Evaluation of the anti-cervical cancer effect of a prodrug ：CBZ-AAN-DOX with hypoxic cell culture and tumor-bearing zebrafish models.

BACKGROUND: Prodrugs are medications or compounds that, after administration, can be converted into pharmacologically active drugs through metabolism. Unlike conventional drugs, prodrugs have reduced adverse or unintended effects, which could become critical limitations in treatments such as chemotherapy. Previously through computer-aided drug design and chemical synthesis, we have obtained and examined a prodrug N-benzyloxycarbonyl-Ala-Asn-Doxorubicin (CBZ-AAN-DOX). CBZ-AAN-DOX is essentially Doxorubicin that is chemically-modified with tripeptides to target Legumain, a highly expressed protein in cancer cells and is involved in tumor metastasis and tumor microvessel formation. The difficulty to test the safety and efficacy of the prodrug (including the pharmacodynamic parameters of CBZ-AAN-DOX on metastasis and invasion of tumors, as well as cardiac and vascular toxicity) primarily comes from the lack of appropriate experimental models. METHODS: Human cervical cancer cell lines CaSki under hypoxic conditions were used to evaluate the cell viability by CCK-8 assay after the prodrug treatment. Western blotting method was performed for Legumain protein determination in the cell culture. Wound healing and transwell invasion assays were performed to determine the effects of the prodrug on tumor metastasis and invasion, respectively. Zebrafish models were constructed for toxicity and angiogenesis visual analysis after in vivo treatment with the prodrug. RESULTS: The CCK-8 results showed that CBZ-AAN-DOX exhibits an IC50 of 28.7 µM in 48 h on CaSki cells that had a lower cell inhibition rate than DOX 80.3 µM for 24 h. Legumain expression was significantly increased in a time-dependent manner in 48 h under hypoxia conditions. The results also showed that 13.9 µM of the prodrug significantly inhibited the migration and invasion of cells and the effects were significantly stronger than that of 41.8 µM of DOX under hypoxia conditions after 48 h. The effects of 160 µM of the prodrug on the survival rate of zebrafish after 72 h and heart-toxicity showed no obvious abnormalities. Cell metastasis and angiogenesis were also inhibited in tumor-bearing zebrafish model. CONCLUSION: The findings in this study demonstrated that CBZ-AAN-DOX is a promising chemotherapy candidate with low toxicity and high efficiency for cervical cancer. Remarkably, the hypoxic culture model together with the zebrafish model serve as a good system for the evaluation of the toxicity, targeting and impact of the prodrug on tumor invasion and metastasis.

Isolation and complete genome sequence of a novel cyanophage, S-B05, infecting an estuarine Synechococcus strain: insights into environmental adaptation.

A new cyanophage, S-B05, infecting a phycoerythrin-enriched (PE-type) Synechococcus strain was isolated by the liquid infection method, and its morphology and genetic features were examined. Phylogenetic analysis and morphological observation confirmed that S-B05 belongs to the family Myoviridae of the order Caudovirales. Its genome was fully sequenced, and found to be 208,857 bp in length with a G + C content of 39.9%. It contained 280 potential open reading frames and 123 conserved domains. Ninety-eight functional genes responsible for cyanophage structuring and packaging, DNA replication and regulation, and photosynthesis were identified, as well as genes encoding 172 hypothetical proteins. The genome of S-B05 is most similar to that of Prochlorococcus phage P-TIM68. Homologues of open reading frames of S-B05 can be found in various marine environments, as revealed by comparison of the S-B05 genome sequence to sequences in marine viral metagenomic databases. The presence of auxiliary metabolic genes (AMGs) related to photosynthesis, carbon metabolism, and phosphorus assimilation, as well as the phylogenetic relationships based on AMGs and the complete genome sequence, reflect the phage-host interaction mechanism or the specific adaptation strategy of the host to environmental conditions. The genome sequence information reported here will provide an important basis for further study of the adaptive evolution and ecological role of cyanophages and their hosts in the marine environment.

Process of immunogenic cell death caused by disulfiram as the anti-colorectal cancer candidate.

BACKGROUND: Disulfiram (DSF), a drug widely used to control alcoholism, which has anticancer activity by inducing apoptosis in a copper (Cu)-dependent manner. Numerous evidences from mouse experiments indicated that some anti-cancer agents of chemotherapeutic drugs favor the induction of immunogenic cancer cell death (ICD) leading to tumor-specific immune responses. However, whether DSF could induce the colorectal tumor cells death and the mechanism involved in ICD regulatory remains elusive. The main objective of this study was to elucidate the effect of DSF/Cu on the apoptosis of colorectal cancer (CRC) cells and the expression of the two major ICD markers in CRC cells: calreticulin (CRT) and heat shock proteins (HSP) 70. METHODS: Firstly, the toxicity of DSF/Cu in HCT116, SW620 and HCT8 cells was assayed by MTT. Flow cytometry was utilized to detect the apoptosis effects. The effects of DSF/Cu on the expression of ICD-related molecules in tumor tissues were further verified in the CRC xenograft mouse model. RESULTS: The results showed that DSF/Cu increase apoptosis of these three cells in a dose dependent manner and significantly inhibited the proliferation at the concentration range from 0.05 to 1.6 µM. Furthermore, the expression of CRT and HSP70 on the cell surface also increased. The rate of transplanted tumors grew slowly, and the expression of CRT and HSP70 in colorectal cancer tissues was increased after treated with DSF/Cu. CONCLUSION: In conclusion, our results show that DSF/Cu exerts anti-colorectal cancer and its underlying mechanisms are associated with the enhancement of molecules expression of cell ICD. These results provide experimental evidence and theory basis of therapy for developing the DSF/Cu as the drug for CRC.

The Genome Sequence of a Novel Cyanophage S-B64 from the Yellow Sea, China.

A novel cyanophage, S-B64, which can infect marine Synechococcus WH8102, was isolated from the coastal waters of the Yellow Sea using the liquid serial dilution method. Morphological study by transmission electron microscopy revealed that the cyanophage belongs to Podovirus. It's genome, which was completely sequenced, contains a 151,867 bp DNA molecule with a G+C content of 41.78% and 186 potential open reading frames. The functions of the genes include cyanophage structure, cyanophage packaging, DNA replication and regulation. After primary characterization, it was found that the latent period is about 3 h, and it lysed after 8 h, the burst size is about 23 virions per cell. This information will provide an important benchmark for further research on the interaction between cyanophages and their hosts.

Isolation and Complete Genome Sequence of a Novel Marinobacter Phage B23.

We used the double-agar layer method to isolate a novel Marinobacter marina bacteriophage, B23, from the surface water sample of the Bohai sea of China. There is some work to better understand the phage. The result of transmission electron microscopy revealed that B23 belongs to the family Siphoviridae with a head of 80 nm in diameter and a tail of 230 nm. Microbiological characterization evidenced that phage B23 is stable at the temperatures from - 25 to 60 °C, and showed vigorous vitality at pH between 4.0 and 12.0. One-step growth experiment showed that it had a longer latent period and higher lysis efficiency. Furthermore, the complete genome of B23 was sequenced and analyzed, which consists of a 35132 bp DNA with a G + C content of 59.8% and 50 putative open reading frames. The genome was divided into five parts, consisting of DNA replication and regulation, phage packaging, phage structure, host lysis and hypothetical protein.

Characterization and Complete Genome Sequence of a Novel Siphoviridae Bacteriophage BS5.

A novel Siphoviridae family Phage BS5, which infects Pseudoalteromonas atlantica, was isolated from the surface waters of the Yellow Sea. Morphological study by transmission electron microscopy revealed that the novel phage belongs to Siphoviridae. The complete genome sequence of PBS5 contained a linear, double-strand 39949-bp DNA molecule with a G + C content of 40.6% and 65 putative open reading frames. Twelve conserved domains were detected by BLASTP in NCBI, and of these the functions of 5 were known. The genome was grouped into four modules as follows: phage structure, phage packaging, DNA replication and regulation, and some additional functions.16 S rDNA sequence analysis was also applied to identify the host bacteria. After initial characterization of bacteriophage PBS5, it was found that the optimal pH was 7.0, the optimal temperature was 30 °C, and the burst size was about 95 virions per cell. This information will provide an important benchmark for further research on the interaction between bacteriophages and their hosts.

Morusin inhibits breast cancer-induced osteolysis by decreasing phosphatidylinositol 3-kinase (PI3K)-mTOR signalling.

Bone metastases caused by breast cancer pose a major challenge to the successful treatment of breast cancer patients. Many researchers have suggested that herbal medicines are extremely effective at preventing and treating cancer-associated osteolysis. Previous studies have revealed that Morusin (MOR) is cytotoxic to many cancer cells ex vivo. Nevertheless, how MOR contributes to osteolysis induced by breast cancer is still unknown, and the potential mechanism of action against osteolysis is worthy of further study. The protective effect and molecular mechanism of MOR in inhibiting breast cancer cell-induced osteolysis were verified by experiments and network pharmacology. Cell function was assessed by cell proliferation, osteoclast (OC) formation, bone resorption, and phalloidin staining. Tumour growth was examined by micro-CT scanning in vivo. To identify potential MOR treatments, the active ingredient-target pathway of breast cancer was screened using network pharmacology and molecular docking approaches. This study is the first to report that MOR can prevent osteolysis induced by breast cancer cells. Specifically, our results revealed that MOR inhibits RANKL-induced osteoclastogenesis and restrains the proliferation, invasion and migration of MDA-MB-231 breast cells through restraining the PI3K/AKT/MTOR signalling pathway. Notably, MOR prevented bone loss caused by breast cancer cell-induced osteolysis in vivo, indicating that MOR inhibited the development of OCs and the resorption of bone, which are essential for cancer cell-associated bone distraction. This study showed that MOR treatment inhibited osteolysis induced by breast cancer in vivo. MOR inhibited OC differentiation and bone resorption ex vivo and in vivo and might be a potential drug candidate for treating breast cancer-induced osteolysis.

Lymphatic-localized Treg-mregDC crosstalk limits antigen trafficking and restrains anti-tumor immunity.

The tumor microenvironment (TME) has a significant impact on tumor growth and immunotherapy efficacies. However, the precise cellular interactions and spatial organizations within the TME that drive these effects remain elusive. Using advanced multiplex imaging techniques, we have discovered that regulatory T cells (Tregs) accumulate around lymphatic vessels in the peripheral tumor stroma. This localized accumulation is facilitated by mature dendritic cells enriched in immunoregulatory molecules (mregDCs), which promote chemotaxis of Tregs, establishing a peri-lymphatic Treg-mregDC niche. Within this niche, mregDCs facilitate Treg activation, which in turn restrains the trafficking of tumor antigens to the draining mesenteric lymph nodes, thereby impeding the initiation of anti-tumor adaptive immune responses. Disrupting Treg recruitment to mregDCs inhibits tumor progression. Our study provides valuable insights into the organization of TME and how local crosstalk between lymphoid and myeloid cells suppresses anti-tumor immune responses.

Correction: Cepharanthine hydrochloride reverses the mdr1 (P-glycoprotein)-mediated esophageal squamous cell carcinoma cell cisplatin resistance through JNK and p53 signals.

[This corrects the article DOI: 10.18632/oncotarget.22676.].

Characterization and Genomic Analysis of Marinobacter Phage vB_MalS-PS3, Representing a New Lambda-Like Temperate Siphoviral Genus Infecting Algae-Associated Bacteria.

Marinobacter is the abundant and important algal-associated and hydrocarbon biodegradation bacteria in the ocean. However, little knowledge about their phages has been reported. Here, a novel siphovirus, vB_MalS-PS3, infecting Marinobacter algicola DG893(T), was isolated from the surface waters of the western Pacific Ocean. Transmission electron microscopy (TEM) indicated that vB_MalS-PS3 has the morphology of siphoviruses. VB_MalS-PS3 was stable from -20 to 55°C, and with the latent and rise periods of about 80 and 10 min, respectively. The genome sequence of VB_MalS-PS3 contains a linear, double-strand 42,168-bp DNA molecule with a G + C content of 56.23% and 54 putative open reading frames (ORFs). Nineteen conserved domains were predicted by BLASTp in NCBI. We found that vB_MalS-PS3 represent an understudied viral group with only one known isolate. The phylogenetic tree based on the amino acid sequences of whole genomes revealed that vB_MalS-PS3 has a distant evolutionary relationship with other siphoviruses, and can be grouped into a novel viral genus cluster with six uncultured assembled viral genomes from metagenomics, named here as Marinovirus. This study of the Marinobacter phage vB_MalS-PS3 genome enriched the genetic database of marine bacteriophages, in addition, will provide useful information for further research on the interaction between Marinobacter phages and their hosts, and their relationship with algal blooms and hydrocarbon biodegradation in the ocean.

Genomic analysis of Synechococcus phage S-B43 and its adaption to the coastal environment.

Synechococcus dominate picocyanobacterial communities in coastal environments. However, only a few Synechococcus phages have been described from the coastal seas of the Northwest Pacific Ocean. Here a new Synechococcus phage, S-B43 was isolated from the Bohai Sea, a semi-closed coastal sea of the Northwest Pacific Ocean. S-B43 is a member of Myoviridae, containing 275 predicted open reading frames. Fourteen auxiliary metabolic genes (AMG) were identified from the genome of S-B43, including five photosynthetic associated genes and several AMGs related to its adaption to the high turbidity and eutrophic coastal environment with a low ratio of phosphorus to nitrogen (HNLP). The occurrences of 31 AMGs among 34 cyanophage genomes indicates that AMGs zwf, gnd, speD, petF and those coding for FECH and thioredoxin were more common in coastal areas than in the open ocean and AMGs pebS and ho1 were more prevalent in the open ocean. The occurrence of cyanophage AMGs in different environments might be a reflection of the environmental adaption of their hosts. This study contributes to our understanding of the interactions between cyanobacteria and cyanophages and their environmental adaption to the coastal environment.

Magnolol attenuates the inflammation and enhances phagocytosis through the activation of MAPK, NF-κB signal pathways in vitro and in vivo.

Magnolol is a natural extract and the main bioactive component from Chinese medicine-Magnolia. We speculate that it's functional action might be associated with the anti-inflammatory effects of magnolol. Herein, the main purpose was to elucidate the phagocytic immune function and anti-inflammatory activities associated. The toxicity of magnolol on U937 and LO-2 cells was assayed by MTT, flow cytometry and laser scanning confocal microscope was utilized to detect the phagocytosis effect on U937 cells, C57BL/6 mice and the follow-up hematoxylin-eosin staining methods were used to evaluate its bioactivity in vivo. The results showed that magnolol had dose dependent effects on enhancement of phagocytosis ability and significantly inhibited the NO production at the concentration range from10 to 40 µM. Furthermore, Magnolol significantly reduced the gene expression and protein release of IL-1ß and TNF-α. However, the p-ERK1/2 in MAPK signaling pathway was not significantly affected by magnolol, whereas p-JNK and p-P38 were down-regulated. Magnolol also inhibited the expression of p-IκBα and p-P65 of NF-κB signaling pathways. The loss of body weight and the shorter length of colon were significantly improved in DSS-treated colitis C57BL/6 mice after the administration of magnolol. The cytokines of pro-inflammatory factors TNF-α, IL-6 and IL-1ß attenuated significantly in a concentration dependent manner. The histopathological manifestations of 5-20 mg/kg after the treatment magnolol were markedly improved in the DSS-treated mice. These findings showed that magnolol exerted an anti-inflammatory effect through immunoregulatory phagocytosis, MAPK and NF-κB signaling pathways. Our results provide experimental evidence and theory basis for research on anti-inflammatory effects for magnolol as a potentially anti-inflammatory drug candidate.

Viral Diversity and Its Relationship With Environmental Factors at the Surface and Deep Sea of Prydz Bay, Antarctica.

A viral metagenomic analysis of five surface and two bottom water (878 meters below surface, mbs, and 3,357 mbs) samples from Prydz Bay, was conducted during February-March 2015. The results demonstrated that most of the DNA viruses were dsDNA viruses (79.73-94.06%, except at PBI1, 37.51%). Of these, Caudovirales (Siphoviridae, Myoviridae, and Podoviridae) phages were most abundant in surface seawater (67.67-71.99%), while nucleocytoplasmic large DNA viruses (NCLDVs) (Phycodnaviridae, Mimiviridae, and Pandoraviridae accounted for >30% of dsDNA viruses) were most abundant in the bottom water (3,357 mbs). Of the ssDNA viruses, Microviridae was the dominant family in PBI2, PBI3, PBOs, and PBI4b (57.09-87.55%), while Inoviridae (58.16%) was the dominant family in PBI1. Cellulophaga phages (phi38:1 and phi10:1) and Flavobacterium phage 11b, infecting the possible host strains affiliated with the family Flavobacteriaceae of the phylum Bacteroidetes, were abundant in surface water dsDNA viromes. The long contig (PBI2_1_C) from the viral metagenomes were most similar to the genome architectures of Cellulophaga phage phi10:1 and Flavobacterium phage 11b from the Arctic Ocean. Comparative analysis showed that the surface viral community of Prydz Bay could be clearly separated from other marine and freshwater environments. The deep sea viral community was similar to the deep sea viral metagenome at A Long-term Oligotrophic Habitat Assessment Station (ALOHA, at 22°45'N, 158°00'W). The multivariable analysis indicated that nutrients probably played an important role in shaping the local viral community structure. This study revealed the preliminary characteristics of the viral community in Prydz Bay, from both the surface and the deep sea. It provided evidence of the relationships between the virome and the environment in Prydz Bay and provided the first data from the deep sea viral community of the Southern Ocean.

Cepharanthine hydrochloride reverses the mdr1 (P-glycoprotein)-mediated esophageal squamous cell carcinoma cell cisplatin resistance through JNK and p53 signals.

Esophageal squamous cell carcinoma (ESCC) is an aggressive malignancy that is often resistant to therapy. Nowadays, chemotherapy is still one of the main methods for the treatment of ESCC. However, the multidrug resistance (MDR)-mediated chemotherapy resistance is one of the leading causes of death. Exploring agents able to reverse MDR, which thereby increase the sensitivity with clinical first-line chemotherapy drugs, could significantly improve cancer treatment. Cepharanthine hydrochloride (CEH) has the ability to reverse the MDR in ESCC and the mechanism involved have not been reported. The aim of the study was to investigate the potential of CEH to sensitize chemotherapeutic drugs in ESCC and explore the underlying mechanisms by in vitro and in vivo studies. Our data demonstrated that CEH significantly inhibited ESCC cell proliferation in a dose-dependent manner, induced G2/M phase cell cycle arrest and apoptosis, and increased the sensitivity of cell lines resistant to cisplatin (cDDP). Mechanistically, CEH inhibited ESCC cell growth and induced apoptosis through activation of c-Jun, thereby inhibiting the expression of P-gp, and enhancing p21 expression via activation of the p53 signaling pathway. In this study, we observed that growth of xenograft tumors derived from ESCC cell lines in nude mice was also significantly inhibited by combination therapy. To our knowledge, we demonstrate for the first time that CEH is a potentially effective MDR reversal agent for ESCC, based on downregulation of the mRNA expression of MDR1 and P-gp. Together, these results reveal emphasize CEH putative role as a resistance reversal agent for ESCC.