scholarly journals Berberine Sensitizes Human Hepatoma Cells to Regorafenib via Modulating Expression of Circular RNAs

2021 ◽  
Vol 12 ◽  
Author(s):  
Kunyuan Wang ◽  
Ganxiang Yu ◽  
Jiaen Lin ◽  
Zhilei Wang ◽  
Qianting Lu ◽  
...  

Regorafenib resistance is a key limiting factor in the treatment of advanced hepatocellular carcinoma (HCC). Increasing evidence has demonstrated that Berberine (BBR) can synergistically enhance the therapeutic effect of various chemotherapeutic agents. However, the contribution of BBR on regorafenib therapy remains unclear. The purpose of this study was to explore the combined treatment effect of berberine and regorafenib in HCC. We found that BBR enhanced the cytotoxicity of regorafenib in HCC cells. Compared with regorafenib alone, the combined treatment of BBR and regorafenib significantly inhibited the proliferation of HCC cells and induced cellular apoptosis. Meanwhile, the combined treatment group with BBR (10mg/kg/day) and regorafenib (5mg/kg/day) had a dramatic inhibitory effect on the growth of HCC xenograft tumors in nude mice. The increased apoptosis of xenograft tumors was seen in the combined treatment group. Moreover, a comprehensive circular RNA sequencing was performed to identify differentially expressed circRNAs in HCC cells after exposure to 100µM BBR and 5µM regorafenib. The volcano plot and scatter plot analyses revealed that there were 58 up-regulated and 19 down-regulated differentially expressed circRNAs between the combination treatment and control groups. Among them, the expression of hsa_circ_0032029 and hsa_circ_0008928 were up-regulated in HCC cells after treatment with 100µM BBR and 5µM regorafenib. Taken together, this study demonstrated that BBR enhanced the anti-HCC effect of regorafenib both in vitro and in vivo. The synergistic anti-tumor effect of BBR and regorafenib might be related to the up-regulation of hsa_circ_0032029 and hsa_circ_0008928 in HCC cells.

2020 ◽  
Vol 38 (4_suppl) ◽  
pp. 551-551
Author(s):  
Shiri Davidi ◽  
Catherine Tempel-Brami ◽  
Mijal Munster ◽  
Karnit Gotlib ◽  
Einav Zeevi ◽  
...  

551 Background: Hepatocellular carcinoma (HCC) is a leading global cause of cancer-related mortality. Sorafenib (oral multikinase inhibitor) is approved in patients with advanced HCC, yet survival benefit is limited. Tumor Treating Fields (TTFields) are an effective, anti-neoplastic treatment modality delivered via noninvasive, low intensity (1-3 V/cm), intermediate frequency (100-500 kHz), alternating electric fields. The study aim was to explore in vitro and in vivo effects of TTFields alone and combined with sorafenib for HCC treatment. Methods: HCC (HepG2 and Huh-7D12) cells were TTFields treated with at frequencies of 100-400 kHz for 72 hr using the inovitro system. Efficacy of TTFields and sorafenib combined treatment was tested at optimal frequency with various sorafenib concentrations. Cell counts, apoptosis induction, and clonogenic potential were determined. Healthy rats were used to assess safety of TTFields applied to the abdomen. N1S1 HCC cells were injected into the left hepatic lobe of Sprague Dawley rat; after 1 week, TTFields (1.2 V/cm) and sorafenib (10 mg/kg) were applied for 6 days. Tumor growth was evaluated using MRI. Results: The optimal TTFields frequency was 150 kHz in HepG2 and Huh-7D12 HCC cells. TTFields 150 kHz treatment (1.0 - 1.7 V/cm, 72 hr) led to cell count reductions (53-55%) and further decreases in clonogenic potential (65-69%). TTFields and sorafenib combination treatment led to a significant reduction in cell count (2-way ANOVA, P < 0.05) vs either treatment alone. Also, tumor growth was significantly reduced in the combined treatment group vs the control group (student t test, P < 0.01). Tumor volume (fold increase) in the combination treatment group (1.6) was significantly lower vs control (5.9, P < 0.0001), TTFields alone (3.3, P < 0.01), and sorafenib alone (2.3, P < 0.05) groups. Safety studies did not reveal any TTFields related adverse events with delivery to the rat abdomen. Conclusions: In vitro and in vivo data demonstrated efficacy and safety of TTFields in HCC; and improved efficacy in combination with sorafenib. A phase 2 study (HEPANOVA; NCT03606590) will explore the clinical potential of TTFields 150 kHz plus sorafenib.


2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Ying Xie ◽  
Xiaofeng Hang ◽  
Wensheng Xu ◽  
Jing Gu ◽  
Yuanjing Zhang ◽  
...  

Abstract Background Most of the biological functions of circular RNAs (circRNAs) and the potential underlying mechanisms in hepatocellular carcinoma (HCC) have not yet been discovered. Methods In this study, using circRNA expression data from HCC tumor tissues and adjacent tissues from the Gene Expression Omnibus database, we identified out differentially expressed circRNAs and verified them by qRT-PCT. Functional experiments were performed to evaluate the effects of circFAM13B in HCC in vitro and in vivo. Results We found that circFAM13B was the most significantly differentially expressed circRNA in HCC tissue. Subsequently, in vitro and in vivo studies also demonstrated that circFAM13B promoted the proliferation of HCC. Further studies revealed that circFAM13B, a sponge of miR-212, is involved in the regulation of E2F5 gene expression by competitively binding to miR-212, inhibits the activation of the P53 signalling pathway, and promotes the proliferation of HCC cells. Conclusions Our findings revealed the mechanism underlying the regulatory role played by circFAM13B, miR-212 and E2F5 in HCC. This study provides a new theoretical basis and novel target for the clinical prevention and treatment of HCC.


2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Junjie Xu ◽  
Lin Ji ◽  
Yuelong Liang ◽  
Zhe Wan ◽  
Wei Zheng ◽  
...  

AbstractSorafenib is the first-line chemotherapeutic therapy for advanced hepatocellular carcinoma (HCC). However, sorafenib resistance significantly limits its therapeutic efficacy, and the mechanisms underlying resistance have not been fully clarified. Here we report that a circular RNA, circRNA-SORE (a circular RNA upregulated in sorafenib-resistant HCC cells), plays a significant role in sorafenib resistance in HCC. We found that circRNA-SORE is upregulated in sorafenib-resistant HCC cells and depletion of circRNA-SORE substantially increases the cell-killing ability of sorafenib. Further studies revealed that circRNA-SORE binds the master oncogenic protein YBX1 in the cytoplasm, which prevents YBX1 nuclear interaction with the E3 ubiquitin ligase PRP19 and thus blocks PRP19-mediated YBX1 degradation. Moreover, our in vitro and in vivo results suggest that circRNA-SORE is transported by exosomes to spread sorafenib resistance among HCC cells. Using different HCC mouse models, we demonstrated that silencing circRNA-SORE by injection of siRNA could substantially overcome sorafenib resistance. Our study provides a proof-of-concept demonstration for a potential strategy to overcome sorafenib resistance in HCC patients by targeting circRNA-SORE or YBX1.


2018 ◽  
Vol 51 (3) ◽  
pp. 1389-1398 ◽  
Author(s):  
Lili Zhu ◽  
Tingting Ren ◽  
Zixin Zhu ◽  
Mingliang  Cheng ◽  
Qiuju Mou ◽  
...  

Background/Aims: Hepatic stellate cells (HSCs) are the primary cell type responsible for liver fibrosis. Our study proved that thymosin beta 4 (Tβ4) has anti-fibrogenic effects in HSCs through PI3K/AKT pathway. However, the underlying mechanisms are not fully elucidated. Circular RNAs (circRNAs) play important roles in fine-tuning gene expression and are often deregulated in cancers. However, the expression profile and clinical significance of in liver fibrosis is still unknown. Therefore, we hypothesize that Tβ4 influences circRNAs in liver fibrosis. Methods: Circular RNA microarray was conducted to identify Tβ4-related circRNAs. Pathway analysis and miRNA response elements analysis was conducted to predict the potential roles of differentially expressed circRNAs in liver fibrosis. CCK8 assays and flow cytometric assays were conducted to clarify the role of circRNA in liver fibrosis. Bioinformatics analysis and in vitro experiments were conducted to clarify the mechanism of circRNA-mediated gene regulation in liver fibrosis. Results: A total of 644 differentially expressed circRNAs were identified between the Tβ4-depleted LX-2 cells and the control LX2 cells. The expression of circRNA-0067835 was significantly increased in the Tβ4-depleted LX-2 cells compared with control. Knockdown of circRNA-0067835 observably decreased LX-2 cell proliferation by causing G1 arrest and promoting apoptosis. Bioinformatics online programs predicted that circRNA-0067835 acted as miR-155 sponge to regulate FOXO3a expression, which was validated using luciferase reporter assay. Conclusion: Our experiments showed that circRNA-0067835 regulated liver fibrosis progression by acting as a sponge of miR-155 to promote FOXO3a expression, indicating that circRNA-0067835 may serve as a potential therapeutic target for patients with liver fibrosis.


2019 ◽  
Vol 10 (10) ◽  
Author(s):  
Rahul Sreekumar ◽  
Muhammad Emaduddin ◽  
Hajir Al-Saihati ◽  
Karwan Moutasim ◽  
James Chan ◽  
...  

Abstract Epithelial–mesenchymal transition (EMT) is a process by which tumour cells lose epithelial characteristics, become mesenchymal and highly motile. EMT pathways also induce stem cell features and resistance to apoptosis. Identifying and targeting this pool of tumour cells is a major challenge. Protein kinase C (PKC) inhibition has been shown to eliminate breast cancer stem cells but has never been assessed in hepatocellular cancer (HCC). We investigated ZEB family of EMT inducer expression as a biomarker for metastatic HCC and evaluated the efficacy of PKC inhibitors for HCC treatment. We showed that ZEB1 positivity predicted patient survival in multiple cohorts and also validated as an independent biomarker of HCC metastasis. ZEB1-expressing HCC cell lines became resistant to conventional chemotherapeutic agents and were enriched in CD44high/CD24low cell population. ZEB1- or TGFβ-induced EMT increased PKCα abundance. Probing public databases ascertained a positive association of ZEB1 and PKCα expression in human HCC tumours. Inhibition of PKCα activity by small molecule inhibitors or by PKCA knockdown reduced viability of mesenchymal HCC cells in vitro and in vivo. Our results suggest that ZEB1 expression predicts survival and metastatic potential of HCC. Chemoresistant/mesenchymal HCC cells become addicted to PKC pathway and display sensitivity to PKC inhibitors such as UCN-01. Stratifying patients according to ZEB1 and combining UCN-01 with conventional chemotherapy may be an advantageous chemotherapeutic strategy.


Biomedicines ◽  
2020 ◽  
Vol 8 (12) ◽  
pp. 531
Author(s):  
Vivian P. Wagner ◽  
Manoela D. Martins ◽  
Esra Amoura ◽  
Virgilio G. Zanella ◽  
Rafael Roesler ◽  
...  

The brain-derived neurotrophic factor (BDNF)/tyrosine receptor kinase B (TrkB) pathway was previously associated with key oncogenic outcomes in a number of adenocarcinomas. The aim of our study was to determine the role of this pathway in mucoepidermoid carcinoma (MEC). Three MEC cell lines (UM-HMC-2, H253 and H292) were exposed to Cisplatin, the TrkB inhibitor, ANA-12 and a combination of these drugs. Ultrastructural changes were assessed through transmission electron microscopy; scratch and Transwell assays were used to assess migration and invasion; and a clonogenic assay and spheroid-forming assay allowed assessment of survival and percentage of cancer stem cells (CSC). Changes in cell ultrastructure demonstrated Cisplatin cytotoxicity, while the effects of ANA-12 were less pronounced. Both drugs, used individually and in combination, delayed MEC cell migration, invasion and survival. ANA-12 significantly reduced the number of CSC, but the Cisplatin effect was greater, almost eliminating this cell population in all MEC cell lines. Interestingly, the spheroid forming capacity recovered, following the combination therapy, as compared to Cisplatin alone. Our studies allowed us to conclude that the TrkB inhibition, efficiently impaired MEC cell migration, invasion and survival in vitro, however, the decrease in CSC number, following the combined treatment of ANA-12 and Cisplatin, was less than that seen with Cisplatin alone; this represents a limiting factor.


2021 ◽  
Vol 22 (24) ◽  
pp. 13247
Author(s):  
Tugce Batur ◽  
Ayse Argundogan ◽  
Umur Keles ◽  
Zeynep Mutlu ◽  
Hani Alotaibi ◽  
...  

AXL, a member of the TAM family, is a promising therapeutic target due to its elevated expression in advanced hepatocellular carcinoma (HCC), particularly in association with acquired drug resistance. Previously, RNA interference was used to study its role in cancer, and several phenotypic changes, including attenuated cell proliferation and decreased migration and invasion, have been reported. The mechanism of action of AXL in HCC is elusive. We first studied the AXL expression in HCC cell lines by real-time PCR and western blot and showed its stringent association with a mesenchymal phenotype. We then explored the role of AXL in mesenchymal SNU475 cells by CRISPR-Cas9 mediated gene knock-out. AXL-depleted HCC cells displayed drastic phenotypic changes, including increased DNA damage response, prolongation of doubling time, G2 arrest, and polyploidization in vitro and loss of tumorigenicity in vivo. Pharmacological inhibition of AXL by R428 recapitulated G2 arrest and polyploidy phenotype. These observations strongly suggest that acute loss of AXL in some mesenchymal HCC cells is lethal and points out that its inhibition may represent a druggable vulnerability in AXL-high HCC patients.


2021 ◽  
Author(s):  
Ruhua Wang ◽  
Yunong Fu ◽  
Menglin Yao ◽  
Xiaomeng Cui ◽  
Yan Zhao ◽  
...  

Abstract Background: The oxaliplatin-based chemotherapy has revealed an encouraging therapeutic efficacy for advanced hepatocellular carcinoma patients. However, the development of resistance limits its clinical utilization. In addition, the chemotherapy resistance in HCC is usually accompanied with other malignant phenotypes, such as cell proliferation and metastasis, which together result in poor prognosis of HCC patients. Therefore, efforts should be made to explore potential regulators which fuel multiple events of HCC progression.Methods: The qRT-PCR, western blot, immunohistochemistry and immunofluorescence were performed to measure mRNA and protein expression. MTT assay, colony formation and Transwell assay were performed to evaluate cell proliferation and metastasis. Flow cytometry was performed to test cell apoptosis. Alkaline Comet assay was performed to measure DNA lesions. Transmission electron microscope analysis provided potent testimony of autophagy. The role of HN1 on the malignant phenotypes of hepatoma carcinoma was demonstrated in vitro and in vivo.Results: The immunohistochemistry analysis of HCC patient tissues revealed that the expression of HN1 was higher in HCC tissues compared to adjacent tissues and was associated with worse prognosis. In vitro, HN1 knockdown inhibited proliferation and metastasis of HCC cells, whereas HN1 overexpression promoted their proliferation and metastasis. In addition, we found that HN1 knockdown sensitized HCC cells to oxaliplatin, which is companied with deteriorated DNA damage and increased cell apoptosis in oxaliplatin-treated HCC cells. In vivo, HN1 knockdown inhibited the tumor growth and metastasis, and promoted the anti-cancer efficiency of oxaliplatin. Mechanically, HN1 prevented HMGB1 from ubiquitination and degradation via autophagy-lysosome pathway, which is related to its interaction with TRIM28, and overexpression of HMGB1 can restore the malignant phenotypes of HN1 knockdown in HCC cells. Furthermore, we found that HN1 can regulate cellular autophagy via HMGB1, which is important to tumor-promoting effect of HN1.Conclusions: In conclusion, we systemically revealed the multiple functions of HN1 in HCC progression and the underlying molecular mechanism, which indicated that HN1 could be a promising therapeutic target for HCC treatment.


2021 ◽  
Vol 11 ◽  
Author(s):  
Xi He ◽  
Huiwei Sun ◽  
Qiyu Jiang ◽  
Yantao Chai ◽  
Xiaojuan Li ◽  
...  

Increasing evidence has shown that the metabolism and clearance of molecular targeted agents, such as sorafenib, plays an important role in mediating the resistance of HCC cells to these agents. Metabolism of sorafenib is performed by oxidative metabolism, which is initially mediated by CYP3A4. Thus, targeting CYP3A4 is a promising approach to enhance the sensitivity of HCC cells to chemotherapeutic agents. In the present work, we examined the association between CYP3A4 and the prognosis of HCC patients receiving sorafenib. Using the online tool miRDB, we predicted that has-microRNA-4277 (miR-4277), an online miRNA targets the 3’UTR of the transcript of cyp3a4. Furthermore, overexpression of miR-4277 in HCC cells repressed the expression of CYP3A4 and reduced the elimination of sorafenib in HCC cells. Moreover, miR-4277 enhanced the sensitivity of HCC cells to sorafenib in vitro and in vivo. Therefore, our results not only expand our understanding of CYP3A4 regulation in HCC, but also provide evidence for the use of miR-4277 as a potential therapeutic in advanced HCC.


2020 ◽  
Author(s):  
Yaoting Chen ◽  
Huiqing Li ◽  
Dong Chen ◽  
Xiongying Jiang ◽  
Weidong Wang ◽  
...  

Abstract Background : Although arsenic trioxide (ATO) is used in the treatment of advanced hepatocellular carcinoma (HCC) in clinical trials, it is not satisfactory in terms of improving HCC patients’ overall survival. Intratumoral hypoxia and overexpression of hypoxia-inducible-1α (HIF-1α) may result in ATO-resistance and tumor progression. We investigated the mechanisms involving HIF-1α expression and acquired ATO chemoresistance in HCC cells and mice. Methods: The therapeutic effects of ATO in normoxic and hypoxic HCC cells were assessed using cell viability and apoptosis assays in vitro and a xenograft model in vivo . mRNA and protein expression of HIF-1α, P-glycoprotein, and VEGF were measured by qRT-PCR and western blotting. HIF-1α inhibition was performed to investigate the mechanism of ATO-resistance. VEGF secretion was tested using ELISA and tube-formation assays. Results : Compared to normoxic cells, hypoxic HCC cells were more resistant to ATO, with higher IC 50 values and less apoptosis, and upregulated HIF-1α protein expression, accompanied with the enhancement of P-glycoprotein and VEGF synthesis after ATO treatment. VEGF secretion was elevated in the supernatant of ATO-treated HCC cells, and this change can potentiate angiogenesis in vitro . HIF-1α inhibition attenuated ATO-resistance and angiogenesis, and promoted the anticancer effects of ATO both in vitro and in vivo by downregulating therapy-induced P-glycoprotein and VEGF overexpression. Conclusions : Hypoxic HCC cells acquire ATO resistance by upregulating HIF-1α levels; thus, combining ATO with a HIF-1α-targeting agent may lead to enhanced antitumor effects in HCC.


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