Lei Zhao

The multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster as a therapeutic suicide gene of breast cancer cells.

The multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster (Dm‐dNK) was investigated for its broader substrate specificity and higher catalytic rate as a suicide gene in a combined gene/chemotherapy of cancer.

Synergistic therapeutic effect in gastric cancer cells produced by oncolytic adenovirus encoding Drosophila melanogaster deoxyribonucleoside kinase

Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (Dm-dNK), a novel suicide kinase, was applied as a cancer gene therapeutic approach. To improve the antitumor effect of Dm-dNK and its substrate with the selectivity and safety control in consideration, the conditionally replicative gene-viral system ZD55-dNK, which includes the replicationselective adenovirus ZD55 armed with the Dm-dNK, was used to further explore the potential of this approach. When ZD55-dNK was combined with BVDU it produced a synergistic inhibitive effect of adenovirus replication in vitro while maintaining specific cancer cell killing activity. From a clinical standpoint, this approach is promising for its considerably low toxic side effects.

Bufalin inhibits TGF-β-induced epithelial-to-mesenchymal transition and migration in human lung cancer A549 cells by downregulating TGF-β receptors.

The epithelial-to-mesenchymal transition (EMT) is a well-known prerequisite for cancer cells to acquire the migratory and invasive capacity, and to subsequently metastasize. Bufalin is one of the major active components of the traditional Chinese medicine Chan Su, and accumulating evidence has shown its anticancer effect in multipe types of cancer. However, the role of bufalin in transforming growth factor-β (TGF-β)-induced EMT and migration remains unclear. In the present study, the effect of bufalin on TGF-β-induced EMT and migration was investigated in human lung cancer A549 cells. TGF-β induced EMT in A549 cells and increased their migratory ability, which were markedly suppressed by bufalin. Additionally, TGF-β-induced upregulation of Twist2 and zinc finger E-box binding homeobox 2 (ZEB2), as well as the phosphorylation of Smad2 and Smad3 were also inhibited by bufalin. However, the Smad-independent signaling pathways were not affected. Further analysis showed that the TGF-β receptor I (TβRI) and TGF-β receptor II (TβRII) were downregulated in the presence of bufalin. Pretreatment with SB431542, a potent inhibitor of the phosphorylation of TβRI, significantly attenuated TGF-β-induced EMT, mimicking the effect of bufalin on A549 cells. Taken together, these results suggest that bufalin suppresses TGF-β-induced EMT and migration by downregulating TβRI and TβRII in A549 cells.

The combined effect of survivin-targeted shRNA and emodin on the proliferation and invasion of ovarian cancer cells

Survivin has been shown to be highly expressed in ovarian cancers, but not normal ovarian tissue, which makes it an attractive target for ovarian cancer treatment. Emodin is a traditional Chinese medicine that has been found to inhibit proliferation and induce apoptosis in ovarian cancer cells. Thus, in our study, we combined survivin-targeted shRNA (sur-shRNA) with emodin and tested the effects of this combination on ovarian cancer cells to identify more effective therapeutics against ovarian cancer. A sur-shRNA plasmid was constructed and transfected into the ovarian cancer cell lines SKOV3 and HO8910, and the cells were cultured for 24 h. The cells were then treated with emodin for specific time periods and assessed for viability and apoptosis using the MTT assay and flow cytometry, respectively. Cell invasion was also measured using a Matrigel invasion assay. The shRNA specific for survivin effectively reduced the expression of survivin at the mRNA and protein levels in SKOV3 and HO8910 cells. Both emodin and shRNA-mediated knockdown of survivin significantly inhibited cell proliferation, induced apoptosis, and suppressed invasion in SKOV3 and HO8910 cells (P<0.05). Moreover, the combination of the agents significantly enhanced these effects (P<0.05). We found that the combination of sur-shRNA and emodin could be effective in the treatment of ovarian cancer.

Potent anticancer effects of lentivirus encoding a Drosophila melanogaster deoxyribonucleoside kinase mutant combined with brivudine.

OBJECTIVE Deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK) mutants have been reported to exert suicide gene effects in combined gene/chemotherapy of cancer. Here, we aimed to further evaluate the capacity of the mutanted enzyme and its potential for inhibiting cancer cell growth. METHODS We altered the sequence of the last 10 amino acids of Dm-dNK to perform site-directed mutagenesis and constructed active site mutanted Dm-dNK (Dm-dNKmut), RT-PCR and western bloting studies were used to reveal the expression of lentivirus mediated Dm-dNKmut in a breast cancer cell line (Bcap37), a gastric cancer cell line (SGC7901) and a colorectal cancer cell line (CCL187). [3H]-labeled substrates were used for enzyme activity assays, cell cytotoxicity was assessed by MTT assays, cell proliferation using a hemocytometer and apoptosis induction by thenannexin-V-FITC labeled FACS method. In vivo, an animal study was set out in which BALB/C nude mice bearing tumors were treated with lentivirus mediated expression of Dm-dNKmut with the pyrimidine nucleoside analog brivudine (BVDU, (E)-5-(2-bromovinyl)-(2-deoxyuridine). RESULTS The Dm-dNKmut could be stably expressed in the cancer cell lines and retained its enzymatic activity. Moreover, the cells expressing Dm-dNKmut exhibited increased sensitivity in combination with BVDU, with induction of apoptosis in vitro and in vivo. CONCLUSION These findings underlined the importance of BVDU phosphorylated by Dm-dNKmut in transduced cancer cells and the potential role of Dm-dNKmut as a suicide gene, thus providing the basis for future intensive research for cancer therapy.

Src promotes EGF-induced epithelial-to-mesenchymal transition and migration in gastric cancer cells by upregulating ZEB1 and ZEB2 through AKT: Src in EGF-induced EMT and migration

Epithelial‐to‐mesenchymal transition (EMT) plays important roles in the migration, invasion, and metastasis of cancer cells. However, the role of Src in epidermal growth factor (EGF)‐induced EMT and migration in gastric cancer cells remains to be clarified. In the current study, the effect of Src on EGF‐stimulated EMT and migration was explored in gastric cancer cells. EGF induced EMT in gastric cancer cells and increased their migratory ability, which was accompanied by the phosphorylation of Src. PP2, the Src inhibitor, markedly suppressed EGF‐mediated EMT and migration in gastric cancer cells. Additionally, EGF‐stimulated upregulation of zinc finger E‐box binding homeobox 1 (ZEB1) and zinc finger E‐box binding homeobox 2 (ZEB2) was significantly repressed by PP2. Further analysis showed that EGF‐stimulated phosphorylation of protein kinase B (AKT) was almost completely abolished by PP2, whereas that of extracellular signal‐regulated kinase (ERK), signal transducer and activator of transcription 3 (STAT3) was only mildly suppressed. Moreover, LY294002, the AKT inhibitor, significantly inhibited EGF‐induced upregulation of ZEB1 and ZEB2 as well as EMT and migration stimulated by EGF in gastric cancer cells. However, neither ERK inhibitor nor STAT3 inhibitor repressed EGF‐induced EMT‐related changes. Taken together, these results suggest that Src promotes EGF‐stimulated EMT and migration by upregulation of ZEB1 and ZEB2 through AKT signaling pathway in gastric cancer cells.