic approaches became the focus of intense scientific and clinical research. Herein, preclinical studies showed promising results with high response rates and improvement of overall survival. However, results of phase III clinical trials revealed that only a subfraction of hepatocellular carcinoma (HCC) patients respond to therapy and display only moderate objective response rates. Further, predictive molecular characteristics are largely missing. In consequence, suitable trial design has emerged as a crucial factor for the success of a novel compound. In addition, increasing knowledge from translational studies indicate the importance of targeting the tumor immune environment to overcome resistance to immunotherapy. Thus, combination of different immunotherapies with other treatment modalities including antibodies, tyrosine kinase inhibitors, or local therapies is highly promising. However, the mechanisms of failure to respond to immunotherapy in liver cancer are still not fully understood and the modulation of the immune system and cellular tumor composition is particularly relevant in this context. Altogether, it is increasingly clear that tailoring of immunotherapy and individualized approaches are required to improve efficacy and patient outcome in liver cancer. This review provides an overview of the current knowledge as well as translational considerations to overcome therapy resistance in immunotherapy of primary liver cancer.

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