High-expressing Cells Research Articles

Methylation-mediated silencing of the LIM homeobox 6 (LHX6) gene promotes cell proliferation in human pancreatic cancer

Epigenetic gene silencing by aberrant DNA methylation leads to loss of key cellular pathways in tumorigenesis. DNA methylation-mediated silenced genes in pancreatic cancer were searched for using the methyl-CpG targeted transcriptional activation (MeTA) method, and LHX6 (LIM homeobox 6), a transcription factor involved in embryogenesis and head development, was selected as a strong candidate gene. LHX6 was downregulated in most of the pancreatic cancer cell lines (83%, 10/12), mainly through promoter hypermethylation and histone deacetylation. Furthermore, LHX6 was methylated in primary pancreatic cancer specimens (57%, 16/28) in a tumor-specific manner. Re-expression of LHX6 inhibited colony formation and proliferation in LHX6 low-expressing pancreatic cancer cell lines, PK-1 and PK-9. In contrast, knockdown of LHX6 accelerated cell proliferation in LHX6 high-expressing pancreatic cancer cell lines, PCI-35 and MIA PaCa-2. In order to analyze LHX6 downstream genes, we performed microarray analyses using LHX6 inducible PK-1 and PK-9 and found that LHX6 induction upregulated several genes that had tumor suppressive functions. Among these, we focused on TFPI2 (Tissue factor pathway inhibitor-2) and found that TFPI2 was greatly downregulated in all twelve pancreatic cancer cell lines. Our present results suggest that epigenetic inactivation of LHX6 plays an important role in pancreatic tumorigenesis by promoting cell proliferation through aberrant transcriptional regulation of several cancer-related genes.

Cold atmospheric plasma induces GSDME-dependent pyroptotic signaling pathway via ROS generation in tumor cells

Cold atmospheric plasma (CAP) has been proposed as a novel promising anti-cancer treatment modality. Apoptosis and necrosis have been revealed in CAP-induced cell death, but whether CAP induces pyroptosis, another kind of programmed cell death is still unknown. In the present study, we first reported that CAP effectively induced pyroptosis in a dose-dependent manner in Gasdermin E (GSDME) high-expressed tumor cell lines. Interestingly, the basal level of GSDME protein was positively correlated with the sensitivity to CAP in three selected cancer cell lines, implying GSDME might be a potential biomarker of prognosis in the forthcoming cancer CAP treatment. Moreover, our study revealed that CAP-induced pyroptosis depended on the activation of mitochondrial pathways (JNK/cytochrome c/caspase-9/caspase-3) and the cleavage of GSDME but not Gasdermin D (GSDMD). ROS generation induced by CAP was identified to initiate the pyroptotic signaling. These results complemented our knowledge on CAP-induced cell death and provide a strategy to optimize the effect of CAP cancer treatment.

H1R mediates local anesthetic-induced vascular permeability in angioedema

Angioedema may occur during local anesthetic (LA) injection in the perioperative period. Histaminergic angioedema is the most common form of angioedema. It has been reported that LA is a potential exogenous ligand for histamine receptor 1 (H1R). Whether H1R participates in LA-induced angioedema is still controversial. By using a constructed H1R high-expressed cell model, siRNA transfection, pharmacologic means, and genetically modified animal models, here we showed that H1R mediated LA-induced hyperpermeability. LA with uncycled N-methyl scaffold in the side chain (procaine, tetracaine and lidocaine) had a better strength of drug-H1R affinity than that for LA with cycled N atom (bupivacaine and ropivacaine) by the molecular docking assay and equilibrium dissociation constant (KD values) obtained from the cell membrane chromatography (CMC) relative standard method. Procaine, tetracaine, and lidocaine triggered big calcium mobilization in H1R-HEK293 cells and human umbilical vein endothelial cells (HUVECs) but much weaker in NC-HEK293 cells or H1R knockdown HUVECs. Besides, the results of transendothelial resistance measurement, paracellular flux assay and immunofluorescence showed that procaine induced H1R-dependent hyperpermeability, which involved in PLCγ/IP3R/PKC, ERK1/2, Akt signaling pathways, downstream vascular endothelial cadherin (VE-cad) destabilization. Furthermore, H1R gene knockout prevented paw swelling and vascular leakage caused by procaine, tetracaine, and lidocaine in vivo. This study supported a key role of H1R in LA-induced angioedema, and suggested that in the design of LA structure, the ring formation of the N-methyl scaffold on the side chain can properly avoid the angioedema.

Inhibitory Effect of Hesperidin on the Expression of Programmed Death Ligand (PD-L1) in Breast Cancer.

Programmed death ligand 1 (PD-L1) is overexpressed in the most aggressive breast cancer subtype, triple-negative breast cancer (TNBC), assisting the eradication of antitumor immunity, and thereby enhancing the survival of the tumor. This study explored how hesperidin affects PD-L1 expression, and thereby cancer progression in breast cancer cells. We found that MDA-MB231, the triple-negative breast adenocarcinoma cancer cell line, (high aggressiveness) has higher expression, in both mRNA and protein, of PD-L1 than that of the other breast cancer cell line, MCF-7 (low aggressiveness). Hesperidin inhibited cell proliferation in MDA-MB231 cells. Additionally, high expression of PD-L1 (both mRNA and protein) in aggressive cancer cells was strongly inhibited by hesperidin through inhibition of Akt and NF-κB signaling. Moreover, hesperidin treatment, by inhibiting activation of matrix metalloproteinases such as MMP-9 and MMP-2, suppressed the metastatic phenotype and cell migration in the PD-L1 high-expressing MDA-MB231 cells. In summary, hesperidin inhibits breast cancer cell growth through the inhibition of the expression of PD-L1 via downregulation of Akt and NF-κB signaling in TNBC. Moreover, hesperidin significantly suppresses cell migration of MDA-MB231 cells. Our findings reveal fresh insights into the anticancer effects of hesperidin which might have potential clinical implications.

Tumor microenvironment dictates regulatory T cell phenotype: Upregulated immune checkpoints reinforce suppressive function

BackgroundRegulatory T (Treg) cells have an immunosuppressive function in cancer, but the underlying mechanism of immunosuppression in the tumor microenvironment (TME) is unclear.MethodsWe compared the phenotypes of T cell subsets, including Treg cells, obtained from peripheral blood, malignant effusion, and tumors of 103 cancer patients. Our primary focus was on the expression of immune checkpoint (IC)-molecules, such as programmed death (PD)-1, T-cell immunoglobulin and mucin-domain containing (TIM)-3, T cell Ig and ITIM domain (TIGIT), and cytotoxic T lymphocyte antigen (CTLA)-4, on Treg cells in paired lymphocytes from blood, peritumoral tissue, and tumors of 12 patients with lung cancer. To identify the immunosuppressive mechanisms acting on tumor-infiltrating Treg cells, we conducted immunosuppressive functional assays in a mouse model.ResultsCD8+, CD4+ T cells, and Treg cells exhibited a gradual upregulation of IC-molecules the closer they were to the tumor. Interestingly, PD-1 expression was more prominent in Treg cells than in conventional T (Tconv) cells. In lung cancer patients, higher levels of IC-molecules were expressed on Treg cells than on Tconv cells, and Treg cells were also more enriched in the tumor than in the peri-tumor and blood. In a mouse lung cancer model, IC-molecules were also preferentially upregulated on Treg cells, compared to Tconv cells. PD-1 showed the greatest increase on most cell types, especially Treg cells, and this increase occurred gradually over time after the cells entered the TME. PD-1 high-expressing tumor-infiltrating Treg cells displayed potent suppressive activity, which could be partially inhibited with a blocking anti-PD-1 antibody.ConclusionsWe demonstrate that the TME confers a suppressive function on Treg cells by upregulating IC-molecule expression. Targeting IC-molecules, including PD-1, on Treg cells may be effective for cancer treatment.

Structure-based virtual screening and biological evaluation of novel non-bisphosphonate farnesyl pyrophosphate synthase inhibitors

Farnesyl pyrophosphate synthase (FPPS) is known to participate in a variety of disease-related cell signaling pathway and bisphosphonates (BPs) are served as FPPS inhibitors. However, the high polarity of BPs often induces a series of side effects, limiting their applications. In the present study, novel non-BP FPPS inhibitors were discovered by in silico screening and experimental validation. From the structure-based virtual screening (SBVS) strategy combining molecular docking, pharmacophore and binding affinity prediction, 10 hits with novel scaffolds were filtered. The inhibition activity of hits against FPPS was identified and 7 hits showed comparable or higher inhibition activity than Zoledronate. The hit VS-4 with higher lipophilicity (XlogP = 1.81) and binding affinity (KD = 14.3 ± 2.63 μM) to FPPS was selected for further study on cancer cells with different FPPS expression level. Experimental results revealed that VS-4 could better target the FPPS high-expressing colon LoVo and HCT116 cancer cell lines with IC50 of 51.772 ± 0.473 and 43.553 ± 1.027 μM, respectively, whereas the IC50 value against FPPS low expressing MDA-MB-231 cells was >100 μM. The mechanism of VS-4 against colon cancer cells was investigated by flow cytometry and the results indicated that VS-4 induced cell apoptosis by increasing the intracellular reactive oxygen species (ROS) level. Taken together, the SBVS strategy could be used to discover promising non-BP FPPS inhibitors and the lead compound VS-4 might shed a light on designing more potent inhibitors as novel anticancer drugs.

A human expression system based on HEK293 for the stable production of recombinant erythropoietin

Mammalian host cell lines are the preferred expression systems for the manufacture of complex therapeutics and recombinant proteins. However, the most utilized mammalian host systems, namely Chinese hamster ovary (CHO), Sp2/0 and NS0 mouse myeloma cells, can produce glycoproteins with non-human glycans that may potentially illicit immunogenic responses. Hence, we developed a fully human expression system based on HEK293 cells for the stable and high titer production of recombinant proteins by first knocking out GLUL (encoding glutamine synthetase) using CRISPR-Cas9 system. Expression vectors using human GLUL as selection marker were then generated, with recombinant human erythropoietin (EPO) as our model protein. Selection was performed using methionine sulfoximine (MSX) to select for high EPO expression cells. EPO production of up to 92700 U/mL of EPO as analyzed by ELISA or 696 mg/L by densitometry was demonstrated in a 2 L stirred-tank fed batch bioreactor. Mass spectrometry analysis revealed that N-glycosylation of the produced EPO was similar to endogenous human proteins and non-human glycan epitopes were not detected. Collectively, our results highlight the use of a human cellular expression system for the high titer and xenogeneic-free production of EPO and possibly other complex recombinant proteins.

P2.14-06 The Study of Rac3 Molecular Mechanism Through ERK Signaling Pathway in Lung Adenocarcinoma

To find out the relationship between Rac3 gene and prognosis of lung adenocarcinoma, the role of Rac3 in the proliferation and migration of lung adenocarcinoma cells, and the mechanism of Rac3 promoting the proliferation and migration of lung adenocarcinoma cells. 542 patients with lung adenocarcinoma in the TCGA database were selected for analysis, the expression of Rac3 in cancer tissues and normal lung tissues were compared. Kaplan-Meier Plotter database was selected to analyze the relationship between Rac3 expression and 5-year overall survival rate of patients by Kaplan-Meier method. Human lung adenocarcinoma cell lines A549 and H1299 were used to build Rac3 knockdown and high-expression cell lines. The role of Rac3 in the proliferation and migration of lung adenocarcinoma cells was detected by cell proliferation and healing-wound test. The Rac3 knockdown cell lines were used to detect the changes of intracellular signaling pathways by serum starvation test and Western Blot experiment, screening out the signaling pathways with significant differences and study the biological function of them. The results of TCGA database analysis showed that Rac3 expression in 542 patients with lung adenocarcinoma was significantly higher than that in normal lung tissues, and the result is statistically significant (P<0.01). Kaplan-Meier survival analysis showed that patients with high Rac3 expression had a significantly shorter 5-year overall survival than those with low Rac3 expression (P<0.0001). In A549 and H1299 cell lines, Rac3 shRNA is used to build Rac3 knockdown and high expression cell lines by lentivirus infection. The proliferation and migration of lung adenocarcinoma cells were detected by cell proliferation test and healing-wound test. Compared with the control group, the proliferation and migration ability of Rac3-shRNA group was significantly decreased, while that of Rac3 high expression group was significantly increased (P <0.01). Serum starvation test and Western Blot results showed that in A549 cell lines, the phosphorylation of ERK pathway was significantly reduced after Rac3 expression was knocked down by shRNA compared with the control group. Rac3 is highly expressed in lung adenocarcinoma. The overall survival (OS) of patients with high Rac3 expression was significantly shorter than those with low Rac3 expression. Rac3 promotes the proliferation of lung adenocarcinoma cells. Rac3 promotes the migration of lung adenocarcinoma cells. Rac3 probably regulates the ERK signaling pathway to promote the proliferation in lung adenocarcinoma cells.

Temozolomide has anti-tumor effects through the phosphorylation of cPLA2 on glioblastoma cells

Temozolomide is an alkylating agent used as the first line of treatment for glioblastoma. However, chemoresistance to temozolomide is common in glioma patients. In addition, there are likely many unknown mechanisms for the anti-tumor effects of temozolomide. It is known that an alkylating agent, sulfur mustard, activates cytosolic phospholipase A2 (cPLA₂) releasing arachidonic acid to suppress tumors. The present study was performed to elucidate the involvement of cPLA2 in the anti-tumor mechanisms of temozolomide.In three glioblastoma cell lines (GL261, U251MG and T98G), we performed several evaluations including cell viability, cell migration and apoptosis, to study temozolomide-induced anti-tumor effects. Further, we evaluated tumor size in the murine orthotropic glioblastoma model after oral administration of temozolomide. Finally, we investigated the phosphorylation of cPLA2 in GL261 cells treated with temozolomide, and clarified whether phosphorylation of cPLA2 affects cell growth.Temozolomide suppressed cell growth and cell migration in glioblastoma cells in vitro and showed anti-tumor effect in the murine orthotopic glioblastoma model in vivo. Furthermore, temozolomide increased phosphorylation of cPLA2, which was associated with suppression of cell growth. However, in MGMT high-expressing glioblastoma T98G cells, temozolomide could not suppress cell growth or cause phosphorylation of cPLA2.These findings indicate that temozolomide suppressed cell growth partly by phosphorylation of cPLA2 in glioblastoma cells. In addition, because temozolomide did not cause phosphorylation of cPLA2 in MGMT high-expressing glioblastoma T98G cells, phosphorylation of cPLA2 may be caused by DNA alkylation of temozolomide.

Stereoselective Anti-Cancer Activities of Ginsenoside Rg3 on Triple Negative Breast Cancer Cell Models.

Ginsenoside Rg3 (Rg3) has two epimers, 20(S)-ginsenoside Rg3 (SRg3) and 20(R)-ginsenoside Rg3 (RRg3), and while Rg3 itself has been reported to have anti-cancer properties, few studies have been reported on the anti-cancer effects of the different epimers. The aim was to investigate the stereoselective effects of the Rg3 epimers on triple negative breast cancer (TNBC) cell lines, tested using cell-based assays for proliferation, apoptosis, cell cycle arrest, migration and invasion. Molecular docking showed that Rg3 interacted with the aquaporin 1 (AQP1) water channel (binding score −9.4 kJ mol−1). The Xenopus laevis oocyte expression system was used to study the effect of Rg3 epimers on the AQP1 water permeability. The AQP1 expression in TNBC cell lines was compared with quantitative-polymerase chain reaction (PCR). The results showed that only SRg3 inhibited the AQP1 water flux and inhibited the proliferation of MDA-MB-231 (100 μM), due to cell cycle arrest at G0/G1. SRg3 inhibited the chemoattractant-induced migration of MDA-MB-231. The AQP1 expression in MDA-MB-231 was higher than in HCC1143 or DU4475 cell lines. These results suggest a role for AQP1 in the proliferation and chemoattractant-induced migration of this cell line. Compared to SRg3, RRg3 had more potency and efficacy, inhibiting the migration and invasion of MDA-MB-231. Rg3 has stereoselective anti-cancer effects in the AQP1 high-expressing cell line MDA-MB-231.

The effects of Sestrin2 on apoptosis of heat-exposed lung epithelial cells and its mechanism

To investigate the protective effects of Sestrin2 protein on lung epithelial Beas-2B cells in the heat-exposure environment and its mechanism. Lung epithelial Beas-2B cells were cultured at 37℃, 39℃, 40℃ and 41℃ respectively. Cells were harvested at different times (0, 3, 6 and 12 h) after pancreatin digestion. The expressions of Sestrin2, superoxide dismutase(SOD), reactive oxygen species(ROS), cell mitochondrial membrane potential and apoptosis rate of cells were detected by Western blot, fluorescence spectrophotometer and flow cytometry, respectively. Gene expression sequence was cloned into high expression plasmid pcDNA3.1+. Beas-2B cells were transfected by Lipfectamine 2000 to construct Sestrin2 and SOD high expression cells. The changes of mitochondrial membrane potential and cell apoptosis were observed in the Sestrin2 and SOD high expression cells. With the increase of temperature, the expression level of Sestrin2 protein in heat treatment group was decreased compared with the control group. When Beas-2B cells were exposed to 41℃, the ROS level was increased, mitochondrial membrane potential was decreased significantly and apoptosis rate was increased at different time points. After high expression of Sestrin2 and SOD in the Beas-2B cells, the expression level of ROS was decreased and the change tendency of mitochondrial membrane potential was decreased, and the apoptosis rate was reduced at 41℃ exposure. Sestrin2 can alleviate the apoptosis of lung epithelial cells induced by heat exposure through mitochondrial membrane potential and SOD, which has protective effect on lung epithelial Beas-2B cells.

Abstract 2963: CD44+/CD133+ colorectal cancer stem cells are sensitive to trifluridine

Abstract Background: Cancer stem cells (CSCs) are involved in disease recurrence, metastases, and therapeutic resistance. However, anticancer agents that target CSCs are not currently available. Trifluridine (FTD)/tipiracil (TPI), a novel oral antitumor drug, was approved for the treatment of patients with metastatic colorectal cancer who had been previously treated with anti-vascular endothelial growth factors; anti-epidermal growth factor receptors (in patients with ras wild-type genes); or fluoropyrimidine-, oxaliplatin-, irinotecan-based chemotherapies. FTD/TPI improves overall survival, and FTD is the key component of FTD/TPI. Here, we demonstrated the efficacy of FTD against CD44 and CD133 high-expressing (CD44+CD133+) colorectal cancer cells that possess CSC-like properties. Method: CD44+CD133+ and other populations of DLD-1, a colorectal cell line, were separated by fluorescence-activated cell sorting (FACS). The sphere-forming activity of each population, and anti- sphere-forming effects of FTD and fluorouracil (5-FU) on CD44+CD133+ cells, were measured. In addition, DLD-1 and HCT-116, two colorectal cell lines, were treated with FTD for 5 months. The CSC markers CD44 and CD133 were measured by FACS, and the cells' in vitro and in vivo tumor-initiating properties were evaluated. Results: CD44+CD133+ DLD-1 cells formed significantly more spheres than did CD44-CD133- cells (sphere ratio CD44+CD133+/CD44-CD133- = 4.0) and CD44+CD133- cells (sphere ratio CD44+CD133+/CD44+CD133- = 1.7). In the in vitro proliferation assay, CD44+CD133+ cells had greater 5-FU resistance, but not higher FTD resistance, than did unsorted cells. In addition, treatment of CD44+CD133+ DLD-1 cells with subtoxic concentrations of FTD (1 µM) disrupted sphere formation, which was superior to the effect of treatment with 1 µM 5-FU. The inhibition rates for FTD and 5-FU were 59.5% and 14.3%, respectively. DLD-1 and HCT-116 cell lines treated with FTD for 5 months had smaller CD44+CD133+ populations and lower sphere-forming activity than did untreated cell lines; population decline rates were 93.8% and 74.7%, respectively. Furthermore, 5 months of in vitro exposure to FTD reduced in vivo tumor formation potential in both cell lines. These results suggest that FTD is effective against CSC-like cells, and that treatment with FTD might reduce CSC-like populations. Conclusion: FTD had a direct effect on the sphere-forming activity of CSC-like CD44+CD133+ cells that was superior to that of treatment with 5-FU. Its effectiveness against CSC-like CD44+CD133+ cells suggests that FTD might be useful for CSC-targeted chemotherapy in tumors that highly express CD44 and CD133. Citation Format: Kenta Tsunekuni, Masamitsu Konno, Jun Koseki, Ayumu Asai, Kazuaki Matsuoka, Teiji Takechi, Yuichiro Doki, Masaki Mori, Hideshi Ishii. CD44+/CD133+ colorectal cancer stem cells are sensitive to trifluridine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2963.

Abstract 2010: SKI-G-801, an AXL kinase inhibitor, blocks metastasis and induces anti-tumor immune responses in various syngeneic cancer models

Abstract Introduction: Aberrant AXL expression plays a critical role in cancer cell migration, metastasis and drug resistance. Researchers have revealed that AXL signaling is also over expressed on cells associated with tumor microenvironment. These findings highlight AXL as an attractive drug candidate for targeting tumor evasion and metastasis. Here we present SKI-G-801, a small molecule inhibitor that targets phosphorylation of AXL (IC50 = 20 nM) and its downstream signals. Methods: Inhibitory effects of SKI-G-801 on cancer viability (MTT and colony formation assay), invasion and migration (trans-well invasion assay) were examined in AXL high-expressing lung cancer cells in vitro. LLC2 lung and 4T1 breast cancer bearing mouse models were established. in addition, C57BL/6 mice were injected intravenously with B16F10 melanoma cells to establish lung metastasis model. Mice were administrated with 30 mg/kg of SKI-G-801 orally before (metastasis model) or after (syngeneic model) tumor injection. To elucidate the involvement of AXL inhibitor on tumor microenvironment, the population of T cells and myeloid cells was analyzed by flow cytometry from the LLC2 tumor. Results: Treatment of SKI-G-801 showed strong inhibition of cancer migration and invasion, when its direct killing effect on cancer cells was modest. These results were reproduced in vivo test that pretreatment of SKI-G-801 significantly reduced metastatic burden in B16F10 model (p &amp;lt; 0.05). LLC2 and 4T1 tumors were decreased in SKI-G-801 treatment group (p &amp;lt; 0.05), but not in anthemic nude mice. CD3+CD8+ T cell population and memory Tc cells were increased in SKI- G-801 treatment group (p &amp;lt; 0.05). Especially, granzyme B+ Tc and gp70+ tumor specific Tc were increased (p &amp;lt; 0.05). SKI-G-801 increase the helper T cell population; CD3+CD4+ (p &amp;lt; 0.05), and CD44+ memory Th cells (p &amp;lt; 0.01). Conclusion: SKI-G-801 demonstrates great potential in anti-cancer activity though immune responses. The anti-cancer effects lead to a reversal of the metastatic phenotype in animal model. Our results suggest that SKI-G-801 is a promising drug for prevention against metastatic cancer. Citation Format: Chun-Feng Xin, Sung Eun Kim, Kyoung-Ho Pyo, Ha Ni Jo, Jae Seok Cho, Jae Hwan Kim, Wongeun Lee, Hee Kyu Lee, Jung-Ho Kim, Ho-Juhn Song, Jong Sung Koh, Byoung Chul Cho. SKI-G-801, an AXL kinase inhibitor, blocks metastasis and induces anti-tumor immune responses in various syngeneic cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2010.

Increased FLYWCH1 Expression is Negatively Correlated with Wnt/β-catenin Target Gene Expression in Acute Myeloid Leukemia Cells.

Acute myeloid leukaemia (AML) is a heterogeneous clonal malignancy of hematopoietic progenitor cells. The Wnt pathway and its downstream targets are tightly regulated by β-catenin. We recently discovered a new protein, FLYWCH1, which can directly bind nuclear β-catenin. Herein, we studied the FLYWCH1/β-catenin pathway in AML cells using qRT-PCR, Western blot, and immunofluorescence assays. In addition, the stemness activity and cell cycle were analysed by the colony-forming unit (CFU) using methylcellulose-based and Propidium iodide/flow cytometry assays. We found that FLYWCH1 mRNA and protein were differentially expressed in the AML cell lines. C-Myc, cyclin D1, and c-Jun expression decreased in the presence of higher FLYWCH1 expression, and vice versa. There appeared to be the loss of FLYWCH1 expression in dividing cells. The sub-G0 phase was prolonged and shortened in the low and high FLYWCH1 expression cell lines, respectively. The G0/G1 arrest correlated with FLYWCH1-expression, and these cell lines also formed colonies, whereas the low FLYWCH1 expression cell lines could not. Thus, FLYWCH1 functions as a negative regulator of the Wnt/β-catenin pathway in AML.

Discovery of 7-bromo-1,4-dihydrothieno[3’,2’:5,6]thiopyrano[4,3-c]pyrazole-3-carboxamide derivatives as the potential epidermal growth factor receptors for tyrosine kinase inhibitors

Small molecule tyrosine kinase inhibitors (TKIs) targeting at epidermal growth factor receptor (EGFR) in recent years have made great progress in the treatment of advanced non-small cell cancer (NSCLC). Although as the first-line treatment for sensitizing EGFR mutation-positive metastatic NSCLC, gefitinib has also behaved quite a lot of side effect and EGFR tolerance. Herein, a novel series of 7-bromo-1,4-dihydrothieno[3’,2’:5,6]thiopyrano[4,3-c]pyrazole-3-carboxamide derivatives were designed and synthesized, and screened for their inhibitory activity on the EGFR high-expressing human lung adenocarcinoma cell line A549 and human large cell lung cancer cell line NCI-H460 by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide colorimetric assay. The calculated IC50 values were reported. Compound 8h demonstrated the most potent inhibitory activity (IC50 = 9.57 ± 2.20 μmol L–1 for A549 and IC50 = 13.04 ± 1.21 μmol L–1 for NCI-H460), comparable to the positive-control gefitinib (IC50 = 8.58 ± 1.65 μmol L–1 for A549 and IC50 = 18.66 ± 5.01 μmol L–1 for NCI-H460). Conclusively, 7-bromo- 1,4-dihydrothieno[3’,2’:5,6]thiopyrano[4,3-c]pyrazole-3-carboxamide derivatives as the EGFR–TKIs were discovered, and could be used as potential leading compounds for further research.

Expression of long non-coding RNA hypoxia-inducible factor-1α antisense RNA-2 in hepatocellular carcinoma and its effect on proliferation and invasion

Objective To detect the expression of long non-coding RNA hypoxia-inducible factor-1α antisense RNA-2 (lncRNA-HIF1A-AS2) in hepatocellular carcinoma (HCC) and cell lines and its effect on proliferation and invasion of hepatoma cells. Methods Real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) was used to detect the expression of HIF1A-AS2 in 49 HCC tissues and adjacent normal tissues, four HCC cell lines(Huh7, HepG2, SMMC-7721, Bel-7402) and one normal liver cell line L02, then analyze the correlation between HIF1A-AS2 expression and clinicopathological features; Two high-expression HIF1A-AS2 HCC cells were transfected into small interfering RNA (si-HIF1A-AS2) and negative control (si-NC); Cell counting kit 8 (CCK-8) assay and Colony-forming assay were conducted to evaluate the cell proliferation; Transwell assay was conducted to evaluate the cell invasion. Results The expression of HIF1A-AS2 in HCC tissues was higher than that in adjacent tissues (-12.060±0.657 vs. -12.450±0.683, P<0.01); The expression of HIF1A-AS2 was correlated with age, tumor size and tumor differentiation (χ2=4.573, 4.871, 8.353, P<0.05); The expression of HIF1A-AS2 in HCC cell line was also significantly higher than that in normal liver cell line, and the expression was highest in Huh7 and HepG2 (Huh7: 5.349±0.992, HepG2: 5.660±0.726, P<0.01); CCK8 assay and colony-forming assay showed that the cell proliferation ability after transfection of si-HIF1A-AS2 was significantly lower than that of the control group in Huh7 and HepG2 (Huh7: 0.668±0.067 vs. 0.558±0.055, 0.931±0.881 vs. 0.670±0.072, 1.769±0.079 vs. 1.036±0.074; HepG2: 1.063±0.0604 vs. 0.803±0.097, 1.767±0.091 vs. 1.083±0.080, P<0.01) (Huh7: 542.70±25.50 vs. 378.00±19.31, HepG2: 530.00±14.53 vs. 217.30±18.15, P<0.01); Transwell assay showed that the number of cell invasion was significantly lower after transfection of si-HIF1A-AS2 [Huh7: (116.300±6.506) cells vs. (520.000±9.539) cells, HepG2: (88.330±5.686) cells vs. (55.000±5.245) cells, P<0.01]. Conclusion HIF1A-AS2 is highly expressed in HCC and cell lines, and down-regulation of HIF1A-AS2 expression can inhibit the proliferation and invasion ability of HCC cells. Key words: Hepatocellular carcinoma; Long non-coding RNA hypoxia-inducible factor-1α antisense RNA-2; Proliferation; Invasion

Osteopontin Promotes Colorectal Cancer Cell Invasion and the Stem Cell-Like Properties through the PI3K-AKT-GSK/3β-β/Catenin Pathway.

BackgroundOsteopontin (OPN) is a molecule expressed in numerous cancers including colorectal cancer (CRC) that correlates disease progression. The interaction of OPN that promotes CRC cell migration, invasion, and cancer stem-like cells (CSCs) have not been elucidated. Hence, we aimed to investigate the mechanisms that might be involved.Material/MethodsExpression of OPN in tumor tissues derived from patients was monitored with real-time quantitative polymerase chain reaction and western blot. Wound healing and Transwell assay were used to test the differences in migration and invasion in an OPN enriched environment and OPN knockdown condition. Aldehyde dehydrogenase 1 (ALDH1) positive stem cells were isolated using fluorescence-activated cell sorting (FACS) following the protocol of the ALDEFLUOR™ kit. The expression of protein participation in the PI3K-Akt-GSK/3β-β/catenin pathway was detected by western blot.ResultsOPN exhibited increased levels in CRC tumor tissue compared with non-tumor normal tissue and the high level of which correlated with lymphatic metastasis and late TNM stage. Additional rhOPN co-cultured low-expression CRC cells demonstrated more aggressive capability of proliferation, migration, and invasion. For knockdown of OPN in high-expression CRC cells, the bioactivities of proliferation, migration, and invasion were significantly inhibited. Interestingly, the percentage of ALDH1 labeled stem cells was dramatically decreased by OPN inhibition. The phosphorylation of PI3K-Akt-GSK/3β-β/catenin pathway was involved in the OPN signaling. Furthermore, Ly294002, a specific PI3K inhibitor, can reverse the promotion of bioactivities and stem cell proportion among rhOPN treated CRC cells.ConclusionsOPN promoted cell proliferation, migration, and invasion, and was accompanied by upregulation of ALDH1-positive CSC in CRC through activation of PI3K-Akt-GSK/3β-β/catenin pathway.

Development of the 99mTc-Hydroxamamide Complex as a Probe Targeting Carbonic Anhydrase IX.

Carbonic anhydrase IX (CA-IX) is regarded as a favorable target for in vivo imaging because of its specific expression in hypoxic regions of tumors. Hypoxia assists tumor propagation and growth and is resistant to chemotherapy and radiotherapy. Here, we designed and synthesized [99mTc]hydroxamamide ([99mTc]Ham) and [99mTc]methyl-substituted-hydroxamamide ([99mTc]MHam) complexes including a bivalent CA-IX ligand, sulfonamide (SA), and ureidosulfonamide (UR). In a cell binding assay, [99mTc]Ham complexes with bivalent SA ([99mTc]SAB2A and [99mTc]SAB2B) and UR ([99mTc]URB2A and [99mTc]URB2B) showed significantly greater uptake into CA-IX high-expressing (HT-29) cells than that into CA-IX low-expressing cells. Since the binding affinity of [99mTc]URB2A and [99mTc]URB2B for CA-IX was significantly higher than that of [99mTc]SAB2A and [99mTc]SAB2B, we additionally synthesized [99mTc]MURB2 (a [99mTc]MHam complex with bivalent UR) and evaluated the CA-IX-specific binding affinity of [99mTc]URB2A, [99mTc]URB2B, and [99mTc]MURB2. Their uptake into HT-29 cells was reduced by the addition of a CA inhibitor, acetazolamide, suggesting their CA-IX-specific binding affinity. A biodistribution study in HT-29 tumor-bearing mice was carried out using [99mTc]URB2A and [99mTc]MURB2 with the highest specificity for HT-29 cells. [99mTc]URB2A showed moderate tumor uptake and reduction by coinjection with acetazolamide; however, the tumor/blood ratio was insufficient for in vivo imaging. These results provided key information for the design of novel Ham-based imaging probes targeting CA-IX.

Identification and Characterization of the OCT4 Upstream Regulatory Region in Sus scrofa.

OCT4 plays pivotal roles in maintaining pluripotency during early mammalian embryonic development and in embryonic stem cells. It is essential to establish a reporter system based on the OCT4 promoter region to study pluripotency. However, there is still a lack of information about the porcine OCT4 upstream reporter system. To improve our understanding of the porcine OCT4 regulatory region, we identified conserved regions in the porcine OCT4 promoter upstream region by sequence-based comparative analysis using various mammalian genome sequences. The similarity of nucleotide sequences in the 5′ upstream region was low among mammalian species. However, the OCT4 promoter and four regulatory regions, including distal and proximal enhancer elements, had high similarity. Next, a functional analysis of the porcine OCT4 promoter region was conducted. Luciferase reporter assay results indicated that the porcine OCT4 distal enhancer and proximal enhancer were highly activated in mouse embryonic stem cells and embryonic carcinoma cells, respectively. A comparison analysis of naïve and primed state marker gene expression in a dual-reporter assay showed that the expression levels of naïve and primed markers differed in fluorescence signal between high-expressing cells and low-expressing cells. Similar to OCT4 upstream-based reporter systems derived from other species, the porcine OCT4 upstream region-based reporter constructs showed exclusive expression patterns depending on the state of pluripotency. This work provides basic information about the porcine OCT4 upstream region and various porcine OCT4 fluorescence reporter constructs, which can be applied to study species-specific pluripotency in early embryo development and the establishment of embryonic stem cells in pigs.

Synthesis, Characterization, and Biological Activity of a Novel Series of Benzo[4,5]imidazo[2,1-b]thiazole Derivatives as Potential Epidermal Growth Factor Receptor Inhibitors.

Based on the analysis of epidermal growth factor receptor (EGFR) complexes with gefitinib with molecular docking, the scaffold-hopping strategy, combination of the active substructures, and structural optimization of EGFR inhibitors, a novel series of benzo[4,5]imidazo[2,1-b]thiazole derivatives was designed, synthesized, and evaluated for antitumor activity in human cancer cell lines and cellular toxicity against human normal cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay and EGFR inhibitory activities in vitro. Some target compounds such as 2-(benzo[4,5]imidazo[2,1-b]thiazol-3-yl)-N-(2-hydroxyphenyl)acetamide (D04) and 2-(benzo[4,5]imidazo[2,1-b]thiazol-3-yl)-N-(naphthalen-1-yl)acetamide (D08) have shown significant antitumor activity against the EGFR high-expressed human cell line HeLa. All the target compounds showed hardly any antitumor activity against the EGFR low-expressed human cell line HepG2, and nearly no cellular toxicity against the human normal cell lines HL7702 and human umbilical vein endothelial cell lines (HUVEC). The inhibitory activities against EGFR kinase in vitro of the three target compounds were greatly consistent with the anti-proliferative activities. The preliminary structure–activity relationships of the target compounds were summarized. Conclusively, the novel benzo[4,5]imidazo[2,1-b]thiazole derivatives as novel potential EGFR inhibitors may be used as the potential lead compounds for the development of antitumor agents.