Apoptosis In Huh7 Cells Research Articles

Ribonucleic acid-binding protein CPSF6 promotes glycolysis and suppresses apoptosis in hepatocellular carcinoma cells by inhibiting the BTG2 expression

Hepatocellular carcinoma (HCC) is currently the sixth most common malignancy and the second major cause of tumor-related deaths in the world. This study aimed to investigate the role of cleavage and polyadenylation factor-6 (CPSF6) and B-cell translocation gene 2 (BTG2) in regulating the glycolysis and apoptosis in HCC cells. The RNA and protein expression of CPSF6 and BTG2 in normal hepatocyte and HCC were, respectively, detected by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis and Western blot analysis. The viability and apoptosis of transfected Huh-7 cells were, respectively, analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay. The expression of apoptosis-related proteins and HK-2 in transfected Huh-7 cells was also detected by Western blot analysis. The levels of glucose and lactate in the culture supernatant of transfected Huh-7 cells were, respectively, detected with the glucose assay kit and lactate assay kit. The interaction of CPSF6 and BTG2 was confirmed by RNA binding protein immunoprecipitation (RIP) assay. As a result, CPSF6 expression was increased while BTG2 expression was decreased in Huh-7 cells. Interference with CPSF6 suppressed the viability and glycolysis, and promoted the apoptosis of Huh-7 cells. Furthermore, CPSF6 interacted with BTG2 and interference with CPSF6 upregulated the BTG2 expression and inhibited the protein kinase B (AKT)/extracellular signal-regulated kinase (ERK)/nuclear factor (NF)-κB pathway. Interference with BTG2 could partially reverse the above cell changes caused by interference with CPSF6. In conclusion, CPSF6 inhibited the BTG2 expression to promote glycolysis and suppress apoptosis in HCC cells by activating AKT/ERK/NF-κB pathway.

Propofol induces apoptosis of hepatocellular carcinoma cells by upregulating miR-134 expression.

BackgroundPropofol is an anesthetic used in clinical surgery. Many studies have shown that propofol has the potential to kill cancer cells; however, the mechanism by which it kills hepatocellular carcinoma (HCC) cells remains unclear.MethodsTo examine this issue, the apoptosis change of HepG2 and Huh-7 cell lines treated with propofol were observed. Additionally, a quantitative reverse-transcriptase polymerase chain reaction was used to measure the expression level of hsa-miR-134 (miR-134), and a Cell Counting Kit-8 assay and flow cytometry assay were used to observe cell apoptosis. A dual-luciferase assay was used to confirm the binding effect of miR-134 and BCL-2 (B cell lymphoma-2), and a western blot assay was used to detect expression level changes of BCL-2 and cleaved caspase-3.ResultsThe results showed that propofol significantly promoted HepG2 and Huh-7 cell apoptosis, and that miR-134 expression level is related to the concentration of propofol. The dual-luciferase assay showed that miR-134 significantly reduced the luciferase activity of BCL-2-wt, but had no notable effect on BCL-2-mut. In rescue experiments, miR-134 deficiency resulted in a high apoptosis rate, a low BCL-2 expression level, and a high cleaved caspase-3 expression level induced by propofol in HepG2.ConclusionsIn summary, propofol appears to upregulate the expression level of miR-134, decrease the BCL-2 level, and induce HCC cell apoptosis by promoting the cleaved caspase-3 expression level.Trial registrationChinese Clinical Trial Registry ChiCTR18000199

Effect of Synchronous Versus Sequential Regimens on the Pharmacokinetics and Biodistribution of Regorafenib with Irradiation.

This study was performed to evaluate the interaction between conventional or high-dose radiotherapy (RT) and the pharmacokinetics (PK) of regorafenib in concurrent or sequential regimens for the treatment of hepatocellular carcinoma. Concurrent and sequential in vitro and in vivo studies of irradiation and regorafenib were designed. The interactions of RT and regorafenib in vitro were examined in the human hepatoma Huh-7, HA22T and Hep G2 cell lines. The RT–PK phenomenon and biodistribution of regorafenib under RT were confirmed in a free-moving rat model. Regorafenib inhibited the viability of Huh-7 cells in a dose-dependent manner. Apoptosis in Huh-7 cells was enhanced by RT followed by regorafenib treatment. In the concurrent regimen, RT decreased the area under the concentration versus time curve (AUC)regorafenib by 74% (p = 0.001) in the RT2 Gy × 3 fraction (f’x) group and by 69% (p = 0.001) in the RT9 Gy × 3 f’x group. The AUCregorafenib was increased by 182.8% (p = 0.011) in the sequential RT2Gy × 1 f’x group and by 213.2% (p = 0.016) in the sequential RT9Gy × 1 f’x group. Both concurrent regimens, RT2Gy × 3 f’x and RT9Gy × 3 f’x, clearly decreased the biodistribution of regorafenib in the heart, liver, lung, spleen and kidneys, compared to the control (regorafenib × 3 d) group. The concurrent regimens, both RT2Gy × 3 f’x and RT9Gy × 3 f’x, significantly decreased the biodistribution of regorafenib, compared with the control group. The PK of regorafenib can be modulated both by off-target irradiation and stereotactic body radiation therapy (SBRT).

Folic acid modified TPGS as a novel nano-micelle for delivery of nitidine chloride to improve apoptosis induction in Huh7 human hepatocellular\u2002carcinoma

BackgroundThe development of novel and effective drugs for targeted human hepatocellular carcinoma still remains a great challenge. The alkaloid nitidine chloride (NC), a component of a traditional Chinese medicine, has been shown to have anticancer properties, but doses at therapeutic levels have unacceptable side effects. Here we investigate folic acid modified D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS-FA) as a potential carrier for controlled delivery of the drug.MethodsSynthesized TPGS-FA was characterized by FTIR, UV-visible and 1H NMR spectroscopy, and TPGS loaded with NC was evaluated for its ability to induce apoptosis in Huh7 cells by Annexin V/PI and MTT assays, and observed by laser scanning confocal microscopy and inverted phase contrast microscopy.ResultsTPGS-FA/NC complexes were prepared successfully, and were homogenious with a uniform size of ~ 14 nm diameter. NC was released from the TPGS-FA/NC complexes in a controlled and sustained manner under physiological conditions (pH 7.4). Furthermore, its cytotoxicity to hepatocarcinoma cells was greater than that of free NC.ConclusionsTPGS-FA is shown to be useful carrier for drugs such as NC, and TPGS-FA/NC could potentially be a potent and safe drug for the treatment of hepatocellular carcinoma.

Zika M Oligopeptide ZAMP Confers Cell Death-Promoting Capability to a Soluble Tumor-Associated Antigen through Caspase-3/7 Activation.

Mosquito-borne Zika virus (ZIKV) is an emerging flavivirus of medical concern associated with neurological disorders. ZIKV utilizes apoptosis as a mechanism of cell killing. The structural M protein may play a role in flavivirus-induced apoptosis. The death-promoting capability of M has been restricted to an oligopeptide representing the residues M-32/40. Here, we evaluated the apoptosis inducing ability of the residues M-31/41 of ZIKV. The ZIKV M oligopeptide was associated to a soluble form of GFP (sGFP) and the resulting sGFP-M31/41 construct was assessed in Huh7 cells. Expression of sGFP-M31/41 can trigger apoptosis in Huh7 cells through caspase-3/7 activation. The translocation of sGFP-M31/41 in the endoplasmic reticulum was a prerequisite for apoptosis induction. The residues M-33/35/38 may play a critical role in the death-promoting activity of sGFP-M31/41. The effect of ZIKV M oligopeptide defined as ZAMP (for Zika Apoptosis M Peptide) on expression of a tumor-associated antigen was assayed on megakaryocyte-potentiating factor (MPF). Expression of MPF-ZAMP construct resulted in caspase-associated apoptosis activation in A549 and Huh7 cells. ZIKV has been proposed as an oncolytic virus for cancer therapy. The ability of the Zika M oligopeptide to confer death-promoting capability to MPF opens up attractive perspectives for ZAMP as an innovative anticancer agent.

Atorvastatin potentiates the chemosensitivity of human liver cancer cells to cisplatin via downregulating YAP1.

Atorvastatin is a competitive inhibitor of β-hydroxy β-methylglutaryl-CoA reductase, which is involved in anticancer effects in numerous types of cancer, including in human liver cancer. However, its functions and underlying mechanisms of chemosensitivity in liver cancer remain to be elucidated. The present study investigated the effect of atorvastatin on cisplatin chemosensitivity and its molecular mechanisms, with a focus on the Yes1-associated transcriptional regulator (YAP1) protein. The present study demonstrated that atorvastatin significantly potentiated chemosensitivity to cisplatin in the liver cancer HepG2 and Huh-7 cell lines. Furthermore, cell survival and apoptosis in liver cancer cell lines were analyzed using MTT assay and flow cytometry, respectively. Atorvastatin suppressed HepG2 and Huh-7 cell viability in a dose-dependent manner, similar to cisplatin and paclitaxel. Subtoxic levels of atorvastatin significantly increased cisplatin-induced apoptosis in Huh-7 cells. Atorvastatin-promoted chemosensitivity was predominantly mediated by caspase 3, caspase 9 and poly-(ADP ribose)-polymerase activation, and YAP1 downregulation. Finally, YAP1 overexpression significantly reversed the susceptibility of Huh-7 cells to cisplatin. Overall, the results of the present study suggested the underlying mechanisms of atorvastatin chemosensitivity in inducing liver cancer cell apoptosis via downregulating YAP1 and implicated the potential application of atorvastatin-potentiated chemosensitivity in liver cancer therapy.

Circular RNA ZNF292 affects proliferation and apoptosis of hepatocellular carcinoma cells by regulating Wnt/β-catenin pathway.

The purpose of this study was to explore the function of circular ribonucleic acid (circRNA) zinc finger protein 292 (ZNF292) in hepatocellular carcinoma (HCC). The expression of circRNA ZNF292 in Huh-7 cells was knocked down by small interfering RNAs (siRNAs), and the effect of circRNA ZNF292 knockdown on the proliferation of Huh-7 cells was analyzed by Cell Counting Kit-8 (CCK-8) assay and colony formation assay. Then, flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were adopted to analyze the impacts of circRNA ZNF292 knockdown on the cycle distribution and apoptosis of Huh-7 cells. Besides, the influences of circRNA ZNF292 knockdown on Wnt/β-catenin signaling pathway and its downstream molecules were detected via quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting. Compared with those in siRNA-normal control (NC) group, the proliferation of Huh-7 cells was significantly inhibited and their cloning ability was remarkably weakened (p<0.05), the proportion of cells in S phase was decreased while that in G1 phase was increased (p<0.05), the apoptosis rate of Huh-7 cells was higher and the number of apoptosis was larger in siRNA-2# knockdown group (p<0.05). Besides, in Huh-7 cells with circRNA ZNF292 knockdown, the expressions of Axin, β-catenin, phosphorylated signal transducer and activator of transcription 3 (p-STAT3), p-STAT5, Cyclin A and Cyclin-dependent kinase 2 (CDK2) were down-regulated, while the expressions of STAT3 and STAT5 did not change remarkably. Knock downing circRNA ZNF292 leads to cell cycle arrest in G1 phase, thus suppressing cell proliferation and promoting cell apoptosis. The regulatory mechanism of circRNA ZNF292 may involve the regulation of cell cycle and related genes.

Anti-Hepatoma Compound Determination by the Method of Spectrum Effect Relationship, Component Knock-Out, and UPLC-MS2 in Scheflera heptaphylla (L.)Frodin Harms and Its Mechanism.

Scheflera heptaphylla (L.)Frodin, a kind of Traditional Chinese Medicine, is commonly used in anti-inflammatory, analgesic, anti-viral, anti-tumor, and hemostasis. This study aimed to determine the anti-hepatoma components and its mechanism from the leaves of S. heptaphylla. The spectrum-effect relationships were analyzed by the method of Partial least squares, indicating that P1, P2, and P10 were positively correlated to inhibitory activity of Huh7 cells. Whereas others were negatively correlated. The technologies of component knock-out and UPLC-MS2 were used to determine compounds as 3,4-Dicaffeoylquinic acid (P6), 3,5-Dicaffeoylquinic acid (P7), 3α-Hydroxy-lup-20(29)-ene-23,28-dioic acid (P10, named Compound A). The results forecasted that Compound A had the best correlation with inhibitory activity. The effects of Compound A on the activities of human hepatoma cells (Huh7, SMMC-7721, HepG 2) and normal hepatocytes (L0-2, Chang liver) were evaluated. Cell apoptosis was observed with inverted microscope and flow cytometer. In addition, the proteins, related to apoptosis, were detected by Western blot. The results showed that Compound A (400 nM) could significantly inhibit the activity of three hepatoma cells (P < 0.001) with slight toxicity to normal hepatocytes, and the IC50 values were 285.3 and 315.1 nM, respectively, which were consistent with the prediction of spectrum-effect relationships. After treatment with Compound A, the number of hepatoma cells decreased significantly. And the apoptosis rate of Huh7 cells increased significantly (P < 0.001) in Compound A (200, 400 nM) groups, SMMC-7721 and HepG 2 were directly necrotic. Compound A groups could significantly improve the level of intracellular reactive oxygen species (ROS) (P < 0.05, P < 0.001) in Huh7 with no effect on normal hepatocytes. The content of apoptotic protein (Bax and Bim) in mitochondria was significantly increased in Compound A groups (P < 0.001). On the contrary, the content of anti-apoptotic protein (Bcl-xL and Mcl-1) decreased significantly (P < 0.001). These results demonstrated that Compound A was the main anti-hepatoma active component in the S. heptaphylla leaves. It achieved the effect of promoting apoptosis of Huh7 cells by regulating the levels of ROS and Bcl-2 family protein in mitochondrial apoptosis pathway.

NLRC3 silencing accelerates the invasion of hepatocellular carcinoma cell via IL-6/JAK2/STAT3 pathway activation.

Nucleotide-binding domain, leucine-rich repeat family with a caspase activation and recruitment domain 3 (NLRC3) participates in both immunity and cancer. The aim of this study was to determine the role of NLRC3 in human hepatocellular carcinoma (HCC) and the underlying mechanisms. We collected human liver tissues from nonalcoholic steatohepatitis (NASH), HCC, and adjacent normal tissues to characterize the pattern of NLRC3 expression by real-time quantitative polymerase chain reaction and immunohistochemistry. Then, we used the HCC cell line, HuH-7, transfected with small interfering RNA to silence the NLRC3 expression. 5-Ethynyl-2'-deoxyuridine assay, scratch assay, and transwell invasion assay were used for assessing proliferation, migration, and invasion, respectively. Flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were conducted to assess cell apoptosis. The expression of NLRC3 was reduced in human HCC tissues, compared with normal liver and nonalcoholic steatohepatitis tissues. After knocking down of NLRC3, the proliferation, migration, and invasion were increased in HuH-7 cells. And flow cytometry and TUNEL assay showed that HuH-7 cell apoptosis was suppressed after NLRC3 knockdown. As for the underlying mechanisms, knockdown of NLRC3 in HuH-7 cells was associated with the activation of Janus kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT3) pathway under interleukin-6 (IL-6) stimulation. NLRC3 expression was downregulated in human HCC tissues. NLRC3 silencing in HuH-7 cells can promote the proliferation, migration, and invasion of hepatocellular carcinoma cells. JAK2/STAT3 pathway activation induced by IL-6 may be the underlying mechanism for HCC when NLRC3 expression is silenced. And the invasion of HuH-7 cells was partially suppressed by the STAT3 specific inhibitor (cryptotanshinone). Therefore, NLRC3 may play a significant role in HCC and might be a therapeutic target for the treatment of HCC.

LncRNA-HEIH suppresses hepatocellular carcinoma cell growth and metastasis by up-regulating miR-199a-3p.

The aim of this study was to explore the functional changes of long non-coding ribonucleic acid (lncRNA)-HEIH on hepatocellular carcinoma (HCC) Huh7 and Hep3B cells. The expression changes of HEIH in 18 pairs of HCC tissues and adjacent normal tissues were detected by quantitative real time-polymerase chain reaction (qRT-PCR). According to its expression changes in HCC cells silenced by short hairpin ribonucleic acid (shRNA) transfection in vitro, these cells were divided into sh-HEIH group and sh-NC group. The effects of lowly expressed HEIH on the proliferation, migration and apoptosis of HCC cells were examined through functional assays. Western blotting was adopted to determine the expression changes of epithelial-mesenchymal transition (EMT) proteins, vimentin, matrix metalloproteinase (MMP)-2 and MMP-3. In addition, the role of HEIH downstream effector micro RNA (miR)-199a-3p in HCC was explored. Compared with adjacent normal tissues, HEIH was highly expressed in HCC tissues (p<0.01). HEIH silencing significantly inhibited the proliferation and migration, but induced the apoptosis of Huh7 cells (p<0.05). The expressions of vimentin and MMP-2 in sh-HEIH group were remarkably lower than those in sh-NC group (p<0.05). Furthermore, miR-199a-3p was identified as the downstream effector of HEIH. The expression of miR-199a-3p increased markedly in Huh7 and Hep3B cells with silenced HEIH expression (p<0.01). Moreover, when miR-199a-3p expression was inhibited, the effects of HEIH on Huh7 and Hep3B cells were weakened, manifested as notably enhanced cell proliferation and migration capabilities (p<0.05). LncRNA-HEIH suppresses HCC cell growth and metastasis by up-regulating miR-199a-3p. Our findings suggest that HEIH may be a promising target for HCC treatment.

MiR-328-3p overexpression attenuates the malignant proliferation and invasion of liver cancer via targeting Endoplasmic Reticulum Metallo Protease 1 to inhibit AKT phosphorylation.

BackgroundLiver cancer is one of the most common cancers worldwide. microRNAs (miRNAs) have been recognized as minimally invasive prognostic markers for distinct types of cancer. This study evaluates the mitigation role of miR-328-3p on liver cancer in vitro and in vivo.MethodsLiver cancer cell line Huh-7 and HepG2 were used for in vitro experiments. Compared with the control group, miR-328-3p overexpression inhibited the proliferation, invasion, and promoted apoptosis of Huh-7 cells. miR-328-3p and endoplasmic reticulum metalloprotease 1 (ERMP1) had an excellent targeting relationship. Compared with the pcDNA-ERMP1 transfection group, the ratios of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR in miR-328-3p mimic and pcDNA-ERMP1 co-transfection group were significantly decreased. Animal models were set up using four-week-old immunodeficient BABL/c female nude mice. Huh-7 cells transfected with lentivirus holding miR-328-3p or empty vector were injected into the right dorsal side of BABL/c nude mice, respectively. Tumor volume was measured every five days. After one month, animals were sacrificed, xenograft tumors were dissected and weighed for RT-PCR and immunohistochemical assays.ResultsCompared with control group, miR-328-3p overexpression significantly inhibited tumor weight (0.46±0.07 vs. 0.11±0.05 g, P<0.05) and tumor volume (1876±321 vs. 543±168 mm3, P<0.05) after thirty days. miR-328-3p overexpression significantly downregulated the percentage of Ki67 positive cells, N-cadherin positive cells and vimentin positive cells.ConclusionsThese findings suggested that miR-328-3p could be a new treatment or a novel marker for liver cancer prognosis.

Circ_0001178 regulates miR-382/VEGFA axis to facilitate hepatocellular carcinoma progression

Circular RNAs (circRNAs) have been reported to regulate the gene expression through sponging corresponding microRNAs in multiple malignant tumors, including hepatocellular carcinoma (HCC). Up to now, the effects of circ_0001178 in HCC are barely known. In our current work, we tested circ_0001178 expression in HCC tissues and HCC cells and found it was greatly elevated. Then, we evaluated the function of circ_0001178 on HCC cell proliferation. We found HepG2 and Huh-7 cell proliferation was repressed after circ_0001178 shRNA was infected into the cells. Moreover, flow cytometry evidenced that HepG2 and Huh-7 cell apoptosis was markedly triggered and cell cycle was arrested. Meanwhile, it was shown that HCC cell migration and invasion capacity were markedly inhibited by loss of circ_0001178. Knockdown of circ_0001178 restrained HCC tumor growth in vivo. Then, miR-382 was predicted and confirmed as the target of circ_0001178. Circ_0001178 was demonstrated to modulate miR-382 expression negatively. The effect of circ_0001178 on HCC tumor was rescued by miR-382 overexpression. Furthermore, vascular epithelial growth factor A (VEGFA) is identified in various cancers. Currently, VEGFA was proved to be the downstream target of miR-382. To conclude, this research revealed that circ_0001178 induced HCC progression via modulating miR-382 and VEGFA axis.

Low‑intensity ultrasound enhances the antitumor effects of doxorubicin on hepatocellular carcinoma cells through the ROS‑miR‑21‑PTEN axis.

Hepatocellular carcinoma (HCC) is a type of liver cancer and is a leading cause of cancer-associated mortality. In China, ~466,000 patients are diagnosed with HCC and it is responsible for ~422,000 cases of mortality each year. Surgery is the most effective treatment available; however it is only suitable for patients with early-stage HCC. Chemotherapy has been confirmed as a necessary treatment for patients with advanced HCC, although drug resistance may limit its clinical outcome. Low intensity ultrasound (LIUS) represents a novel therapeutic approach to treat patients with HCC; however, its underlying molecular mechanism remains unclear. In the present study, cell viability, apoptosis and reactive oxygen species (ROS) generation were determined via Cell Counting Kit-8, flow cytometry and 2′,7′-dichlorofluorescein diacetate assays, respectively. The expression of miRNA in HCC cells following exposure to LIUS and doxorubicin (Dox) was analyzed using a microarray and reverse transcription-quantitative polymerase chain reaction analysis. It was revealed treatment with LIUS in combination with Dox was able to induce apoptosis of Huh7 cells, increasing the intracellular levels of reactive oxygen species (ROS) and malondialdehyde. Glutathione peroxidase and superoxide dismutase 1 are ROS-scavenging enzymes, which serve important roles in the oxidative balance, preventing oxidative stress. The protein expression levels of these two enzymes were significantly decreased following treatment with LIUS combined with Dox. The present results suggested that LIUS may decrease Dox resistance in HCC cells and that LIUS may be combined with chemotherapy to treat HCC. By performing microarray analysis, the expression levels of microRNA-21 (miR-21) were decreased following treatment with LIUS combined with Dox. Functional experiments showed that knockdown of miR-21 enhanced the antitumor activity of Dox, whereas overexpression of miR-21 reversed these effects. Phosphatase and tensin homolog (PTEN), a well-known tumor suppressor, was revealed to be a direct target of miR-21, and its translation was suppressed by miR-21. Finally, it was determined that combined treatment of LIUS and Dox induced anticancer effects by blocking the activation of the AKT/mTOR pathway, as demonstrated by the downregulation of phosphorylated (p-)AKT and p-mTOR; N-acetylcysteine, a general ROS inhibitor reversed the suppressive effects on the AKT/mTOR pathway mediated by LIUS and Dox. Collectively, the present results suggested that LIUS increased cell sensitivity to Dox via the ROS/miR-21/PTEN pathway. Chemotherapy combined with LIUS may represent a novel effective therapeutic strategy to treat patients with advanced HCC.

Long Noncoding RNA RP5-833A20.1 Suppresses Tumorigenesis In Hepatocellular Carcinoma Through Akt/ERK Pathway By Targeting miR-18a-5p.

BackgroundPrevious studies indicated that long noncoding RNAs (lncRNAs) played vital roles in the development and progression of hepatocellular carcinoma (HCC). Recently, downregulation of lncRNA RP5‑833A20.1 has been observed in HCC tissues. However, the underlying mechanism by which RP5‑833A20.1 regulates the proliferation and apoptosis in HCC has not been investigated. Thus, this study aimed to investigate the role of RP5‑833A20.1 in the progression of HCC.MethodsThe levels of RP5‑833A20.1 in 30 pairs of HCC tissues and adjacent normal tissues were detected by RT-qPCR. In addition, the effects of RP5‑833A20.1 on cell proliferation, apoptosis and invasion were evaluated by CCK-8, flow cytometric, transwell assays, respectively. Meanwhile, the dual-luciferase reporter system assay was used to explore the interaction of RP5‑833A20.1 and miR-18a-5p in HCC.ResultsThe level of RP5‑833A20.1 was significantly downregulated in HCC tissues and HCC cell lines. Downregulation of RP5‑833A20.1 markedly promoted the proliferation and invasion of Bel-7402 cells. In addition, overexpression of RP5‑833A20.1 notably inhibited the proliferation and invasion of Huh7 cells. Moreover, overexpression of RP5‑833A20.1 obviously induced the apoptosis of Huh7 cells via increasing the levels of Bax and active caspase 3, and decreasing the levels of Bcl-2, p-Akt and p-ERK. Meanwhile, in vivo experiments performed also indicated that overexpression of RP5-833A20.1 could inhibit the tumorigenesis of subcutaneous Huh7 xenograft in nude mice. Furthermore, bioinformatics and luciferase reporter assay identified that RP5-833A20.1 functioned as a competing endogenous RNA (ceRNA) for miR-18a-5p in HCC.ConclusionIn this study, we found that RP5‑833A20.1 was downregulated in HCC tissues. In addition, RP5-833A20.1 could suppress the tumorigenesis in HCC through inhibiting Akt/ERK pathway by acting as a ceRNA for miR-18a-5p. Therefore, RP5-833A20.1 might be a valuable and potential biomarker and therapeutic target for the treatment of HCC.

Design, synthesis and biological evaluation of novel perimidine o-quinone derivatives as non-intercalative topoisomerase II catalytic inhibitors

For the development of novel anticancer agents, we designed and synthesized a total of 37 perimidine o-quinone derivatives containing the o-quinone group at the A or B ring and different substituents (alkyl groups, aryl groups or heterocycles) at the C ring of the compounds. The structure-activity relationships (SARs) were established based on the cytotoxicity data of compounds from the HL-60, Huh7, Hct116, and Hela cell lines. The cytotoxicity results showed that most compounds exhibited potent cytotoxicity. In particular, compound b-12 showed the best anti-proliferative activity (IC50 ≤ 1 μM) against four cancer cell lines and strong potency against the HL-60/MX2 (0.47 μM) cell line, which is resistant to Topo II poisons. Further studies showed that b-12 exhibited potent Topo IIα inhibitory activity (IC50 = 7.54 μM) compared with Topo I, which acted as a class of non-intercalative Topo IIα catalytic inhibitor by inhibiting the ATP binding site of Topo II. Cell apoptosis and cell cycle assays confirmed that b-12 could induce the apoptosis of Huh7 cells in a dose-dependent manner.

Bioactive glycoalkaloides isolated from Solanum melongena fruit peels with potential anticancer properties against hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is progressively increasing tumor with lack of accurate prognosis and inadequate systemic treatment approaches. Solanum sp. (such as Solanum melongena) is a folk herb which is reported to possess anticancer properties. In a continuity for our interest in pursuing the anticancer activity of compounds isolated from the fruit peels of Solanum melongena, the HPLC profiling and ESI-MS assessment for the methanolic extract evidenced the presence of bioactive glycoalkaloids (solasonine, solasodine and solamargine). These glycoalkaloids were isolated, purified and proved to possess in vitro cytotoxicity against human liver cancer cell lines (Huh7 and HepG2). Herein, we investigated the potential mechanism of action of these compounds using DNA content flow-cytometry and apoptosis/necrosis differential anaylsis using annexin-V/FITC staining. Solasonine, solasodine and solamargine inducd significant antiproliferative effect against liver cancer cells (Huh7 and HepG2) which was attributed to cell cycle arrest at S-phase. Solamargine, solasodine and solasonine induced significant apoptosis in Huh7 cells. Only solamargine-induced cell cycle arrest, was reflected as apoptotic cell killing effect against HepG2 cells. In conclusion, glycoalkaloids derived from Solanum melongena and particularly, solamargine are promising antiproliferative agents with potential anticancer effects.

Paeonol-mediated apoptosis of hepatocellular carcinoma cells by NF-κB pathway.

The aim of the study was to investigate the effect of paeonol on the apoptosis of hepatocellular carcinoma cells and to explore the possible mechanism of its effect. During the experiment, the human hepatoma (Huh7) cell line was cultured and treated with different concentrations of paeonol. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect the effects of paeonol at different concentrations on the proliferation of Huh7 cells after 24 h, and the optimal concentration of paeonol was selected for follow-up experiments. Huh7 cells were divided into the blank control group (C group), parthenolide [(an inhibitor of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB)] group (CE group), paeonol group (PO group), and paeonol + tumor necrosis factor-α (TNF-α) (an activator of NF-κB) group (PN group). The effect of paeonol on the apoptosis of Huh7 cells was detected via flow cytometry and Hoechst staining, respectively. The expression levels of NF-κB and protein apoptosis inhibitor-5 (p-API-5) were detected by semi-quantitative polymerase chain reaction (PCR) and western blot analysis, respectively, and the activity of NF-κB in cells was measured by NF-κB p65/50. After determination of the effects of paeonol at different concentrations on Huh7 cells by MTT assay, it was found that paeonol at the concentration of 200–800 µM could inhibit the proliferation of Huh7 cells (P<0.01), with 500 µM phenol being selected as the treatment concentration for follow-up experiments. Results of flow cytometry and Hoechst staining showed that the apoptotic levels of Huh7 cells in the PO and CE groups were significantly increased compared with that in the C group, and that in the PO group was higher than that in the PN group. The differences were statistically significant (P<0.01). Results of semi-quantitative PCR and western blot analysis revealed that the expression levels of NF-κB and p-API-5 in the PO and CE groups were significantly lower than those in the C group, and those in the PO group were lower than those in the PN group. The differences were statistically significant (P<0.01). The expression level of NF-κB p65/50 in the PO group was significantly lower than that in the C group (P<0.01). The results suggest that paeonol can significantly increase the apoptosis rate of Huh7 cells, and the possible mechanism of inducing apoptosis is related to the downregulation of NF-κB and p-API-5 and inhibition of the NF-κB signaling pathway.

Bioactivity-guided isolation of cytotoxic secondary metabolites from the roots of Glycyrrhiza glabra and elucidation of their mechanisms of action

Licorice (Glycyrrhiza glabra L.) is one of the most widely used plants worldwide for its various pharmacological activities. The aim of this study was to isolate the potential cytotoxic secondary metabolites from the MeOH extract prepared from the roots of Glycyrrhiza glabra through bioactivity-guided isolation procedure and to elucidate their mechanisms of action. The crude MeOH extract as well as CHCl3 and EtOAc subextracts significantly inhibited cell proliferation on hepatocelullar (Huh7), breast (MCF7) and colorectal (HCT116) cancer cell lines with IC50 values in the range of 5.6 to 33.6 μg/mL. Chromatographic seperations of the CHCl3 and EtOAc subextracts yielded 13 secondary metabolites. Structures were characterized based on NMR and MS data. Amongst isolates, glabridin (1), 4′-O-methylglabridin (2), β-amyrin (3), kanzonol U (4), glabrene (7) and tetrahydroxymethoxychalcone (10) were established to be responsible for in vitro cytotoxicity of G. glabra, exerting the best activity particularly against Huh7 cells. Further mechanistic studies demonstrated that 2 and 7 induced caspase-dependent apoptosis by increasing cytochrome C release and subsequently cleaved caspase-9 level in Huh7 cells. Moreover, both compounds decreased pRb and p21 levels and thus induced the accumulation of Huh-7 cells in subG1 and G2/M phases. Compound 10 which displayed the most potent activity in Hoechst staining and cell cycle assays through G2/M arrest, caused cell death by apoptosis in Huh7 cells.

MiR-106b regulates the apoptosis and tumorigenesis of hepatocellular carcinoma via targeting Zinc finger and BTB domain-containing protein 7A (Zbtb7a).

MicroRNAs play vital regulatory roles in various type of tumorigenesis. We aimed to explore the functional microRNAs that might play as therapeutic targets in hepatocellular carcinoma (HCC). In this study, our results revealed that microRNA-106b was significantly increased in HCC tumor tissues. However, miR-106b knockdown remarkably suppressed the growth and increased the apoptosis of Hub-7 HCC cells. Biological analysis indicated that miR-106b directly targeted toZinc finger and BTB domain-containing protein 7A (Zbtb7a) to regulate the apoptosis of Hub-7 cells. Extensively, Zbtb7a overexpression reversed Huh-7 cell apoptosis and growth in vitro. Furthermore, in vivo studies confirmed that miR-106b inhibition or Zbtb7a overexpression retarded the growth of Hub-7 xenograft tumor in nude mice. In conclusion, we provide the evidence for the regulatory role of miR-106b in HCC, which is causally linked to targeting of Zbtb7a. This study may provide miR-106b as a potential therapeutic strategy for HCC.

Abstract 2681: Antiproliferative effects of the naturally occurring solamargine and related glycoalkaloids against liver cancer cells

Abstract Hepatocellular carcinoma (HCC) is progressively increasing tumor with lack of accurate prognosis and inadequate systemic treatment approaches. Solanum sp. (such as Solanum melongena) is a folk herb which is reported to possess anticancer properties. In a continuity for our interest in pursuing the anticancer activity of compounds isolated from the fruit peels of Solanum melongena, the HPLC profiling and ESI-MS assessment for the methanolic extract evidenced the presence of bioactive glycoalkaloids (solasonine, solasodine and solamargine). These glycoalkaloids were isolated, purified and proved to possess in vitro cytotoxicity against human liver cancer cell lines (Huh7 and HepG2). Herein, we investigated the potential mechanism of action of these compounds using DNA content flow-cytometry and apoptosis/necrosis differential anaylsis using annexin-V/FITC staining. Solasonine, solasodine and solamargine inducd significant antiproliferative effect against liver cancer cells (Huh7 and HepG2) which was attributed to cell cycle arrest at S-phase. Solamargine, solasodine and solasonine induced significant apoptosis in Huh7 cells. Only solamargine-induced cell cycle arrest, was reflected as apoptotic cell killing effect against HepG2 cells. In conclusion, glycoalkaloids derived from Solanum melongena and particularly, solamargine are promising antiproliferative agents with potential anticancer effects. Citation Format: Mostafa I. Fekry, Shahira M. Ezzat, Maha M. Salama, Ahmed M. Al-Abd. Antiproliferative effects of the naturally occurring solamargine and related glycoalkaloids against liver cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2681.