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

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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Autophagy Overactivity Regulate Terf1 and Terf2 in Positive and Negative-Telomerase Cancer Cell Lines

10.21203/rs.3.rs-1102455/v1 ◽

2021 ◽

Author(s):

mohammad panahi ◽

Saeed Samani ◽

Nasrin Mohajeri ◽

Akram Sadat Tabatabee Bafroee ◽

Kazem Baesi ◽

...

Keyword(s):

Flow Cytometry ◽

Real Time ◽

Cell Lines ◽

Real Time Pcr ◽

Huh7 Cell ◽

Gfp Expression ◽

Transfected Cells ◽

Autophagy Induction ◽

Huh7 Cells ◽

The Relationship

Abstract A recent suggestion for cancer therapy is targeting intracellular homeostatic signaling pathways like autophagy providing the balance between metabolism and cell cycling. Our study focused on investigating the relationship between autophagy activation by Beclin1 transfection and assessing Terf1 and Terf2 expression as shelterin proteins. The beclin1-containing plasmid was introduced to the U-2OS and Huh7 cell lines using Lipofectamine. The LC3-II as an intracellular autophagosomal marker was detected in transfected cells by flow cytometry. Also, the cells were treated with 3-methyladenine and metformin as autophagy inhibitors and inducers, respectively. Finally, the expression levels of Terf1 and Terf2 were analyzed by real-time PCR. Fluorescent images and flow cytometry results proved excellent GFP expression in the transfected cells. The results of real-time PCR demonstrated that autophagy induction by Beclin1 was increased Terf1 expression level in U-2OS cells up to 451%, while Huh7 cells suffered from the decreased expression of Terf1. Altogether, Terf2 expression was enhanced significantly in both cell lines after 48h treatment in comparison with 24h treatment. The obtained data provided that Beclin1-based activation of autophagy leads to overexpression of some protective shelterin proteins.

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Low Expression of hOCT1 May Be a Key Point Mediating Low Intracellular Imatinib Accumulation in Imatinib Mesylate Resistance K562 Cell Line.

Blood ◽

10.1182/blood.v110.11.4547.4547 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4547-4547

Author(s):

Huanling Zhu ◽

Ting Liu ◽

Yongqian Jia

Keyword(s):

Flow Cytometry ◽

Cell Line ◽

Real Time ◽

Cell Lines ◽

Real Time Pcr ◽

K562 Cells ◽

Pcr Analysis ◽

Sensitive Cell ◽

Imatinib Resistance ◽

Low Expression

Abstract Objective To establish an imatinib resistance cell line and to study its resistant principia. Methods K562 cells were cultured in imatinib at gradually increased concentrations to generate their resistance cell line. Clone imatinib resistance cell lines by limited dilution culture. MTT assay, real time PCR and Semi-quantity PCR, flow cytometry and HPLC were used to clarify the possible mechanisms of the resistance. Results Imatinib resistance cell line K562R was successfully induced by continuous culture in the presence of gradually increasing doses of imatinib up to 5μmol/L. K562R cells were maintained in the media containing 5μmol/L imatinib. Proliferation data showed that cell growth of K562R was not inhibited in 5 μmol/L imatinib, whereas the parental sensitive cell was significantly inhibited by up to 2μM imatinib. The IC50 of K562R was about 7.5μmol/L which was ten times higher than that of the parental cell. HPLC revealed that the intracellular imatinib concentration of K562R was strikingly lower than that of the parental cells (up to 27.8-fold). MDR1 were not detected in mRNA (by RT-PCR)and protein(by flow cytometry) levels on K562R cell, whereas hOCT1 level measured by semi-quantity PCR showed lower expression in K562R cell lines than that of parental sensitive cell, indicating that low intracellular imatinib concentration may be due to lower affluence of imatinib by low level of hOCT1. (5) Real time PCR analysis showed no BCR-ABL/G6PD gene amplification and sequence analysis of the 374bp ABL kinase domain showed no mutation in K562R cell lines. Conclusion An imatinib resistance cell line K562R has been successfully established. Low expression of hOCT1 may be a key point mediating low intracellular imaitnib accumulation in K562R cell lines.

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Thiazolidinedione or Rhodanine: A Study on Synthesis and Anticancer Activity Comparison of Novel Thiazole Derivatives

Journal of Pharmacy & Pharmaceutical Sciences ◽

10.18433/j38p9r ◽

2017 ◽

Vol 20 (1) ◽

pp. 415 ◽

Cited By ~ 6

Author(s):

Cigdem Ozen ◽

Meltem Ceylan Unlusoy ◽

Nazanin Aliary ◽

Mehmet Ozturk ◽

Oya Bozdag Dundar

Keyword(s):

Hepatocellular Carcinoma ◽

Anticancer Activity ◽

Cell Line ◽

Cell Lines ◽

Condensation Reaction ◽

Huh7 Cell ◽

Anticancer Activities ◽

Thiazole Derivatives ◽

Ic50 Values ◽

Huh7 Cell Line

Purpose: A new series of thiazolyl-2,4-thiazolidinedione / rhodanine compounds T1-T23 was synthesized and tested for their anticancer activities. Hepatocellular carcinoma cell lines were chosen due to their strong drug resistance to test the new compounds. Methods: All compounds were synthesized via Knoevenagel Condensation reaction and thiazolidinedione ester compounds (T3,T9,T15,T20) were hydrolyzed for obtaining the acidic compounds (T6,T12,T17,T23). All compounds were firstly screened for their anticancer activity against two hepatocellular carcinoma (HCC) cell lines, Huh7 and Plc/Prf/5 (Plc) cell lines by sulforhodamine B assay. Further IC50 values were calculated for three candidates (T4, T15, T21) in five different HCC (Huh7, Plc, Snu449, HepG2, Hep3B) and one breast cancer (Mcf7) cell line. Results: Compounds T4, T15, T21 had very strong anticancer effects even though their 10 µM concentration in Huh7 cell line. According to IC50 values, T21 was the most effective compound with IC50 values in a range from 2 to 16 µM in 6 cancer cell lines. In terms of cytotoxicity T21 mostly affected Huh7 and interestingly it was less effective against Plc. Conclusions: Considering these results it can be suggested that compounds T4, T15 and T21 may lead to the development of more potent anticancer drugs in the future. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.

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Identification of Characteristic Genomic Markers in Human Hepatoma Huh7 and Huh7.5.1-8 Cell Lines

10.1101/2020.02.17.953281 ◽

2020 ◽

Author(s):

Masaki Kawamoto ◽

Toshiyuki Yamaji ◽

Kyoko Saito ◽

Kazuhiro Satomura ◽

Toshinori Endo ◽

...

Keyword(s):

Cell Line ◽

Cell Lines ◽

Huh7 Cell ◽

Host Cells ◽

Human Hepatoma ◽

Loss Of Function ◽

Genomic Deletions ◽

Huh7 Cells ◽

Huh7 Cell Line

AbstractThe human hepatoma-derived Huh7 cell line and its derivatives (Huh7.5 and Huh7.5.1) have been widely used as a convenient experimental substitute for primary hepatocytes. In particular, these cell lines represent host cells suitable for propagating the hepatitis C virus (HCV) in vitro. The Huh7.5.1-8 cell line, a subline of Huh7.5.1, can propagate HCV more efficiently than its parental cells. To provide genomic information for cells’ quality control, we performed whole-genome sequencing of Huh7 and Huh7.5.1-8 and identified their characteristic genomic deletions, some of which are applicable to an in-house test for cell authentication. Among the genes related to HCV infection and replication, 53 genes were found to carry missense or loss-of-function mutations likely specific to the Huh7 and/or Huh7.5.1-8. Eight genes, including DDX58 (RIG-I), BAX, EP300, and SPP1 (osteopontin), contained mutations observed only in Huh7.5.1-8 or mutations with higher frequency in Huh7.5.1-8. These mutations might be relevant to phenotypic differences between the two cell lines and may also serve as genetic markers to distinguish Huh7.5.1-8 cells from the ancestral Huh7 cells.

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DPM1 expression as a potential prognostic tumor marker in hepatocellular carcinoma

PeerJ ◽

10.7717/peerj.10307 ◽

2020 ◽

Vol 8 ◽

pp. e10307

Author(s):

Ming Li ◽

Shengli Xia ◽

Ping Shi

Keyword(s):

Hepatocellular Carcinoma ◽

Overall Survival ◽

Liver Cancer ◽

Real Time ◽

Real Time Pcr ◽

Hepatoma Cells ◽

Genetic Alteration ◽

Quantitative Real Time Pcr ◽

Post Translational Modification

Background Altered glycosylation of proteins contributes to tumor progression. Dolichol phosphate mannose synthase (DPMS), an essential mannosyltransferase, plays a central role in post-translational modification of proteins, including N-linked glycoproteins, O-mannosylation, C-mannosylation and glycosylphosphatidylinositol anchors synthesis. Little is known about the function of DPMS in liver cancer. Methods The study explored the roles of DPMS in the prognosis of hepatocellular carcinoma using UALCAN, Human Protein Atlas, GEPIA, cBioPortal and Metascape databases. The mRNA expressions of DPM1/2/3 also were detected by quantitative real-time PCR experiments in vitro. Results The transcriptional and proteinic expressions of DPM1/2/3 were both over-expressed in patients with hepatocellular carcinoma. Over-expressions of DPMS were discovered to be dramatically associated with clinical cancer stages and pathological tumor grades in hepatocellular carcinoma patients. In addition, higher mRNA expressions of DPM1/2/3 were found to be significantly related to shorter overall survival in liver cancer patients. Futhermore, high genetic alteration rate of DPMS (41%) was also observed in patients with liver cancer, and genetic alteration in DPMS was associated with shorter overall survival in hepatocellular carcinoma patients. We also performed quantitative real-time PCR experiments in human normal hepatocytes and hepatoma cells to verify the expressions of DPM1/2/3 and results showed that the expression of DPM1 was significantly increased in hepatoma cells SMMC-7721 and HepG2. Conclusions Taken together, these results suggested that DPM1 could be a potential prognostic biomarker for survivals of hepatocellular carcinoma patients.

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UNDERSTANDING THE MECHANISM OF DRUG-RESISTANT AND TUMOR RECURRENCE IN LIVER CANCER

Journal of Drug Delivery and Therapeutics ◽

10.22270/jddt.v8i5.1863 ◽

2018 ◽

Vol 8 (5) ◽

pp. 224-229

Author(s):

Vasanthakumar Sekar ◽

Muthu Dhandapani ◽

Babu Balakrishnan ◽

Kaleeswaran Balasubramanian ◽

Sivalingam Azhagu Madhavan ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Flow Cytometry ◽

Liver Cancer ◽

Tumor Recurrence ◽

Morphological Analysis ◽

Multi Drug Resistance ◽

Flow Cytometry Analysis ◽

Spheroid Formation ◽

Entire Study ◽

Huh7 Cells

Chemo-resistant and tumor recurrence are the major hurdle to overcome the cancer patients. Especially in hepatocellular carcinoma (HCC) is notoriously refractory to chemotherapy because of its tendency to develop multi-drug resistance (MDR), through various mechanisms. Aim: The current research is focussed on understanding the mechanism involved in chemo-resistant and tumor recurrence in liver cancer. Methods: Human hepatocellular carcinoma cell line (Huh7) was used entire study. Huh7 cells were cultured with known chemotherapeutic drugs such as 5-FU, Paclitaxel and Cisplatin-based on their Cmax concentration, and then these drug-treated cells were examined for chemoresistant and tumor recurrence properties through flow cytometry analysis, spheroid formation assay, and morphological analysis. Results: In morphological analysis confirm these all the chemo drugs were shown more cytotoxic effete than control, even though there were few viable cells noticed in cisplatin treatment. In flow cytometry analysis cisplatin pre-treated cells were well expressed LCSC marker such as CD133 and stem cell transduction factors like Oct-4 & Nanog than control. In addition to this, all the CD133 expressed cells also expressed to EpCAM. In spheroid formation assay, cisplatin pre-treated cells shown well-defined spheroid than control. Conclusion: LCSC plays a major role in chemoresistant and tumor recurrence through PI3K/Akt/mTOR, wnt-β catenin signaling, NF-kB signaling. So, targeting LCSC through EpCAM targeted therapy along with chemotherapy might be the better option for enhanced prognosis. Keywords: LCSC, Chemoresistant, Tumor recurrence, Hepatocellular carcinoma.

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Amino-functionalized nanoparticles as a platform for mTOR activity modulation in hepatocellular carcinoma Huh7 cell line

Journal of Hepatology ◽

10.1016/s0168-8278(17)31749-x ◽

2017 ◽

Vol 66 (1) ◽

pp. S645-S646

Author(s):

M. Lunova ◽

V. Zablotskii ◽

A. Prokhorov ◽

M. Jirsa ◽

M. Hof ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Line ◽

Huh7 Cell ◽

Functionalized Nanoparticles ◽

Huh7 Cell Line ◽

Mtor Activity

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

BMC Cancer ◽

10.1186/s12885-021-08043-9 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Hui Li ◽

Yujuan Liu ◽

Wei Jiang ◽

Junhui Xue ◽

Yuning Cheng ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Liver Cancer ◽

Cellular Proliferation ◽

Chinese Herb ◽

New Drugs ◽

Phase Iii ◽

P53 Expression ◽

Expression Levels ◽

Cellular Apoptosis ◽

Afp Promoter

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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Anti-Cancer Effects of Glaucarubinone in the Hepatocellular Carcinoma Cell Line Huh7 via Regulation of the Epithelial-To-Mesenchymal Transition-Associated Transcription Factor Twist1

International Journal of Molecular Sciences ◽

10.3390/ijms22041700 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1700

Author(s):

Jihye Seo ◽

Jain Ha ◽

Eunjeong Kang ◽

Haelim Yoon ◽

Sewoong Lee ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Wound Healing ◽

Transcription Factor ◽

Cell Migration ◽

Cell Line ◽

Intracellular Signaling ◽

Epithelial To Mesenchymal Transition ◽

Mesenchymal Transition ◽

Huh7 Cells ◽

Anti Cancer

Hepatocellular carcinoma (HCC), the most common type of liver cancer, is a leading cause of cancer-related deaths. As HCC has a high mortality rate and its incidence is increasing worldwide, understanding and treating HCC are crucial for resolving major public health concerns. In the present study, wound healing screening assays were performed using natural product libraries to identify natural chemicals that can inhibit cancer cell migration. Glaucarubinone (GCB) showed a high potential for inhibiting cell migration. The anti-cancer effects of GCB were evaluated using the HCC cell line, Huh7. GCB showed anti-cancer effects, as verified by wound healing, cell migration, invasion, colony formation, and three-dimensional spheroid invasion assays. In addition, cells treated with GCB showed suppressed matrix metalloproteinase activities. Immunoblotting analyses of intracellular signaling pathways revealed that GCB regulated the levels of Twist1, a crucial transcription factor associated with epithelial-to-mesenchymal transition, and mitogen-activated protein kinase. The invasive ability of cancer cells was found to be decreased by the regulation of Twist1 protein levels. Furthermore, GCB downregulated phosphorylation of extracellular signal-regulated kinase. These results indicate that GCB exhibits anti-metastatic properties in Huh7 cells, suggesting that it could be used to treat HCC.

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