BackgroundPrognosis of pancreatic cancer is poor with a 5-year survival rate of only 7%. Although several new chemotherapy treatments have shown promising results, all patients will eventually progress, and we need to develop newer chemotherapy treatments to improve response rates and overall survival (OS). HF10 is a spontaneously mutated oncolytic virus derived from a herpes simplex virus-1, and it has potential to show strong antitumor effect against malignancies without damaging normal tissue. We aimed to evaluate the safety and anti-tumor effectiveness in phase I dose-escalation trial of direct injection of HF10 into unresectable locally advanced pancreatic cancer under endoscopic ultrasound (EUS)-guidance in combination with erlotinib and gemcitabine administration. The mid-term results have been previously reported and here we report the final results of our study.MethodsThis was a single arm, open-label Phase I trial. HF10 was injected once every 2 weeks and continued up to four times in total unless dose-limiting toxicity (DLT) appears. A total of nine subjects in three Cohorts with dose-escalation were planned to be enrolled in this trial. The primary endpoint was the safety assessment and the secondary endpoint was the efficacy assessment.ResultsTwelve patients enrolled in this clinical trial, and ten subjects received this therapy. Five patients showed Grade III myelosuppression and two patients developed serious adverse events (AEs) (perforation of duodenum, hepatic dysfunction). However, all of these events were judged as AEs unrelated to HF10. Tumor responses were three partial responses (PR), four stable diseases (SD), and two progressive diseases (PD) out of nine subjects who completed the treatment. Target lesion responses were three PRs and six SDs. The median progression free survival (PFS) was 6.3 months, whereas the median OS was 15.5 months. Two subjects from Cohort 1 and 2 showed downstaging and finally achieved surgical complete response (CR).ConclusionsHF10 direct injection under EUS-guidance in combination with erlotinib and gemcitabine was a safe treatment for locally advanced pancreatic cancer. Combination therapy of HF10 and chemotherapy should be explored further in large prospective studies. Trial registration: This study was prospectively registered in UMIN-CTR (UMIN000010150) on March 4th, 2013.Electronic supplementary materialThe online version of this article (10.1186/s12885-018-4453-z) contains supplementary material, which is available to authorized users.

N-Acetylglucosaminyltransferase V (GnT-V) catalyzes the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to ␣-6-D-mannoside to produce the ␤1-6 linked branching of N-glycan oligosaccharides, which controls the polylactosamine content. The expression of N-acetylglucosaminyltransferase V, which contains 17 exons and spans 155 kilobase pairs, is expressed in a tissue-and cell type-specific manner and is regulated at the level of transcription by multiple promoters (Saito, H., Gu, J., Nishikawa, A., Ihara, Y., Fujii, J., Kohgo, Y., and Taniguchi, N. (1995) Eur. J. Biochem. 233, 18 -26). To elucidate the mechanism by which the GnT-V gene is expressed in a cell-and tissue-specific manner, cell-restricted expression was analyzed using the 5-upstream regions of the human GnT-V gene spanning base pairs ؊2760 to ؉23 in a human bile duct carcinoma cell line, HuCC-T1. We characterized two cis-acting elements that are potentially important in HuCC-T1 cell-specific expression. The two elements each contain an Ets-1 binding site, 5-GGA-3. Specific binding of Ets-1 to the respective elements was demonstrated by competition analysis as well as by antibody supershift experiments. Cotransfection of an Ets-1 expression plasmid along with a GnT-V promoter-luciferase reporter plasmid revealed the participation of Ets-1 in the regulation of the GnT-V gene transcription. These data indicated that the transcriptional regulation of the GnT-V gene was mediated by transcription factor Ets-1.Cell surface oligosaccharides linked to asparagine residues of membrane glycoproteins are thought to participate in a variety of specific biological interactions (1). The high branching of N-glycans seemed to be related to malignancy potential, in particular, increased ␤1-6 branching of N-glycans being directly linked to increased tumor metastasis (2-6). This structure is synthesized by UDP-N-acetylglucosamine:␣-6-D-mannoside ␤1-6-N-acetylglucosaminyltransferase (GnT-V) 1 (EC 2.4.1.155), as shown in Scheme 1. The expression of the GnT-V gene is induced by viral and oncogene transfection, transforming growth factor-␤, and phorbol ester (7-10), suggesting that complicated mechanisms for the induction exist. We and another group succeeded in its purification and cloning of its cDNA (11, 12). Messenger RNA was detected as two bands for HepG2 and MCF7 cells, and LEC rat liver, but the level is not always consistent with its enzymatic activity (13-15). To determine the details of the gene regulation, analysis of the 5Ј-upstream regions of GnT-V was needed. Recently, we isolated the human GnT-V genomic DNA, which contains 17 exons and spans 155 kb. Some putative consensus sequences for the tissue-specific transcription factors have been found in two possible promoter regions of the 5Ј-upstream regions of the GnT-V gene (16).The Ets transcription factor family shares a common DNA binding domain that interacts specifically with sequences containing a common core trinucleotide sequence, GGA (17). Ets binding sites have been identified in the regulator...

Alkaline elastase YaB is an extracellular serine protease of the alkalophilic Bacillus strain YaB. We cloned the structural gene, ale, and determined the nucleotide sequence. The mature enzyme (268 amino acids) was preceded by a putative signal sequence and a prosequence (27 and 83 amino acids, respectively). The mature enzyme was 55 % homologous to subtilisin BPN'. Almost all the positively charged residues are predicted to be on the surface of the molecule, which would facilitate binding to elastin. The P1 substrate site-related sequences differed between alkaline elastase YaB and subtilisin BPN'.To identify enzymes that digest elastin efficiently, we screened for microbial enzymes among alkalophilic bacteria, because local denaturation of elastin under alkaline conditions might facilitate its enzymatic digestion. Alkaline elastase YaB secreted by the alkalophilic Bacillus strain YaB was thus selected and purified (25). The enzyme was characterized as a serine protease of about 25,000 daltons. Chemical modification studies revealed that certain histidine and tyrosine residues are involved in the enzymatic activity (21). The enzyme has the peculiar characteristic of a very high optimum pH for digestion of casein and elastin and an elastolytic activity comparable to that of porcine pancreatic elastase (25). The substrate specificity was examined by cutting oxidized insulin A and B chains (23) and by using synthetic peptide substrates (24). The enzyme has a P1 substrate preference for Ala, in contrast to that of subtilisin BPN' for Tyr, and it efficiently hydrolyzes succinyl-AlaAla-Ala-p-nitroanilide, a specific substrate of porcine elastase (24). Furthermore, the enzyme binds to elastin below its isoelectric point (pl 10.6) (22). These features encouraged us to characterize the enzyme as a microbial elastase.To determine the molecular mechanism of the extremely high optimum pH and high elastolytic activity of the enzyme, we cloned the structural gene and analyzed its nucleotide and deduced amino acid sequences.Bacillus subtilis DB104 (nprEI8 nprR2 AaprE3 his-101) (9) has lesions in the structural genes for neutral and alkaline proteases and was a gift from Roy H. Doi and Fujio Kawamura. Escherichia coli MC1061 and JM109 were used as the host strains for cloning. All bacterial strains were propagated at 37°C in nutrient broth (Eiken Chemical Co., Tokyo, Japan) or Luria broth (11) with vigorous aeration or on media solidified with 1.5% agar. Media were s pplemerted with 100 p.g of ampicillin per ml, 10 ,ug of kana.nycin pei :nl, or 20 .'g of tetracycline per ml when necessary. pL"U18 and pHY300PLK were purchased from Takara Shuzo Co. (Kyoto, Japan).To detect the production of alkaline elastase, elastin plates and skim milk plates were used. Elastin plates were made by overlaying 5 ml of 1% elastin (Sigma Chemical Co., St. Louis, Mo.) and 0.7% agar on basal agar plates containing * Corresponding author. 0.8% nutrient broth and 1.5% agar. Skim milk plates contained 0.8% nutrient broth, 1.5% agar, and 1% skim milk....

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