Identification of interleukin-16 production on tumor aggravation in hepatocellular carcinoma by a proteomics approach

BACKGROUND: Cytokines play an important role in the immune response, angiogenesis, cell growth, and differentiation in hepatocellular carcinoma (HCC). OBJECTIVE: We performed a comprehensive study to identify tumor-related cytokines and pathways involved in HCC pathogenesis. METHODS: Cytokine production was evaluated in human HCC tissues and adjacent non-tumor tissues using an antibody-based protein array technique. We compared cytokine expression in HCC tissues with that of hepatic hemangioma (HH), liver metastasis of colorectal cancer, and noncancerous liver tissues from transplantation donors. The protein levels and localization of the candidate cytokines were analyzed by western blotting and immunohistochemistry. RESULTS: Increased expression of interleukin (IL)-1 receptor antagonist, macrophage migration inhibitory factor, and IL-16 was observed in HCC and paired adjacent non-tumor tissues compared with noncancerous livers. In addition, there were increased IL-16 levels in HCC tissues compared with HH. IL-16 treatment significantly increased cell proliferation in vitro. The expression of extracellular signal-regulated kinase (ERK)1/2 and cyclin D1 was markedly increased in cells from two HCC cell lines, Huh7 and HepG2, in a dose- and time-dependent manner. Phosphorylated to total ERK1/2 ratio was increased in Huh7 cells following IL-16 50 ng/ml, but not HepG2 cells. ERK phosphorylation have occurred earlier than protein accumulation at 48 h. Pretreatment with the ERK inhibitor, FR18024, or an anti-IL-16 antibody reduced the increase in IL-16 production in HCC cells. CONCLUSIONS: These results suggest that cell proliferation induced by IL-16 is mediated through the ERK pathway, thus, we identified a new factor associated with HCC tumor growth.

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miR-369-3p serves as prognostic factor and regulates cancer progression of hepatocellular carcinoma

Personalized Medicine ◽

10.2217/pme-2020-0012 ◽

2021 ◽

Author(s):

Can Chen ◽

Yi Zong ◽

Jiaojiao Tang ◽

Ruisheng Ke ◽

Lizhi Lv ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Proliferation ◽

Cancer Progression ◽

Cell Counting ◽

Migration And Invasion ◽

Pcr Analysis ◽

Reverse Transcription Pcr ◽

Huh7 Cells

Background: The aim of this study was to investigate the role of miR-369-3p in hepatocellular carcinoma (HCC). Materials & methods: The expression levels of miR-369-3p were detected using the quantitative real-time reverse transcription-PCR analysis. The cell counting kit-8 and transwell assays were used to explore the effects of miR-369-3p on cell proliferation, migration and invasion of HCC cells. Results: The miR-369-3p expression was downregulated in HCC tissues and cell lines, in comparison to the normal controls, respectively. In vitro, overexpression of miR-369-3p in Hep 3B and Huh7 cells inhibited cell proliferation, migration and invasion. SOX4 was a direct target of miR-369-3p. Conclusion: Our results suggested that miR-369-3p may be a tumor suppressor in HCC by targeting SOX4.

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BEZ235 Increases the Sensitivity of Hepatocellular Carcinoma to Sorafenib by Inhibiting PI3K/AKT/mTOR and Inducing Autophagy

BioMed Research International ◽

10.1155/2021/5556306 ◽

2021 ◽

Vol 2021 ◽

pp. 1-19

Author(s):

Weiya Cao ◽

Xueke Liu ◽

Yinci Zhang ◽

Amin Li ◽

Yinghai Xie ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Cycle ◽

Cell Lines ◽

Acquired Resistance ◽

Inhibitory Effect ◽

Mtor Pathway ◽

Protein Levels ◽

Huh7 Cells

Acquired resistance of hepatocellular carcinoma (HCC) to sorafenib (SFB) is the main reason for the failure of SFB treatment of the cancer. Abnormal activation of the PI3K/AKT/mTOR pathway is important in the acquired resistance of SFB. Therefore, we investigated whether BEZ235 (BEZ) could reverse acquired sorafenib resistance by targeting the PI3K/mTOR pathway. A sorafenib-resistant HCC cell line Huh7R was established. MTT assay, clone formation assay, flow cytometry, and immunofluorescence were used to analyze the effects of BEZ235 alone or combined with sorafenib on cell proliferation, cell cycle, apoptosis, and autophagy of Huh7 and Huh7R cells. The antitumor effect was evaluated in animal models of Huh7R xenografts in vivo. Western blot was used to detect protein levels of the PI3K/AKT/mTOR pathway and related effector molecules. In vitro results showed that the Huh7R had a stronger proliferation ability and antiapoptosis effect than did Huh7, and sorafenib had no inhibitory effect on Huh7R. SFB + BEZ inhibited the activation of the PI3K/AKT/mTOR pathway caused by sorafenib. Moreover, SFB + BEZ inhibited the proliferation and cloning ability, blocked the cell cycle in the G0/G1 phase, and promoted apoptosis in the two cell lines. The autophagy level in Huh7R cells was higher than in Huh7 cells, and BEZ or SFB + BEZ further promoted autophagy in the two cell lines. In vivo, SFB + BEZ inhibited tumor growth by inducing apoptosis and autophagy. We concluded that BEZ235 enhanced the sensitivity of sorafenib through suppressing the PI3K/AKT/mTOR pathway and inducing autophagy. These observations may provide the experimental basis for sorafenib combined with BEZ235 in trial treatment of HCC.

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Mild Chronic Hypoxia-Induced HIF-2α Interacts with c-MYC Through Competition with HIF-1α in Hepatocellular Carcinoma Proliferation

10.21203/rs.3.rs-37458/v1 ◽

2020 ◽

Author(s):

Han Mu ◽

Ge Yu ◽

Huikai Li ◽

Mengmeng Wang ◽

Yunlong Cui ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Proliferation ◽

Target Genes ◽

Chronic Hypoxia ◽

Annexin V ◽

Proximity Ligation Assay ◽

Hypoxic Cell ◽

Tumor Tissues ◽

Mild Hypoxia

Abstract Background: Hepatocellular carcinoma (HCC) has emerged as a major cause of cancer deaths globally, in which hypoxia and activated hypoxia-inducible factors (HIFs) play an important role. The sibling rivalry between HIF-1α and HIF-2α in hypoxic tumor growth and progression is still debated, including in HCC. This may be associated with the regulation of unique target genes, like c-MYC and mTOR. Methods: In the current study, twenty-six corresponding tumor- and non-tumor tissues, taken from patients with HCC, who underwent liver resection, were analyzed. In vitro, co-immunoprecipitation (Co-IP), Western blot, MTT assay, colony formation assay and Annexin V-FITC/PI staining apoptosis Assay were used to elucidate the relationship between HIF-1α and HIF-2α in hypoxic HCC cell proliferation and involved mechanism.Results: HIF-2α, but not HIF-1α, has a positive correlation with the expression of c-MYC in tumor tissues. In vitro, rapid HCC cell proliferation and increased interaction of HIF-2α/c-MYC were observed in mild chronic hypoxia. It was confirmed in situ proximity ligation assay and co-immunoprecipitation assay that although mild hypoxia up-regulated all HIF-1α, HIF-2α, and c-MYC, mTORC2-regulated HIF-2α competed with HIF-1α to bind c-MYC. HIF-2α knockdown, but not HIF-1α knockdown, decreased the expression of downstream c-MYC and mTORC1, suppressed hypoxic cell proliferation, and induced more cell apoptosis. Moreover, the inhibition of upstream protein PI3K by inhibitor Apitolisib counteracted this mechanism of adaptation to mild hypoxia hereby inducing cancer cells to apoptosis in HCC. Conclusions: In summary, this study highlights the role of HIF-2α but not HIF-1α in activating and binding c-MYC in HCC cell proliferation during mild chronic hypoxia response. PI3K/mTORC2/ HIF-2α/c-MYC axis plays a key role in this process. PI3K inhibitor Apitolisib is suggested as a potential treatment option for HCC, especially for rapidly growing HCC in mild chronic hypoxia.

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Apigenin Inhibits the Growth of Hepatocellular Carcinoma Cells by Affecting the Expression of microRNA Transcriptome

Frontiers in Oncology ◽

10.3389/fonc.2021.657665 ◽

2021 ◽

Vol 11 ◽

Author(s):

Shou-Mei Wang ◽

Pei-Wei Yang ◽

Xiao-Jun Feng ◽

Yi-Wei Zhu ◽

Feng-Jun Qiu ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Cycle ◽

Molecular Mechanisms ◽

Target Genes ◽

Dependent Manner ◽

Regulatory Pathways ◽

The Core ◽

Huh7 Cells

BackgroundApigenin, as a natural flavonoid, has low intrinsic toxicity and has potential pharmacological effects against hepatocellular carcinoma (HCC). However, the molecular mechanisms involving microRNAs (miRNAs) and their target genes regulated by apigenin in the treatment of HCC have not been addressed.ObjectiveIn this study, the molecular mechanisms of apigenin involved in the prevention and treatment of HCC were explored in vivo and in vitro using miRNA transcriptomic sequencing to determine the basis for the clinical applications of apigenin in the treatment of HCC.MethodsThe effects of apigenin on the proliferation, cell cycle progression, apoptosis, and invasion of human hepatoma cell line Huh7 and Hep3B were studied in vitro, and the effects on the tumorigenicity of Huh7 cells were assessed in vivo. Then, a differential expression analysis of miRNAs regulated by apigenin in Huh7 cells was performed using next-generation RNA sequencing and further validated by qRT-PCR. The potential genes targeted by the differentially expressed miRNAs were identified using a curated miRTarBase miRNA database and their molecular functions were predicted using Gene Ontology and KEGG signaling pathway analysis.ResultsCompared with the control treatment group, apigenin significantly inhibited Huh7 cell proliferation, cell cycle, colony formation, and cell invasion in a concentration-dependent manner. Moreover, apigenin reduced tumor growth, promoted tumor cell necrosis, reduced the expression of Ki67, and increased the expression of Bax and Bcl-2 in the xenograft tumors of Huh7 cells. Bioinformatics analysis of the miRNA transcriptome showed that hsa-miR-24, hsa-miR-6769b-3p, hsa-miR-6836-3p, hsa-miR-199a-3p, hsa-miR-663a, hsa-miR-4739, hsa-miR-6892-3p, hsa-miR-7107-5p, hsa-miR-1273g-3p, hsa-miR-1343, and hsa-miR-6089 were the most significantly up-regulated miRNAs, and their key gene targets were MAPK1, PIK3CD, HRAS, CCND1, CDKN1A, E2F2, etc. The core regulatory pathways of the up-regulated miRNAs were associated with the hepatocellular carcinoma pathway. The down-regulated miRNAs were hsa-miR-181a-5p and hsa-miR-148a-3p, and the key target genes were MAPK1, HRAS, STAT3, FOS, BCL2, SMAD2, PPP3CA, IFNG, MET, and VAV2, with the core regulatory pathways identified as proteoglycans in cancer pathway.ConclusionApigenin can inhibit the growth of HCC cells, which may be mediated by up-regulation or down-regulation of miRNA molecules and their related target genes.

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Low-Level Laser Irradiation Modulated Viability of Normal and Tumor Human Lymphocytes In Vitro

Journal of lasers in medical sciences ◽

10.34172/jlms.2020.29 ◽

2020 ◽

Vol 11 (2) ◽

pp. 174-180

Author(s):

Hesam Saghaei Bagheri ◽

Seyed Hossein Rasta ◽

Seyedeh Momeneh Mohammadi ◽

Ali Akbar Rahim Rahimi ◽

AliAkbar Movassaghpour ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Line ◽

Laser Irradiation ◽

Caspase 3 ◽

Mononuclear Cells ◽

Human Lymphocytes ◽

Dependent Manner ◽

Ki 67 ◽

Protein Levels

Introduction: Laser radiation is a promising strategy against various malignancies. Recent studies have shown that the application of low-power laser therapy (LPLT) at different doses and exposure times could modulate the growth dynamic of tumor cells. Based on the type of laser, LPLT could potentially trigger cell proliferation, differentiation, and apoptosis in different cell lines. Methods: In this study, MTT assay was used to monitor the effect of low and high laser intensities on the viability of normal and cancer lymphocytes. The protein levels of Ki-67 (a proliferation marker) and Caspase-3 (an apoptosis factor) were measured in human peripheral mononuclear cells (PBMCs) and the B-lymphoblastic cell line (Nalm-6) using flow cytometry after being-exposed to 630-nm LPLT at low (2, 4, 6, and 10 J/cm2 ) and high (15, 30, 60, and 120 J/cm2 ) energy densities in a continuous mode for 48 and 72 hours. Results: By using higher energy densities, 60 and 120 J/cm2 , a significant decrease was shown in the viability of Nalm-6 cells, which reached 6.6 and 10.1% after 48 hours compared to the control cells (P<0.05). Notably, Cell exposure to doses 30, 60, and 120 J/cm2 yielded 7.5, 12.9, and 21.6 cell viability reduction after 72 hours. The collected data showed that the high-intensity parameters of LPLT (15 to 120 J/cm2 ) promoted significant apoptotic changes in the exposed cells coincided with the activation of Caspase-3 compared to the none-treated control cells (P<0.05). The data further showed the stimulation of the Ki-67 factor both in primary PBMCs and the lymphoblastic cell line treated with LPLT at energy densities of 4 and 6 J/cm2 (P<0.05), indicating enhanced cell proliferation. Similar to Nalm-6 cells, primary PBMCs showed apoptosis after 48 hours of being exposed to doses 60, and 120 J/cm2 , indicated by increased Caspase-3 levels (P<0.05). As expected, the Nalm-6 cells were resistant to cytotoxic effects of laser irradiation in the first 48 hours (P>0.05) compared to normal PBMCs. The exposure of Nalm-6 cells to low-intensity laser intensities increased a proliferation rate compared to the PBMCs treated with the same doses. Conclusion: We showed the potency of LPLT in the induction of apoptosis and proliferation in human primary PBMCs and Nalm-6 cells in a dose and time-dependent manner after 72 hours.

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Activation of Tyrosine Metabolism in CD13+ Cancer Stem Cells Drives Relapse in Hepatocellular Carcinoma

Cancer Research and Treatment ◽

10.4143/crt.2019.444 ◽

2020 ◽

Vol 52 (2) ◽

pp. 604-621 ◽

Cited By ~ 4

Author(s):

Li Sun ◽

Lin Zhang ◽

Jun Chen ◽

Chaoqun Li ◽

Hongqin Sun ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Stem Cells ◽

Metabolic Pathways ◽

Isotope Labeling ◽

Chemotherapeutic Agents ◽

Protein Levels ◽

Huh7 Cells ◽

Tyrosine Metabolism

PurposeCancer stem cells (CSCs) are naturally resistant to chemotherapy, explaining why tumor relapse frequently occurs after initial regression upon administration of chemotherapeutic agents in most cases. A CSC population characterized by CD13 expression has been identified in hepatocellular carcinoma (HCC). In the current study, we aimed to clarify the molecular mechanism by which it escapes conventional therapies. Materials and MethodsHere, we used flow cytometry to examine the percentage of CD13+ CSCs in HepG2 and HuH7 cells after chemotherapy. Using in vitro isotope labeling technique, we compared metabolic pathways between CD13+ and CD13– subpopulations. Using co-immunoprecipitation and western blotting, we determined the target expressions in protein levels under different conditions. We also performed immunohistochemistry to detect the target proteins under different conditions. Animal models were constructed to verify the potential role of tyrosine metabolism in post-chemotherapeutic relapse in vivo.ResultsWe observed that quiescent CD13+ CSCs are enriched after chemotherapy in HCCs, and serve as a reservoir for recurrence. Mechanistically, CD13+ CSCs were dependent on aerobic metabolism of tyrosine rather than glucose as energy source. Tyrosine metabolism also generated nuclear acetyl-CoA to acetylate and stabilize Foxd3, thereby allowing CD13+ CSCs cells to sustain quiescence and resistance to chemotherapeutic agents.ConclusionThese findings encourage further exploration of eliminating CD13+ cells by targeting specific metabolic pathways to prevent recurrence in HCCs.

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A Traditional Chinese Medicine Herb Mixture Qingjie Fuzheng Granules Inhibits Hepatocellular Carcinoma Cells Growth by Inducing Apoptosis

Journal of Evidence-Based Integrative Medicine ◽

10.1177/2515690x18789632 ◽

2018 ◽

Vol 23 ◽

pp. 2515690X1878963 ◽

Cited By ~ 2

Author(s):

Pingping Zhong ◽

Hong Yang ◽

Shan Lin ◽

Jun Peng ◽

Jiumao Lin

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Proliferation ◽

Body Weight Gain ◽

Xenograft Model ◽

Annexin V ◽

Nuclear Staining ◽

Dependent Manner ◽

Mouse Xenograft Model

In this study, hepatocellular carcinoma (HCC) mouse xenograft model, MTT assay, colony formation, nuclear staining, and Annexin-V/PI staining assays were used to evaluate the effect of Qingjie Fuzheng granules (QFG) on cell proliferation and apoptosis of HCC cell in vivo and in vitro. Furthermore, Western blotting was performed to detect the expression of Fas, FasL, Bcl-2, Bax, and the activation of caspase-3/-8/-9. The results showed that QFG reduced tumor weight ( P < .05) but had no effect on body weight gain in HCC mice in vivo. QFG significantly reduced HCC cell viability and attenuated cell proliferation in a dose-dependent manner ( P < .05). QFG increased the expression of Fas, FasL, and Bax ( P < .05). QFG downregulated the expression of Bcl-2 and promoted the activation of caspase-8, -9, and -3 ( P < .05). These results suggested that QFG possessed anticancer effects, and the mechanisms of action may involve the death receptor pathway and mitochondrion-dependent pathway-mediated apoptosis.

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Curcumin Inhibits Hepatocellular Carcinoma via Regulating miR-21/TIMP3 Axis

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2020/2892917 ◽

2020 ◽

Vol 2020 ◽

pp. 1-13 ◽

Cited By ~ 1

Author(s):

Jingtao Li ◽

Hailiang Wei ◽

Yonggang Liu ◽

Qian Li ◽

Hui Guo ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Proliferation ◽

Signaling Pathway ◽

Luciferase Reporter ◽

Dependent Manner ◽

Anticancer Activities ◽

Anticancer Effects ◽

Cell Proliferation Inhibition

Background/Aim. Curcumin exhibits anticancer effects against various types of cancer including hepatocellular carcinoma (HCC). miR-21 has been reported to be involved in the malignant biological properties of HCC. However, whether miR-21 plays a role in curcumin-mediated treatment of HCC is unknown. The purpose of this study was to identify the potential functions and mechanisms of miR-21 in curcumin-mediated treatment of HCC. Methods. The anticancer effects of curcumin were assessed in vivo and in vitro. The underlying mechanism of miR-21 in curcumin-mediated treatment of HCC was assessed by quantitative real-time PCR (RT-qPCR), western blot, and Dual-Luciferase Reporter assays. Results. The present study revealed that curcumin suppressed HCC growth in vivo and inhibited HCC cell proliferation and induced cell apoptosis in a dose-dependent manner in vitro. Meanwhile, the curcumin treatment can downregulate miR-21 expression, upregulate TIMP3 expression, and inhibit the TGF-β1/smad3 signaling pathway. miR-21 inhibition enhanced the effect of curcumin on cell proliferation inhibition, apoptosis, and TGF-β1/smad3 signaling pathway inhibition in HepG2 and HCCLM3 cells. It demonstrated that TIMP3 was a direct target gene of miR-21. Interestingly, the effect of miR-21 inhibition on cell proliferation, apoptosis, and TGF-β1/smad3 signaling pathway in HepG2 and HCCLM3 cells exposed to curcumin was attenuated by TIMP3 silencing. Conclusion. Taken together, the present study suggests that miR-21 is involved in the anticancer activities of curcumin through targeting TIMP3, and the mechanism possibly refers to the inhibition of TGF-β1/smad3 signaling pathway.

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Effects of Lidocaine-Mediated CPEB3 Upregulation in Human Hepatocellular Carcinoma Cell Proliferation In Vitro

BioMed Research International ◽

10.1155/2018/8403157 ◽

2018 ◽

Vol 2018 ◽

pp. 1-7 ◽

Cited By ~ 2

Author(s):

Hongjun Liu ◽

Yiru Wang ◽

Bing Chen ◽

Xia Shen ◽

Wenxian Li

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Proliferation ◽

Antitumor Activity ◽

Cell Viability ◽

Hepg2 Cell ◽

Hepg2 Cells ◽

Human Hepatocellular Carcinoma ◽

Dependent Manner ◽

Real Time Quantitative Pcr

Lidocaine displays antitumor activity by inducing apoptosis and suppressing tumor growth in human hepatocellular carcinoma (HepG2) cells in vitro. However, the molecular mechanism underlying lidocaine-mediated antitumor activity is unclear. In this study, HepG2 cells were treated with lidocaine, and cell proliferation and colony-forming ability were assessed. The expression level of cytoplasmic polyadenylation element binding protein 3 (CPEB3) was detected by real-time quantitative PCR and western blot. Lidocaine treatment resulted in decreased HepG2 cell viability and colony formation in a dose-dependent manner. In hepatocellular carcinoma patient samples, CPEB3 was downregulated and was associated with poor prognosis and high-grade malignancy. Additionally, CPEB3 was a critical mediator of lidocaine-induced repression of HepG2 cell proliferation. These results demonstrated that lidocaine decreased cell viability and colony-forming ability of HepG2 cells by upregulating CPEB3 expression.

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Fraxetin Suppresses Cell Proliferation and Induces Apoptosis through Mitochondria Dysfunction in Human Hepatocellular Carcinoma Cell Lines Huh7 and Hep3B

Pharmaceutics ◽

10.3390/pharmaceutics13010112 ◽

2021 ◽

Vol 13 (1) ◽

pp. 112

Author(s):

Jisoo Song ◽

Jiyeon Ham ◽

Taeyeon Hong ◽

Gwonhwa Song ◽

Whasun Lim

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Proliferation ◽

Cell Lines ◽

Underlying Mechanism ◽

Mitogen Activated Protein Kinase ◽

Anticancer Effects ◽

Huh7 Cells ◽

Mitogen Activated Protein ◽

Hcc Cells

Fraxetin is a coumarin scaffold compound extracted from Fraxinus rhynchophylla. It has antioxidant, anti-inflammatory, hepatoprotective, and antifibrotic effects. Furthermore, fraxetin has anticancer effects in breast and lung cancer. We aimed to evaluate whether fraxetin has anticancer activity in hepatocellular carcinoma (HCC) cells and its underlying mechanism. We demonstrated the anticancer effects of fraxetin in the HCC cell lines Huh7 and Hep3B. We confirmed that fraxetin inhibited cell proliferation (42% ± 10% Huh7; 52% ± 7% Hep3B) by arresting the cell cycle and inducing apoptosis in both cell lines. Moreover, fraxetin increased reactive oxygen species production (221% ± 55% Huh7; 460% ± 73% Hep3B), depolarized the mitochondrial membranes (ΔΨm) (345% ± 160% Huh7; 462% ± 140% Hep3B), and disrupted calcium homeostasis in both HCC cell lines. Chelating calcium ions with BAPTA-AM restored proliferation in fraxetin-treated Huh7 cells but not in Hep3B cells. Fraxetin did not affect the phosphorylation of extracellular-signal-regulated kinase 1/2, whereas it decreased JNK and phosphoinositide 3-kinase signaling. Furthermore, fraxetin and mitogen-activated protein kinase pharmacological inhibitors had synergistic antiproliferative effects on HCC cells. Although our study was limited to in vitro data that require validation, we suggest that fraxetin is a potential therapeutic agent against HCC progression.

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