[Preparation and characterization of Ad-ERα-36-Fc-GFP].

ERα-36 is a novel subtype of estrogen receptor α which promotes tumor cell proliferation, invasion and drug resistance, and it serves as a therapeutic target. However, only small-molecule compounds targeting ERα-36 are under development as anticancer drugs at present. Gene therapy approach targeting ERα-36 can be explored using recombinant adenovirus armed with decoy receptor. The recombinant shuttle plasmid pDC316-Ig κ-ERα-36-Fc-GFP was constructed via genetic engineering to express an Ig κ-signaling peptide-leading secretory recombinant fusion protein ERα-36-Fc. The recombinant adenovirus Ad-ERα-36-Fc-GFP was subsequently packaged, characterized and amplified using AdMaxTM adenovirus packaging system. The expression of fusion protein and functional outcome of Ad-ERα-36-Fc-GFP transduction were further analyzed with triple-negative breast cancer MDA-MB-231 cells. Results showed that the recombinant adenovirus Ad-ERα-36-Fc-GFP was successfully generated. The virus effectively infected MDA-MB-231 cells which resulted in expression and secretion of the recombinant fusion protein ERα-36-Fc, leading to significant inhibition of EGFR/ERK signaling pathway. Preparation of the recombinant adenovirus Ad-ERα-36-Fc-GFP provides a basis for further investigation on cancer gene therapy targeting ERα-36.

Effective inhibition of cancer cells by recombinant adenovirus expressing EGFR-targeting artificial microRNA and reversed-caspase-3.

The EGFR-targeting cancer therapies are commonly facing drug resistance, mostly due to mutations. Gene therapy with artificial microRNA targeting EGFR conserved sequence may avoid such problem. In this study, we constructed a recombinant adenovirus expressing EGFR-targeting artificial microRNA and active revCASP3 (Ad-EC), under the control of tumor-specific SLPI promoter, and evaluated its inhibitory effect on HEP-2 cancer cells both in vitro and in vivo. MTT assay showed that cell growth inhibition rate at 72h was 44.0% in Ad-EC group at MOI 50, while the rate was 7.7% in the control virus Ad-GFP group and 3.6% in Cetuximab (500 µg/ml) group respectively. Flow cytometry analysis revealed the late apoptotic cells rate was 36.1% in Ad-EC group, significantly higher than 6.5% of Ad-GFP group (p < 0.001). When Ad-EC (MOI 50) was combined with CDDP (0.25 µg/ml), late apoptotic cells rate increased to 61.2%, significantly higher than each monotherapy group (P < 0.001). The real-time xCELLigence system recorded an effective cell growth inhibition in Ad-EC and CDDP groups, and more enhanced effect in Ad-EC plus CDDP group. Western blot revealed that Ad-EC could inhibit the activation of AKT pathway and ERK1/2 pathway, while Cetuximab had the AKT pathway over-activated. In vivo experiments with HEP-2 xenograft in nude mice confirmed the tumor inhibition in Ad-EC, CDDP and Ad-EC plus CDDP groups compared with PBS group (P < 0.01). Collectively, these data support the effective inhibition of cancer cells by this novel gene therapy strategy.

[Construction, expression and purification of a mammalian secretory recombinant fusion protein rPC].

Drugs targeting immune checkpoint are used for cancer treatment, but resistance to single drug may occur. Combination therapy blocking multiple checkpoints simultaneously can improve clinical outcome. Therefore, we designed a recombinant protein rPC to block multiple targets, which consists of extracellular domains of programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). The coding sequence was inserted into expression vector and stably transfected into HEK293 cells. The culture supernatant was collected and rPC was affinity-purified. Real-time quantitative PCR was used to evaluate the expression levels of ligands for PD-1 and CTLA-4 in several human cancer cell lines. The binding of rPC with cancer cells was examined by immunofluorescence cell staining, the influence of rPC on cancer cell growth was assayed by CCK-8. The results showed that rPC could be expressed and secreted by stably transfected HEK293 cells, the purified rPC could bind to lung cancer NCI-H226 cells which have high levels of ligands for PD-1 and CTLA-4, no direct impact on cancer cell growth could be observed by rPC treatment. The recombinant protein rPC can be functionally assayed further for developing novel immunotherapeutic drugs for cancer.

ERα36 is an effective target of epigallocatechin-3-gallate in hepatocellular carcinoma.

Epigallocatechin-3-gallate (EGCG) is a natural product with potential anti-cancer property whose direct target has not been identified. This study intended to investigate ERα36, a new isoform of estrogen receptor alpha (ERa), as a therapeutic target of EGCG in hepatocellular carcinoma (HCC). In this work, we examined the expression level of ERs in HCC cell lines and a normal human liver cell line, and evaluated inhibition effect of EGCG on these cells in vitro, and further on Hep3B in vivo. The results showed that ERα36 was the main ER in HCC cells and served as a biomarker of responsiveness to EGCG inhibition, and there was a positive correlation between ERα36 expression level and inhibitory effect of EGCG as indicated by IC50. In vivo experiments also showed dose-dependent inhibition of EGCG on ERα36 high-expressing Hep3B. EGCG exerted inhibition on Hep3B cells by both anti-proliferation and pro-apoptosis. ERα36-EGFR-Her-2 feedback loop, PI3K/Akt and MAPK/ERK pathways were inhibited, while caspase 3 was activated by EGCG in Hep3B cells, with p-ERK accumulated in cytoplasm. The inhibitory effect of EGCG was significantly attenuated when ERα36 was pre-activated. This is the first evidence that EGCG exerts its anti-cancer effect by inhibiting ERα36, followed with inhibition of the ERα36-EGFR-Her-2 feedback loop and PI3K/Akt, MAPK/ERK pathway, activation of caspase 3, and accumulation of p-ERK in cytoplasm. It suggests that ERα36 might be an efficient target of EGCG in HCC.

A novel multikinase inhibitor R8 exhibits potent inhibition on cancer cells through both apoptosis and autophagic cell death.

Chemotherapy is an important treatment for cancer patients, especially for those with unresectable lesions. Targeted therapy of cancer by specific inhibition of aberrant tyrosine kinase activities in cancer cells with chemically synthesized tyrosine kinase inhibitors (TKIs), shows better responses while less side effects than traditional chemotherapeutic drugs. It is common that cancer cells often exhibit deregulation of several tyrosine kinases simultaneously, multikinase TKIs (MKIs) therefore have greater advantages over single-target TKIs. Currently more MKIs are under developing for better efficacy for different types of cancer. In the present work, we evaluated the in vitro therapeutic potential of a novel MKI, namely R8, with comparison to the clinically available MKI Sunitinib. Results showed that R8 has stronger inhibition on six different types of cancer cell lines with lower IC50 than Sunitinib does. Cell cycle analysis showed that R8 induced significant G0/G1 arrest phase of lung cancer A549 and NCI-H226 cells. The inhibition was also confirmed by colony formation and migration assays in both lung cancer cell lines in a dose-dependent manner. R8 could significantly inhibit the phosphorylation of multiple receptor tyrosine kinases (RTKs) included PDGFRß, VEGFR2, EGFR and C-Kit, leading to the down-regulation of PI3K-Akt-mTOR signaling. Further analysis revealed that R8 treatment induced more significant apoptosis than Sunitinib did, which might be the consequence of the autophagic cell death. In conclusion, this work suggested R8 to be a promising novel anticancer MKI, and provided the basis for further in vivo investigation on its potential in treatment of lung cancer.

Sensitization of gastric cancer cells to alkylating agents by glaucocalyxin B via cell cycle arrest and enhanced cell death.

Severe side effects are major problems with chemotherapy of gastric cancer (GC). These side effects can be reduced by using sensitizing agents in combination with therapeutic drugs. In this study, the low/nontoxic dosage of glaucocalyxin B (GLB) was used with other DNA linker agents mitomycin C (MMC), cisplatin (DDP), or cyclophosphamide (CTX) to treat GC cells. Combined effectiveness of GLB with drugs was determined by proliferation assay. The molecular mechanisms associated with cell proliferation, migration, invasion, cell cycle, DNA repair/replication, apoptosis, and autophagy were investigated by immunoblotting for key proteins involved. Cell cycle and apoptosis analysis were performed by flow cytometry. Reactive oxygen species level was also examined for identification of its role in apoptosis. Proliferation assay revealed that the addition of 5 µM GLB significantly sensitizes gastric cancer SGC-7901 cells to MMC, DDP, and CTX by decreasing half-maximal inhibitory concentration (IC50) by up to 75.40%±5%, 45.10%±5%, and 52.10%±5%, respectively. GLB + drugs decreased the expression level of proteins involved in proliferation and migration, suggesting the anticancer potential of GLB + drugs. GLB + MMC, GLB + CTX, and GLB + DDP arrest the cells in G0/G1 and G1/S phase, respectively, which may be the consequence of significant decrease in the level of enzymes responsible for DNA replication and telomerase shortening. Combined use of GLB with these drugs also induces DNA damage and apoptosis by activating caspase/PARP pathways and increased production of reactive oxygen species and increased autophagy in GC cells. GLB dosage sensitizes GC cells to the alkylating agents via arresting the cell cycle and enhancing cell death. This is of significant therapeutic importance in the reduction of side effects associated with these drugs.

[Downregulation of proteinase activated receptor 4 inhibits migration of SW620 human colorectal cancer cells].

OBJECTIVE: To establish the human colorectal cancer cell model SW620/PAR4D with inducible suppression of proteinase activated receptor 4 (PAR4) expression, and investigate the role PAR4 plays in the proliferation and migration of cancer cells. METHODS: A human colorectal cancer cell line with tetracycline-inducible expression regulatory system, namely SW620/Tet-on, was established; inducible expression lentiviral vector with artificial microRNA targeting PAR4, pLVX-Tight-Puro-PAR4-miR, was constructed and transfected into SW620/Tet-on to make an inducible PAR4-suppressed cell model SW620/PAR4D. Western blotting was used to confirm the suppression of PAR4 expression after the doxycycline (DOX) treatment. CCK-8 assay was used to evaluate the impact of suppressed PAR4 expression on cell proliferation, and wound-healing assay was used to analyze the migration of the cells. RESULTS: The SW620/PAR4D cell model was established successfully. Suppression of PAR4 expression by DOX treatment had no significant impact on the growth/proliferation of SW620/PAR4D cells, but markedly inhibited the cell migration. CONCLUSION: Suppression of PAR4 expression has no significant effect on the proliferation of SW620 cells, but can inhibit the migration of the cells.

Overexpression of DCF1 inhibits glioma through destruction of mitochondria and activation of apoptosis pathway.

Gliomas are the most common brain tumors affecting the central nervous system and are associated with a high mortality rate. DCF1 is a membrane protein that was previously found to play a role in neural stem cell differentiation. In the present study, we found that overexpression of dcf1 significantly inhibited cell proliferation, migration, and invasion and dramatically promoted apoptosis in the glioblastoma U251 cell line. DCF1 deletion mutations in the functional region showed that the complete structure of DCF1 was necessary for apoptosis. Furthermore, significantly lower tumorigenicity was observed in athymic nude mice by transplanting U251 cells overexpressing dcf1. To decode the apoptosis induced by dcf1, mitochondrial structure and membrane potential in glioma cells were investigated and the results indicated obvious mitochondrial swelling, destruction of cristae, and a significant decline in membrane potential. Mechanismly, caspase-3 signaling was activated. Finally, endogenous dcf1 silence in U251 cells was investigated. Results showed a highly methylation at -1339 and -1322 position at dcf1 promoter sequence, revealing the causal relationship between dcf1 gene and tumorigencicity. The present study identified a previously unknown cancer apoptosis mechanism involving dcf1 overexpression and provided a novel approach to potentially treat glioma patients.

System approaches reveal the molecular networks involved in neural stem cell differentiation.

The self-renewal and multipotent potentials in neural stem cells (NSCs) maintain the normal physiological functions of central nervous system (CNS). The abnormal differentiation of NSCs would lead to CNS disorders. However, the mechanisms of how NSCs differentiate into astrocytes, oligodendrocytes (OLs) and neurons are still unclear, which is mainly due to the complexity of differentiation processes and the limitation of the cell separation method. In this study, we modeled the dynamics of neural cell interactions in a systemic approach by mining the high-throughput genomic and proteomic data, and identified 8615 genes that are involved in various biological processes and functions with significant changes during the differentiation processes. A total of 1559 genes are specifically expressed in neural cells, in which 242 genes are NSC specific, 215 are astrocyte specific, 551 are OL specific, and 563 are neuron specific. In addition, we proposed 57 transcriptional regulators specifically expressed in NSCs may play essential roles in the development courses. These findings provide more comprehensive analysis for better understanding the endogenous mechanisms of NSC fate determination.