JNC-1043, a Novel Podophyllotoxin Derivative, Exerts Anticancer Drug and Radiosensitizer Effects in Colorectal Cancer Cells.

The objective of this study was to determine whether (5S)-5-(4-benzyloxy-3,5-dimethoxy-phenyl)-5,9-dihydro-8H-furo [3',4':6,7] naphtho [2,3-d] [1,3]dioxol-6-one (JNC-1043), which is a novel chemical derivative of ß-apopicropodophyllin, acts as a novel potential anticancer reagent and radiosensitizer in colorectal cancer (CRC) cells. Firstly, we used MTT assays to assess whether JNC-1043 could inhibit the cell proliferation of HCT116 and DLD-1 cells. The IC50 values of these cell lines were calculated as 114.5 and 157 nM, respectively, at 72 h of treatment. Using doses approximating the IC50 values, we tested whether JNC-1043 had a radiosensitizing effect in the CRC cell lines. Clonogenic assays revealed that the dose-enhancement ratios (DER) of HCT116 and DLD-1 cells were 1.53 and 1.25, respectively. Cell-counting assays showed that the combination of JNC-1043 and γ-ionizing radiation (IR) enhanced cell death. Treatment with JNC-1043 or IR alone induced cell death by 50~60%, whereas the combination of JNC-1043 and IR increased this cell death by more than 20~30%. Annexin V-propidium iodide assays showed that the combination of JNC-1043 and IR increased apoptosis by more 30~40% compared to that induced by JNC-1043 or IR alone. DCFDA- and MitoSOX-based assays revealed that mitochondrial ROS production was enhanced by the combination of JNC-1043 and IR. Finally, we found that suppression of ROS by N-acetylcysteine (NAC) blocked the apoptotic cell death induced by the combination of JNC-1043 and IR. The xenograft model also indicated that the combination of JNC-1043 and IR increased apoptotic cell death in tumor mass. These results collectively suggest that JNC-1043 acts as a radiosensitizer and exerts anticancer effects against CRC cells by promoting apoptosis mediated by mitochondrial ROS.

Radiosensitizer Effect of ß-Apopicropodophyllin against Colorectal Cancer via Induction of Reactive Oxygen Species and Apoptosis.

ß-apopicropodophyllin (APP), a derivative of podophyllotoxin (PPT), has been identified as a potential anti-cancer drug. This study tested whether APP acts as an anti-cancer drug and can sensitize colorectal cancer (CRC) cells to radiation treatment. APP exerted an anti-cancer effect against the CRC cell lines HCT116, DLD-1, SW480, and COLO320DM, with IC50 values of 7.88 nM, 8.22 nM, 9.84 nM, and 7.757 nM, respectively, for the induction of DNA damage. Clonogenic and cell counting assays indicated that the combined treatment of APP and γ-ionizing radiation (IR) showed greater retardation of cell growth than either treatment alone, suggesting that APP sensitized CRC cells to IR. Annexin V-propidium iodide (PI) assays and immunoblot analysis showed that the combined treatment of APP and IR increased apoptosis in CRC cells compared with either APP or IR alone. Results obtained from the xenograft experiments also indicated that the combination of APP and IR enhanced apoptosis in the in vivo animal model. Apoptosis induction by the combined treatment of APP and IR resulted from reactive oxygen species (ROS). Inhibition of ROS by N-acetylcysteine (NAC) restored cell viability and decreased the induction of apoptosis by APP and IR in CRC cells. Taken together, these results indicate that a combined treatment of APP and IR might promote apoptosis by inducing ROS in CRC cells.

Isolation, characterization, and genomic analysis of the novel T4-like bacteriophage ΦCJ20.

Pathogenic Escherichia coli infections have been consistently reported annually. The basic characteristics and genome of the newly isolated ΦCJ20 from swine feces was analyzed. To determine basic characteristics, dotting assays and double-layer agar assays were conducted. Bacteriophage particles were analyzed via transmission electron microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed to determine the sizes of major structural proteins. The complete genome of the phage was analyzed. Bacteriophage particles were identified as Myoviridae, with a head measuring 110.57 ± 1.89 nm and a contractile tail measuring 107.97 ± 3.20 nm and were found to infect E. coli. Major structural proteins of ΦCJ20 showed two well-pronounced bands of approximately 53.6 and 70.9 kDa. The genome size of ΦCJ20 was 169,884 bp, and 118 of 307 open reading frames were annotated. This study provides a baseline for the development of E. coli infection treatment strategies. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10068-021-00906-y.

Radiation-induced IL-1ß expression and secretion promote cancer cell migration/invasion via activation of the NF-κB-RIP1 pathway.

Here, we demonstrate that interleukin-1ß (IL-1ß) contributes to the γ-ionizing radiation (IR)-induced increase of migration/invasion in A549 lung cancer cells, and that this occurs via RIP1 upregulation. We initially observed that the protein expression and secreted concentration of IL-1ß were increased upon exposure of A549 cells to IR. We then demonstrated that IR-induced IL-1ß is located downstream of the NF-κB-RIP1 signaling pathway. Treatments with siRNA and specific pharmaceutical inhibitors of RIP1 and NF-κB suppressed the IR-induced increases in the protein expression and secreted concentration of IL-1ß. IL-1Ra, an antagonist of IL-1ß, treatment suppressed the IR-induced epithelial-mesenchymal transition (EMT) and IR-induced invasion/migration in vitro. These results suggest that IL-1ß could regulate IR-induced EMT. We also found that IR could induce the expression of IL-1ß expression in vivo and that of IL-1 receptor (R) I/II in vitro and in vivo. The IR-induced increases in the protein levels of IL-1 RI/II and IL-1ß suggest that an autocrine loop between IL-1ß and IL-1 RI/II might play important roles in IR-induced EMT and migration/invasion. Based on these collective results, we propose that IR concomitantly activates NF-κB and RIP1 to trigger the NF-κB-RIP1-IL-1ß-IL-1RI/II-EMT pathway, ultimately promoting metastasis.

RIP1 Is a Novel Component of γ-ionizing Radiation-Induced Invasion of Non-Small Cell Lung Cancer Cells.

Abstract: Previously, we demonstrated that γ-ionizing radiation (IR) triggers the invasion/migration of A549 cells via activation of an EGFR-p38/ERK-STAT3/CREB-1-EMT pathway. Here, we have demonstrated the involvement of a novel intracellular signaling mechanism in γ-ionizing radiation (IR)-induced migration/invasion. Expression of receptor-interacting protein (RIP) 1 was initially increased upon exposure of A549, a non-small cell lung cancer (NSCLC) cell line, to IR. IR-induced RIP1 is located downstream of EGFR and involved in the expression/activity of matrix metalloproteases (MMP-2 and MMP-9) and vimentin, suggesting a role in epithelial-mesenchymal transition (EMT). Our experiments showed that IR-induced RIP1 sequentially induces Src-STAT3-EMT to promote invasion/migration. Inhibition of RIP1 kinase activity and expression blocked induction of EMT by IR and suppressed the levels and activities of MMP-2, MMP-9 and vimentin. IR-induced RIP1 activation was additionally associated with stimulation of the transcriptional factor NF-κB. Specifically, exposure to IR triggered NF-κB activation and inhibition of NF-κB suppressed IR-induced RIP1 expression, followed by a decrease in invasion/migration as well as EMT. Based on the collective results, we propose that IR concomitantly activates EGFR and NF-κB and subsequently triggers the RIP1-Src/STAT3-EMT pathway, ultimately promoting metastasis.

Characterization and Genomic Analysis of Novel Bacteriophage ΦCS01 Targeting Cronobacter sakazakii.

Cronobacter sakazakii is an opportunistic pathogen causing serious infections in neonates. In this study, a bacteriophage ΦCS01, which infects C. sakazakii, was isolated from swine feces and its morphology, growth parameters, and genomic analysis were investigated. Transmission electron microscopy revealed that ΦCS01 has a spherical head and is 65.74 nm in diameter with a 98.75 nm contracted tail, suggesting that it belongs to the family Myoviridae. The major viral proteins are approximately 71 kDa and 64 kDa in size. The latent period of ΦCS01 was shown to be 60 min, and the burst size was 90.7 pfu (plaque-forming units)/ infected cell. Bacteriophage ΦCS01was stable at 4-60°C for 1 h and lost infectivity after 1 h of heating at 70°C. Infectivity remained unaffected at pH 4-9 for 2 h, while the bacteriophage was inactivated at pH <3 or >10. The double-stranded ΦCS01 DNA genome consists of 48,195 base pairs, with 75 predicted open reading frames. Phylogenetic analysis is closely related to that of the previously reported C. sakazakii phage ESP2949-1. The newly isolated ΦCS01 shows infectivity in the host bacterium C. sakazakii, indicating that it may be a promising alternative to antibacterial agents for the removal of C. sakazakii from powdered infant formulas.

Whole-Genome Sequencing and Genomic Analysis of a Virulent Bacteriophage Infecting Bacillus cereus.

OBJECTIVE: Infants with a weak immune system are prone to infection with Bacillus cereus, which is commonly found in natural environments. With the aim of achieving better control of this pathogenic bacterium, in the present study we characterized a new bacteriophage, ΦBC01. METHODS: Bacteriophage particles were analyzed by transmission electron microscopy. Factors influencing adsorption were identified in a double-layer plaque assay. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was conducted to determine the size of major structural proteins. The complete genome of the phage was analyzed. RESULTS: Bacteriophage particles (105.3 ± 3.1 nm icosahedral head and 208.8 ± 3.6 nm contractile tail) were identified as Myoviridae. ΦBC01 was found to have host specificity to B. cereus. Major structural proteins of ΦBC01 showed 2 well-pronounced bands of 99 and 56 kDa. The 158,385-bp genome sequence of ΦBC01 was determined: 56 of the 239 open reading frames were annotated, indicating involvement in bacteriophage DNA manipulation, cell lysis, packaging, virion structure, and other functions. CONCLUSION: Because of characterization and genotyping of a new bacteriophage from soil samples containing earthworms, this study provides a baseline for the development of alternatives to antibiotics.