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Supplementary Table 1 from Intratumoral Localization of Aromatase and Interaction between Stromal and Parenchymal Cells in the Non–Small Cell Lung Carcinoma Microenvironment

Supplementary Figures 1-2 from Intratumoral Localization of Aromatase and Interaction between Stromal and Parenchymal Cells in the Non–Small Cell Lung Carcinoma Microenvironment

Estrogens produced as a result of intratumoral aromatization has been recently shown to play important roles in proliferation of human non–small cell lung carcinomas (NSCLC), but the details have remained largely unknown. Therefore, in this study, we evaluated the possible roles of intratumoral aromatase in NSCLCs as follows: (a) evaluation of intratumoral localization of aromatase mRNA/protein in six lung adenocarcinoma cases using laser capture microdissection combined with quantitative reverse transcriptase-PCR and immunohistochemistry; (b) examination of the possible effects of isolated stromal cells from lung carcinoma tissues on aromatase mRNA transcript expression in lung carcinoma cell lines (A549 and LK87) through a coculture system; and (c) screening of cytokines derived from stromal LK001S and LK002S cells using cytokine antibody arrays and subsequent evaluation of effects of these cytokines on aromatase expression in A549 and LK87. Both aromatase mRNA and protein were mainly detected in intratumoral carcinoma cells but not in stromal cells. Aromatase expression of A549 and LK87 was upregulated in the presence of LK001S or LK002S cells. Several cytokines such as interleukin-6 (IL-6), oncostatin M, and tumor necrosis factor-α, all known as inducible factors of aromatase gene, were detected in conditioned media of LK001S and LK002S cells. Treatment of both oncostatin M and IL-6 induced aromatase gene expression in A549 an LK87, respectively. These results all indicated that intratumoral microenvironments, especially carcinoma-stromal cell interactions, play a pivotal role in the regulation of intratumoral estrogen synthesis through aromatase expression in human lung adenocarcinomas. Cancer Res; 70(16); 6659–69. ©2010 AACR.

Supplementary Figure Legends 1-2 from Intratumoral Localization of Aromatase and Interaction between Stromal and Parenchymal Cells in the Non–Small Cell Lung Carcinoma Microenvironment

Fungi-derived flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenases (FADGDHs) are the most popular and advanced enzymes for SMBG sensors because of their high substrate specificity toward glucose and oxygen insensitivity. However, this type of FADGDH hardly shows direct electron transfer (DET) ability. In this study, we developed a new DET-type FADGDH by harboring Cytochrome b562 (cyt b562) derived from Escherichia coli as the electron transfer domain. The structural genes encoding fusion enzymes composed of cyt b562 at either the N- or C-terminus of fungal FADGDH, (cyt b562-GDH or GDH-cyt b562), were constructed, recombinantly expressed, and characteristics of the fusion proteins were investigated. Both constructed fusion enzymes were successfully expressed in E. coli, as the soluble and GDH active proteins, showing cyt b562 specific redox properties. Thusconstructed fusion proteins showed internal electron transfer between FAD in FADGDH and fused cyt b562. Consequently, both cyt b562-GDH and GDH-cyt b562 showed DET abilities toward electrode. Interestingly, cyt b562-GDH showed much rapid internal electron transfer and higher DET ability than GDH-cyt b562. Thus, we demonstrated the construction and production of a new DET-type FADGDH using E.coli as the host cells, which is advantageous for future industrial application and further engineering.

Glucose oxidase (GOx) has been widely utilized for monitoring glycemic levels due to its availability, high activity, and specificity toward glucose. Among the three generations of electrochemical glucose sensor principles, direct electron transfer (DET)-based third-generation sensors are considered the ideal principle since the measurements can be carried out in the absence of a free redox mediator in the solution without the impact of oxygen and at a low enough potential for amperometric measurement to avoid the effect of electrochemically active interferences. However, natural GOx is not capable of DET. Therefore, a simple and rapid strategy to create DET-capable GOx is desired. In this study, we designed engineered GOx, which was made readily available for single-step modification with a redox mediator (phenazine ethosulfate, PES) on its surface via a lysine residue rationally introduced into the enzyme. Thus, PES-modified engineered GOx showed a quasi-DET response upon the addition of glucose. This strategy and the obtained results will contribute to the further development of quasi-DET GOx-based glucose monitoring dedicated to precise and accurate glycemic control for diabetic patient care.

One of the most prominent glucose dehydrogenases (GDHs) capable of direct electron transfer with electrodes is the FADGDH complex derived from Burkholderia cepacia. This FADGDH complex comprises the following three distinct subunits: the catalytic subunit (α subunit) that has an FAD cofactor in its redox center, a hitch-hiker protein from the bacterial TAT secretion system (γ subunit), and the electron transfer subunit (β subunit). The electron transfer subunit (β subunit) of the FADGDH complex is a three-heme c containing cytochrome c like molecule (heme 1, heme 2 and heme 3 from the N-terminal). In this study, an FADGDH complex harboring a truncated electron transfer subunit composed of only heme 3 was constructed, and its enzymatic activity and electrochemical properties were investigated to elucidate the role of heme 3 and its region. A truncated electron transfer subunit, trβ subunit, was designed using the 3D structures of homologous cytochrome c proteins. The designed trβ subunit was expressed as soluble and functional heme c molecules forming complexes with γα catalytic complexes. Thus, the formed FADGDH complex has inter-molecular electron transfers from the FAD to the trβ subunit, and from the trβ subunit to the external electron acceptor, showing electron transfer subunit-mediated characteristic dye-mediated dehydrogenase activity with a Ru-complex. Therefore, heme 3 in the electron transfer subunit is responsible for accepting the electron from the γα catalytic complex. Moreover, the FADGDH complex harboring the trβ subunit showed DET activity toward the electrode. Spectroelectrochemical observations revealed that the trβ subunit possessed a lower formal potential than any of the 3 hemes in the whole electron transfer subunit. These unexpected electrochemical properties of the heme in the trβ subunit may potentially result in the construction of a DET principle-based glucose sensor, which can be operated at a much lower potential than those achieved using the FADGDH complex with a whole electron transfer subunit.

Fungi-derived flavin adenine dinucleotide glucose dehydrogenases (FADGDHs) are currently the most popular and advanced enzymes for self-monitoring of blood glucose sensors; however, the achievement of direct electron transfer (DET) with FADGDHs is difficult. In this study, a designer FADGDH was constructed by fusing Aspergillus flavus derived FADGDH (AfGDH) and a Phanerochaete chrisosporium CDH (PcCDH)-derived heme b-binding cytochrome domain to develop a novel FADGDH that is capable of direct electron transfer with an electrode. A structural prediction suggested that the heme in the CDH may exist in proximity to the FAD of AfGDH if the heme b-binding cytochrome domain is fused to the AfGDH N-terminal region. Spectroscopic observations of recombinantly produced designer FADGDH confirmed the intramolecular electron transfer between FAD and the heme. A decrease in pH and the presence of a divalent cation improved the intramolecular electron transfer. An enzyme electrode with the immobilized designer FADGDH showed an increase in current immediately after the addition of glucose in a glucose concentration-dependent manner, whereas those with wild-type AfGDH did not show an increase in current. Therefore, the designer FADGDH was confirmed to be a novel GDH that possesses electrode DET ability. The difference in the surface electrostatic potentials of AfGDH and the catalytic domain of PcCDH might be why their intramolecular electron transfer ability is inferior to that of CDH. These relevant and consistent findings provide us with a novel strategic approach for the improvement of the DET properties of designer FADGDH. (241 words).

Background Epidermal growth factor receptor (EGFR)–tyrosine kinase inhibitor (TKI) has demonstrated a promising therapeutic response in lung adenocarcinoma patients with EGFR gene mutations. However, the predictive factors for this therapy have not been established, except for the EGFR gene mutation status of carcinoma cells. Methods We first performed microarray analysis in EGFR-TKI–sensitive lung adenocarcinoma cell lines. The results indicated anterior gradient 2 (AGR2) as a potential surrogate marker of EGFR-TKI. Therefore, we then evaluated the correlation between the status of AGR2 immunoreactivity and clinicopathological factors including overall survival (OS), progression-free survival (PFS) and clinical response to EGFR-TKI, in 147 cases of surgically resected lung adenocarcinoma. The biological significance of AGR2 was further evaluated by transfecting small interfering RNA (siRNA) against AGR2 in these cells. Results The status of AGR2 immunoreactivity was significantly higher in lung adenocarcinoma cases with EGFR gene mutations than in those with the wild type (pConclusions AGR2 could serve as an adjunctive surrogate protein marker possibly reflecting EGFR gene mutations in lung adenocarcinoma patients. Results from in vitro analysis indicated that AGR2 could be a potential clinical biomarker of EGFR-TKI therapeutic sensitivity in lung adenocarcinoma cells.

To improve the stability of E. coli-produced non-glycosylated fungal FAD-glucose dehydrogenase induced a disulfide bond by site-directed mutagenesis based on structural comparisons with glucose oxidases.The FAD-glucose dehydrogenase (GDH) mutant Val149Cys/Gly190Cys, which was constructed based on a comparison with the three dimensional structure of glucose oxidase, showed a 110 min half-life of thermal inactivation at 45 °C, which is 13-fold greater than that of the wild-type enzyme. The considerable increase in thermal stability was further supported by Eyring plot analysis. The kinetic parameters of Val149Cys/Gly190Cys (k cat = 760 s(-1), Km = 35 mM, and catalytic efficiency (k cat/Km) = 22 s(-1 )mM(-1)) were almost identical to those of the wild-type enzyme (k cat = 780 s(-1), Km = 35 mM, k cat/Km = 22 s(-1 )mM(-1)). The substrate specificity of Val149Cys/Gly190Cys is indistinguishable from that of the wild type.The constructed mutant, Val149Cys/Gly190Cys, had significantly increased structural stability without changing the catalytic activity and kinetic parameters of FAD-GDH, including its characteristic substrate specificity.

Fungal FAD-dependent glucose dehydrogenases (FADGDHs) are considered to be superior enzymes for glucose sensor strips because of their insensitivity to oxygen and maltose. One highly desirable mediator for enzyme sensor strips is hexaammineruthenium(III) chloride because of its low redox potential and high storage stability. However, in contrast to glucose oxidase (GOx), fungal FADGDH cannot utilize hexaammineruthenium(III) as electron acceptor. Based on strategic structure comparison between FADGDH and GOx, we constructed a mutant of Aspergillus flavus-derived FADGDH, capable of utilizing hexaammineruthenium(III) as electron acceptor: AfGDH-H403D. In AfGDH-H403D, a negative charge introduced at the pathway-entrance leading to the FAD attracts the positively charged hexaammineruthenium(III) and guides it into the pathway. The corresponding amino acid in wild-type GOx is negatively charged, which explains the ability of GOx to utilize hexaammineruthenium(III) as electron acceptor. Electrochemical measurements showed a response current of 46.0 μA for 10 mM glucose with AfGDH-H403D and hexaammineruthenium(III), similar to that with wild-type AfGDH and ferricyanide (47.8 μA). Therefore, AfGDH-H403D is suitable for constructing enzyme electrode strips with hexaammineruthenium(III) chloride as sole mediator. Utilization of this new, improved fungal FADGDH should lead to the development of sensor strips for blood glucose monitoring with increased accuracy and less stringent packing requirements.

In this study, a novel fungus FAD dependent glucose dehydrogenase, derived from Aspergillus niger (AnGDH), was characterized. This enzyme's potential for the use as the enzyme for blood glucose monitor enzyme sensor strips was evaluated, especially by investigating the effect of the presence of xylose during glucose measurements. The substrate specificity of AnGDH towards glucose was investigated, and only xylose was found as a competing substrate. The specific catalytic efficiency for xylose compared to glucose was 1.8%. The specific activity of AnGDH for xylose at 5mM concentration compared to glucose was 3.5%. No other sugars were used as substrate by this enzyme. The superior substrate specificity of AnGDH was also demonstrated in the performance of enzyme sensor strips. The impact of spiking xylose in a sample with physiological glucose concentrations on the sensor signals was investigated, and it was found that enzyme sensor strips using AnGDH were not affected at all by 5mM (75mg/dL) xylose. This is the first report of an enzyme sensor strip using a fungus derived FADGDH, which did not show any positive bias at a therapeutic level xylose concentration on the signal for a glucose sample. This clearly indicates the superiority of AnGDH over other conventionally used fungi derived FADGDHs in the application for SMBG sensor strips. The negligible activity of AnGDH towards xylose was also explained on the basis of a 3D structural model, which was compared to the 3D structures of A. flavus derived FADGDH and of two glucose oxidases.

Molecular markers predicting sensitivity to anticancer drugs are important and useful not only for selecting potential responders but also for developing new combinations. In the present study, we analyzed the difference in the sensitivity of xenograft models to capecitabine (Xeloda®), 5'-deoxy-5-fluorouridine (5'-DFUR, doxifluridine, Furtulon®) and 5-FU by comparing the mRNA levels of 12 pyrimidine nucleoside-metabolizing enzymes. Amounts of mRNA in the tumor tissues of 80 xenograft models were determined by real-time RT-PCR and mutual correlations were examined. A clustering analysis revealed that the 12 enzymes were divided into two groups; one group consisted of 8 enzymes, including orotate phosphoribosyl transferase (OPRT), TMP kinase (TMPK) and UMP kinase (UMPK), and was related to the de novo synthesis pathway for nucleotides, with mRNA expression levels showing significant mutual correlation. In the other group, 4 enzymes, including thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD), were involved in the salvage/degradation pathway of the nucleotides, and the mRNA levels of this group were dispersed more widely than that of the de novo group. Antitumor activity was assessed in 24 xenograft models for each drug. The antitumor activity of capecitabine and 5'-DFUR correlated significantly with the mRNA levels of TP and with the TP/DPD ratio, whereas the activity of 5-FU correlated significantly with OPRT, TMPK, UMPK and CD. In a stepwise regression analysis, TP and DPD were found to be independent predictive factors of sensitivity to capecitabine and 5'-DFUR, and UMPK was predictive of sensitivity to 5-FU. These results indicate that the predictive factors for sensitivity to capecitabine and 5'-DFUR in xenograft models may be different from those for 5-FU, suggesting that these drugs may have different responders in clinical usage.

Erlotinib, an epidermal growth factor receptor tyrosine kinase inhibitor, has been shown to have benefits for non-small cell lung cancer and pancreatic cancer patients; however, almost all patients develop progressive disease during the therapy. On the other hand, it has been reported that a tumor continues to express epidermal growth factor receptor even after developing progressive disease. To demonstrate the clinical relevance of erlotinib treatment after progressive disease, we investigated whether continuous administration of erlotinib in combination with chemotherapy has a useful effect on progressive disease development during erlotinib treatment. For this purpose, we examined the antitumor effect of a combination therapy of a chemotherapeutic agent with erlotinib using two types of erlotinib-resistant tumor xenograft models: a non-small cell lung cancer model, in which EBC-1, H1975 and HCC827TR3 tumors were implanted, and an HPAC pancreatic cancer cell xenograft which generates erlotinib-resistant tumors in vivo. As a result, the combination therapy showed a significantly higher antitumor activity compared with chemomonotherapy in all xenograft models except the H1975 xenografts. Furthermore, erlotinib alone suppressed the phosphorylation of epidermal growth factor receptor in HPAC tumors and the two non-small cell lung cancer cell lines other than H1975. Therefore, combination therapy which uses erlotinib can be considered effective if epidermal growth factor receptor phosphorylation is inhibited by erlotinib, even in erlotinib-resistant tumor xenograft models. Our results suggest that the continuous inhibition of epidermal growth factor receptor phosphorylation by erlotinib after progressive disease enhances the antitumor activity of chemotherapy.

Aspergillus-derived FAD-dependent glucose dehydrogenases (FADGDHs) were screened from fungal genomic databases, primarily by searching for putative homologues of the Aspergillus niger-derived glucose oxidase (GOD). Focusing on a GOD active-site motif, putative proteins annotated as belonging to the glucose methanol choline (GMC) oxidoreductase family were selected. Phylogenetic analysis of these putative proteins produced a GOD clade, which includes the A. niger and Penicillium amagasakiens GODs, and a second clade made up of putative proteins showing 30-40% homology with GOD. The genes encoding the proteins from the second clade were functionally expressed in Escherichia coli, resulting in dye-mediated glucose dehydrogenase (GDH) activity but not GOD activity. These results suggest that the putative proteins belonging to the second clade are FADGDHs. The 3D structure models of these FADGDHs were compared with the 3D structure of GOD.

Estrogens produced as a result of intratumoral aromatization has been recently shown to play important roles in proliferation of human non–small cell lung carcinomas (NSCLC), but the details have remained largely unknown. Therefore, in this study, we evaluated the possible roles of intratumoral aromatase in NSCLCs as follows: (a) evaluation of intratumoral localization of aromatase mRNA/protein in six lung adenocarcinoma cases using laser capture microdissection combined with quantitative reverse transcriptase-PCR and immunohistochemistry; (b) examination of the possible effects of isolated stromal cells from lung carcinoma tissues on aromatase mRNA transcript expression in lung carcinoma cell lines (A549 and LK87) through a coculture system; and (c) screening of cytokines derived from stromal LK001S and LK002S cells using cytokine antibody arrays and subsequent evaluation of effects of these cytokines on aromatase expression in A549 and LK87. Both aromatase mRNA and protein were mainly detected in intratumoral carcinoma cells but not in stromal cells. Aromatase expression of A549 and LK87 was upregulated in the presence of LK001S or LK002S cells. Several cytokines such as interleukin-6 (IL-6), oncostatin M, and tumor necrosis factor-α, all known as inducible factors of aromatase gene, were detected in conditioned media of LK001S and LK002S cells. Treatment of both oncostatin M and IL-6 induced aromatase gene expression in A549 an LK87, respectively. These results all indicated that intratumoral microenvironments, especially carcinoma-stromal cell interactions, play a pivotal role in the regulation of intratumoral estrogen synthesis through aromatase expression in human lung adenocarcinomas. Cancer Res; 70(16); 6659–69. ©2010 AACR.

It has been reported that bevacizumab in combination with paclitaxel significantly prolongs progression-free survival compared with paclitaxel alone in the initial treatment for metastatic breast cancer. To understand how bevacizumab enhances the efficacy of paclitaxel, we investigated the mechanism in a MX-1 human breast cancer xenograft model. The antitumor activity of bevacizumab at 5 mg/kg in combination with paclitaxel at 20 or 30 mg/kg was significantly higher than that of either agent alone. First, we measured the paclitaxel concentration in tumor to see whether bevacizumab enhances the activity by increasing the tumor concentration of paclitaxel. When given in combination with bevacizumab, the levels of paclitaxel in the tumor increased. Paclitaxel at 30 mg/kg with bevacizumab showed a similar tumor concentration as paclitaxel alone at either 60 or 100 mg/kg, with a similar degree of tumor growth inhibition. In contrast, no remarkable differences in paclitaxel concentration in the plasma or liver were observed between the paclitaxel monotherapy group and the paclitaxel plus bevacizumab group. An increase in paclitaxel concentration by bevacizumab was also found in another model, A549. In the same MX-1 model, vascular permeability in the tumor was significantly decreased by treatment with bevacizumab. There was no difference in microvessel density between the bevacizumab alone group and the combination group. Results suggest that the synergistic antitumor activity of paclitaxel and bevacizumab in combination may be a result of the increase in paclitaxel concentration in tumor resulting from the downregulation of vascular permeability when co-administered with bevacizumab.

Erlotinib treatment in combination with gemcitabine is a standard therapy for patients with locally advanced pancreatic cancer in many countries, including the US and the EU. Since mutations of the K-ras oncogene (KRAS) occur in approximately 90% of pancreatic cancers, we examined the antitumor activity of erlotinib in combination with gemcitabine in KRAS-mutated pancreatic cancer cell lines, HPAC and Capan-1, which have the KRAS mutation G12D and G12V, respectively. We analyzed the mode of inhibition of in vitro tumor cell proliferation by means of a combination index and found that a combination treatment of erlotinib plus gemcitabine had an additive effect in the two cell lines. We then examined the effect of erlotinib and gemcitabine on the phosphorylation of epidermal growth factor receptor (EGFR). Erlotinib strongly suppressed, while gemcitabine augmented the phosphorylation of EGFR, which was completely blocked by erlotinib in the two cell lines. An in vivo tumor growth inhibition test was then performed using the HPAC tumor xenograft model. The combination therapy of erlotinib and gemcitabine resulted in a significant inhibition of tumor growth compared with erlotinib or gemcitabine monotherapy. To the best of our knowledge, this is the first study to show the combination effect of erlotinib and gemcitabine in vivo using a xenograft model of a KRAS-mutated pancreatic cancer cell line.

Expression of the estrogen receptor (ER), the progesterone receptor (PgR) or the human epidermal growth factor receptor-2 (HER2) in tumors is a good prognostic marker for breast cancer patients. However, approximately 15–20% of breast cancer patients have triple-negative breast cancer (TNBC; negative for ER, PgR and HER2), and efficient therapeutic modalities for these patients are under investigation. We focused on thymidine phosphorylase (TP), an enzyme metabolizing 5′-DFUR, an intermediate of capecitabine, to 5-fluorouracil in order to investigate the application of well-known therapeutics for TNBC. Results of a gene expression analysis showed that TP expression in TNBC and basal-like breast cancer (BLBC) was higher than that of other subtypes. Immunohistochemically, the high expression of TP in TNBC and BLBC reflected expression in stromal but not tumor cells. Notably, a high TP expression was observed in the stromal cells of EGFR- and/or CK5/6-positive breast tumors. Our present results showing a high expression of TP in BLBC indicate that capecitabine-based chemotherapy would be of benefit for patients with TNBC.

The clinical relevance of prolonged trastuzumab administration in combination therapy beyond progressive disease (PD) has been suggested. Here, we examined whether trastuzumab treatment is effective in combination after failing to show antitumor activity as monotherapy in HER2-positive human breast cancer xenograft models.We established trastuzumab PD models with HER2-positive breast cancer xenograft models and compared the antitumor activity of trastuzumab in combination with a taxane versus monotherapy with a taxane in the models subsequent to tumor progression under trastuzumab monotherapy.We established trastuzumab PD model using the HER2-positive human breast cancer line MDA-MB-361 and KPL-4 in in vivo. In these models, trastuzumab at the same dose as the initial treatment showed no significant antitumor activity at 3 weeks after start of treatment. Re-inoculated tumor tissues showing PD regained sensitivity to trastuzumab. In the trastuzumab PD models, the HER2 status of the tumor tissues did not decrease. Also, the pAKT level continued to decrease, as with the initial treatment, and IGF-1R was not found to be up-regulated. Instead, differences were observed in the gene-expression profiles of the tumor tissues showing PD. Trastuzumab in combination with G-CSF, which is expected to enhance antibody-dependent cellular cytotoxicity (ADCC), showed significant antitumor activity, even though the single agents alone showed no antitumor activity in the PD model. In the MDA-MB-361 trastuzumab PD model, the combination of trastuzumab with paclitaxel showed significantly more potent antitumor activity compared with paclitaxel or docetaxel monotherapy. In the KPL-4 trastuzumab PD model as well, trastuzumab showed significant antitumor activity in combination with taxanes or capecitabine after PD had developed in response to trastuzumab monotherapy.We established in vivo trastuzumab PD models, in which trastuzumab monotherapy ceases to have antitumor activity during the treatment. The mechanisms of PD with trastuzumab are considered to involve both reversible changes in the gene expression profiles in tumor tissues and a decrease of ADCC activity in the host. Our present results demonstrated that trastuzumab showed antitumor activity in combination with taxanes or capecitabine even though it showed no antitumor activity as a monotherapy, suggesting a clinical relevance of treatment with trastuzumab as a combination therapy beyond PD.

We established models of cancer-related anemia in mice from subcutaneous inoculation of two IL-6-producing cancer cell lines, human lung cancer cell line LC-06-JCK and murine colon26 clone 5 colon cancer cells. In both models, elevated levels of IL-6 were detected in sera and hemoglobin levels significantly decreased compared with non-tumor-bearing mice. In the LC-06-JCK model, serum albumin levels also decreased with elevated levels of human IL-6 in sera. On the other hand, serum levels of EPO increased, although anemia developed and did not improve. The development of cancer-related anemia was prevented by the administration of a rat anti-mouse IL-6 receptor antibody, MR16-1, in the LC-06-JCK model. It is therefore suggested that IL-6 causes anemia independent of a reduction in EPO levels. Our preclinical models should be useful for exploring new modalities for the treatment of cancer-related anemia.

We report the first three-dimensional structure of fungus-derived glucose dehydrogenase using flavin adenine dinucleotide (FAD) as the cofactor. This is currently the most advanced and popular enzyme used in glucose sensor strips manufactured for glycemic control by diabetic patients. We prepared recombinant nonglycosylated FAD-dependent glucose dehydrogenase (FADGDH) derived from Aspergillus flavus (AfGDH) and obtained the X-ray structures of the binary complex of enzyme and reduced FAD at a resolution of 1.78 Å and the ternary complex with reduced FAD and D-glucono-1,5-lactone (LGC) at a resolution of 1.57 Å. The overall structure is similar to that of fungal glucose oxidases (GOxs) reported till date. The ternary complex with reduced FAD and LGC revealed the residues recognizing the substrate. His505 and His548 were subjected for site-directed mutagenesis studies and these two residues were revealed to form the catalytic pair, as those conserved in GOxs. The absence of residues that recognize the sixth hydroxyl group of the glucose of AfGDH and the presence of significant cavity around the active site may account for this enzyme activity toward xylose. The structural information will contribute to the further engineering of FADGDH for use in more reliable and economical biosensing technology for diabetes management.

To clarify the potential of parathyroid hormone-related protein (PTHrP) and interleukin-6 (IL-6) as cachectic factors in a colon 26 model and the effects of capecitabine on cancer cachexia as determined by plasma levels of IL-6 and PTHrP and body weight loss.From two colon 26 sublines-cancer cachectic clone20 and non-cachectic clone5 plasma levels of PTHrP protein and mRNA expression levels in tumor tissues were compared. An IL-6 neutralizing antibody, a PTHrP neutralizing antibody, and capecitabine were administered into mice bearing clone20 and their anticachectic effects evaluated.The plasma level of PTHrP protein in mice bearing clone20 was higher than that in mice bearing clone5. The expression level of PTHrP mRNA was 49-fold higher in tumor tissues of clone20 than of clone5, according to GeneChip analysis. PTHrP antibody as well as IL-6 antibody suppressed wasting of the body and gastrocnemius and adipose tissue weights. PTHrP antibody suppressed the induction of hypercalcemia but not hypoglycemia or elevation of IL-6, whereas IL-6 antibody suppressed the induction of hypoglycemia but not hypercalcemia or elevation of PTHrP. Capecitabine, a fluorinated pyrimidine anticancer agent, improved body wasting of mice bearing clone20 at a low dose with no reduction of tumor volume. Furthermore, capecitabine lowered the levels of PTHrP and IL-6 in plasma and suppressed hypoglycemia and hypercalcemia in this model. Capecitabine also showed anticachectic effects on cachexia in a cancer model induced by human cervical cancer cell line Y (also known as Yumoto).PTHrP and IL-6 were found to be factors in the development of cachexia in a colon 26 cancer model, and capecitabine improved cancer cachexia by suppressing the plasma levels of IL-6 and PTHrP in colon 26 and Y cachectic models.

A hyperthermophilic beta-1,4 endoglucanase was identified in Pyrococcus horikoshii, a hyperthermophilic archaeon. In order to clarify the function of the protein in detail, structural and catalytic site studies were performed using protein engineering. By removing some of the C-terminal sequence of the ORF of the endoglucanase (PH1171), two types of recombinant proteins were expressed from one ORF, using Escherichia coli. One exhibited endoglucanase activity, and the other did not. An SD-like sequence was identified in the ORF of the endoglucanase. By removing the SD-like sequence without changing the amino acid sequence of the endoglucanase, one recombinant endoglucanase was prepared effectively from E. coli. From the analysis of the N- and C-terminal regions of the ORF, this endoglucanase appears to be a secreted and membrane-binding enzyme of P. horikoshii. A mutation analysis of the endoglucanase, using the synthetic substrate, indicated that Glu342 is a candidate for the active center and plays a critical role in the activity of the enzyme. Additional catalytic amino acid residues were not found. These results indicate that the catalytic residue of the enzyme is different from that of typical family 5 endoglucanase, even though it has a high homology to the endoglucanase from Acidothermus celluloliticus. The activity of the enzyme, using carboxy methylcellulose and crystalline cellulose as the substrates, was increased, but not for a synthetic low-molecular substrate when a carbohydrate-binding module of chitinase from P. furiosus was added to the C-terminal region.

The development of therapeutic resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs, ie erlotinib or gefitinib) has been the major clinical problem when treating lung adenocarcinoma patients with these agents. However, its mechanisms have not necessarily been well studied to this date. Autophagy has been recently considered to play pivotal roles in escaping from the effects of anti-neoplastic agents. Therefore, in this study, we examined its roles in the development of resistance to EGFR-TKIs in lung adenocarcinoma. We first established erlotinib-resistant cell lines (PC9/ER) from parental PC9 cells by exposing the cells to erlotinib. In PC9/ER, autophagy-related LC3A expression came to be up-regulated and constitutive activation of LC3A-mediated autophagy became more pronounced through the process of acquiring therapeutic resistance. In addition, inhibition of LC3A or autophagy restores sensitivity to EGFR-TKIs in PC9/ER. LC3A was also activated at the transcriptional level in de novo resistant cells via demethylation of the MAP1LC3A gene. We then evaluated the status of LC3A in 169 lung adenocarcinoma patients using immunohistochemistry. LC3A immunoreactivity was only detected in carcinoma cells (89/169 patients), not in non-tumoural cells. In addition, LC3A immunoreactivity was significantly correlated with progression-free survival (p = 0.0039) and overall survival (p = 0.0040) of 35 patients treated with EGFR-TKIs. The results of our present study demonstrated that LC3A-mediated autophagy in carcinoma cells was involved in the development of resistance to EGFR-TKIs, and that LC3A could serve as a promising therapeutic target for overcoming resistance to EGFR-TKIs and a novel predictor of response to EGFR-TKIs in lung adenocarcinoma patients. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd

AXL (anexelekto) has been explored as a potential novel therapeutic target for non-small cell lung carcinoma (NSCLC) but its activation status has not been evaluated in NSCLC.We first immunolocalized the phosphorylated form of AXL in 112 lung adenocarcinoma cases and subsequently evaluated the anti-neoplastic effects of monoclonal antibody AXL in two lung adenocarcinoma cell lines, PC9 and A549.Phospho-AXL immunoreactivity was detected in 59.8% of adenocarcinoma cases examined and tended correlate significantly with larger tumor size (p=0.08) and with overall survival of the patients (p=0.041). Results of in vitro analysis revealed that the monoclonal antibody to AXL significantly inhibited cell proliferation of PC9 and A549, lung adenocarcinoma cell lines, which was caused by an inhibition of extracellular signal-regulated kinase (ERK) activation.AXL-targeted therapy, possibly through inhibiting ERK activation of carcinoma cells, could confer clinical benefits on patients with lung adenocarcinoma.

Experimental and clinical studies have shown that human breast cancer is an angiogenesis-dependent neoplasm. In fact, several authors have demonstrated that the determination in primary tumors of the degree of vascularization (microvessel counts) as well as of some angiogenic peptides is of prognostic value. However, which are the most important mediators of angiogenesis and their relationship with other relevant biological markers needs further investigation. In the series of 260 women with node-negative breast cancer (NNBC) on which we previously assessed vascular endothelial growth factor (VEGF), we have now also determined thymidine phosphorylase (TP) protein as well as p53 protein and Cathepsin-D cytosolic levels using immunometric methods. The median concentrations of TP, p53 and Cathepsin-D were 105.4U/mg (range 1.2–843.1), 0.22 ng/mg (range 0.0–41.65) and 33.80nmol/mg (range 4.20–216.0), respectively. We found that TP concentrations were associated with Cathepsin-D and p53, but not with VEGF. VEGF (p

The development of therapeutic resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs, ie erlotinib or gefitinib) has been the major clinical problem when treating lung adenocarcinoma patients with these agents. However, its mechanisms have not necessarily been well studied to this date. Autophagy has been recently considered to play pivotal roles in escaping from the effects of anti-neoplastic agents. Therefore, in this study, we examined its roles in the development of resistance to EGFR-TKIs in lung adenocarcinoma. We first established erlotinib-resistant cell lines (PC9/ER) from parental PC9 cells by exposing the cells to erlotinib. In PC9/ER, autophagy-related LC3A expression came to be up-regulated and constitutive activation of LC3A-mediated autophagy became more pronounced through the process of acquiring therapeutic resistance. In addition, inhibition of LC3A or autophagy restores sensitivity to EGFR-TKIs in PC9/ER. LC3A was also activated at the transcriptional level in de novo resistant cells via demethylation of the MAP1LC3A gene. We then evaluated the status of LC3A in 169 lung adenocarcinoma patients using immunohistochemistry. LC3A immunoreactivity was only detected in carcinoma cells (89/169 patients), not in non-tumoural cells. In addition, LC3A immunoreactivity was significantly correlated with progression-free survival (p = 0.0039) and overall survival (p = 0.0040) of 35 patients treated with EGFR-TKIs. The results of our present study demonstrated that LC3A-mediated autophagy in carcinoma cells was involved in the development of resistance to EGFR-TKIs, and that LC3A could serve as a promising therapeutic target for overcoming resistance to EGFR-TKIs and a novel predictor of response to EGFR-TKIs in lung adenocarcinoma patients.

The antitumor activity of cytostatic 5'-deoxy-5-fluorouridine (5'-dFUrd) depends on its being converted to 5-fluorouracil (5-FUra) by the enzyme thymidine phosphorylase (dThdPase, EC 2.42.4). We prepared mouse anti-human dThdPase monoclonal antibodies to serve as tools for clinical studies with this drug. Partially purified dThdPase obtained form HCT116 human colon cancer cells grown in athymic mice was used as and antigen for the immunization of mice. Six hybridomas were cloned which produced anti-human dThdPase antibodies, as detected by Western blot analysis with human dThdPase. With these antibodies, we developed an ELISA method sensitive enough to measure dThdPase levels, even in tumor tissue samples weighing as little as 10 mg. In addition, one monoclonal antibody was suitable for immunologically staining the enzyme in tumor tissues. Thus, these anti-human dThdPase monoclonal antibodies could be used to measure levels of the enzyme in tumor cells, which is essential for the activation of 5'-dFUrd.

[Introduction] Self-monitoring of blood glucose (SMBG) is inevitable for the glycemic control of the diabetic patients, and plays an indispensable role in the current diabetes management. The current most popular principle for the sensor for SMBG is based on the so-called “second generation” type that are the use of the dye mediated dehydrogenase reactions. The principle employs the artificial electron acceptor and monitoring the concentration/quantity of thus reacted (reduced) artificial electron acceptor on the electrode. Fungi-derived flavin adenine dinucleotide dependent glucose dehydrogenases (FADGDHs) have recently been paid significant attention, focusing its narrow specificity, especially as the enzyme not active toward maltose. However, interaction between FADGDHs and artificial electron acceptor has not yet been investigated. Recently, we have reported on the first 3D structures of FADGDH, using Aspergillus flavus derived FADGDH (AfGDH). Here we report the investigation of interaction between mediator and enzyme by elucidating the 3D structure of AfGDH complex with potassium ferricyanide. Mutagenesis studies were also carried out to investigate the role of residues elucidated to interact with ferricyanide. [Methods] The structural gene of AfGDH was inserted into an expression vector and the enzyme was recombinantly expressed using E. coli BL21 (DE3). Soluble AfGDH was harvested and purified by hydrophobic interaction chromatography and anion exchange chromatography. Purified protein was concentrated, and crystallized by sitting-drop vapor-diffusion method. In order to obtain a ternary complex structure with FAD and ferricyanide, a single crystal was soaked in the potassium ferricyanide solution. X-ray diffraction data were collected and structure was determined. Based on the 3D structure, several mutants were constructed by site-directed mutagenesis and evaluated GDH activity, which was determined using either phenazine methosulphate/2,6-dichlorophenol indophenol (PMS/DCIP) or potassium ferricyanide as the electron acceptors. [Results & Discussion] A ternary complex structure of AfGDH with FAD and ferricyanide complex was obtained. Two potassium ferricyanide molecules were observed in the protein structure. One potassium ferricyanide interacted with one lysine residue, whereas the other molecules interacted with two lysine residues. In order to elucidate the role of these lysine residues, mutant AfGDHs were constructed by substituting lysine residues with alanine. The structural gene of AfGDH mutants were inserted into an expression vector and the enzyme was recombinantly prepared using E. coli BL21 (DE3). All mutants were expressed at soluble fraction. These mutants were subjected for anion exchange chromatography for purification. Interestingly, the mutant enzymes were eluted at different salt concentration than those eluted in the purification of wild type enzyme, suggesting that surface net charge was significantly changed by these mutations. Some of the mutant AfGDH showed unique property in the specificity toward electron acceptors. The GDH activities were compared by using either PMS/DCIP or ferricyanide as the electron acceptor, for both wild type and mutant enzymes. Although the alanine substitutions to these lysine residues did not significantly influenced PMS/DCIP based dye mediated GDH activity, but some mutation affected the ratio of dye mediated GDH activities of those using PMS/DCIP and of using ferricyanide. These results, together with the results of the change in the elution properties during anion exchange chromatography, suggested the lysine residues, we focused, play important role in the affinity toward ferricyanide during oxidative half reaction. (1) K. Mori, et al., Biotechnol Lett. 33(11) 2255-63(2011) (2) G. Sakai, et al., Biotechnol Lett. 37(5) 1091-99(2015) (3) H. Yoshida, et al. Sci. Rep. 5:13498. doi:10.1038/srep13498 (2015)

【Introduction】 The fungi derived flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenases (GDHs) are flavoproteins that catalyze the oxidation of first hydroxyl group of glucose and other sugar molecules, using FAD as the primary electron acceptor (systematic name; D-glucose:acceptor 1-oxidoreductase). Recently, fungi-derived FADGDHs have received attention as the enzymes for blood glucose monitoring in diabetes patients, especially for the use in the self-monitoring of blood glucose (SMBG), because of their oxygen insensitivity and relatively narrow substrate specificity, in particular their lack of activity toward maltose. However, unlike glucose oxidase, a gold standard enzyme for blood glucose monitoring, especially for the strictly narrow specificity toward glucose, fungi derived FADGDHs react with xylose as the substrate. We have previously reported on the characterization of Aspergillus flavus derived FADGDH (AfGDH) from genome database 1, and also the stabilization of AfGDH by introducing a disulfide bond 2. Recently we have reported on the first 3D structure of fungus derived FADGDH3. In order to construct an engineered enzyme meeting with the current increasing demands in the accuracy of sensors for SMBG, further advance in our understanding of the mechanism of substrate recognition of this enzyme is necessary. In this study, we report the site directed mutagenesis studies toward the residues expected to influence the binding of substrate, based on the structural analyses of AfGDH crystal structure (PDB ID：4ynu). 【Methods】 The residues responsible for the substrate binding and/or recognition of AfGDH and Aspergillus niger derived GOx (AnGOx), were predicted based on the reported crystal structure (PDB ID:4ynu, 1cf3). According to this prediction, several mutant AfGDHs were designed and constructed by the sited directed mutagenesis. AfGDH wild type (WT) and mutants were prepared using recombinant Escherichia coli. The enzyme activities and substrate specificities were determined by measuring dye mediated dehydrogenase activity, spectrometrically, using phenazine methosulphate as the electron mediator and 2,6-dichlorophenolindophenol(DCIP) as the final electron acceptor, and by monitoring decrease in the absorbance of oxidized DCIP at 600nm. 【Results and Discussion】 Several residues existing within the distance which may interact with glucono-δ-lactone by their side chain were observed from the ternary complex structure of AfGDH (4ynu). Assuming that this side chain of each residue may recognize and interact with glucose, AfGDH structure was compared with AnGOx. As results, residues, possibly recognize 1st, 2nd, 3rd or 4th hydroxyl group of glucose are conserved within AfGDH and AnGOx. Especially, two residues recognize 1st hydroxyl group of glucose are completely conserved. Mutational analyses resulted in the complete loss of the catalytic activity, thus suggesting that these residues are the catalytic residues. The residue interacting 4th hydroxyl group is conserved, and has been recognized that plays an important role in discrimination of monosaccharide from disaccharides, such as maltose. Interestingly, the introduction of mutation on this residue did not result in the significant change in the substrate specificity, especially toward maltose. The results suggested that the predicted residue may have a limited role in the determination of substrate specificity, and other residues may have more crucial roles. Significant difference in the substrate interaction was observed at the position where may recognize 6th hydroxyl group, which would be the reason of the AfGDH substrate specificity reacting with xylose. Further mutational analyses will be reported. (1) K. Mori, et al., Biotechnol Lett. 33(11) 2255-63(2011) (2) G. Sakai, et al., Biotechnol Lett. 37(5) 1091-99(2015) (3) H. Yoshida, et al., Sci. Rep. 5:13498 doi: 10.1038/srep13498(2015)

To investigate the mechanism causing cachexia and its association with shorter patient survival, we cloned weight-loss-inducing and -non-inducing sublines of murine colon 26 carcinoma (colon 26). One clone, clone 20, induced substantial weight loss, wasting of adipose tissue and muscle, and hypogly-cemia in mice with a minimum tumor burden of 0.3 g (2 g per 100 g body weight). Clone-20-bearing mice had a median survival of 24.5 days with average tumor weight of 0.4 g. In contrast, clone 5 induced neither severe weight loss, wasting of adipose tissue and muscle, nor hypoglycemia; clone-5-bearing mice survived for a median of 70 days with average tumor weight of 12 g. These results clearly indicate that shorter survival is associated with the degree of cachexia and is independent of tumor size. Using this pair of colon 26 clones, we examined mediators of cachexia. Neither TNFα, IL-1α nor IFNγ was detected in the serum of mice bearing either clone, while IL-6 was detected in mice bearing both clones by ELISA and a bioassay. Administration of anti-IL-6 monoclonal antibody (MAb) partially but significantly suppressed cachexia induction in clone-20-bearing mice. These results point to the involvement of IL-6 in experimental cachexia. However, our finding of the presence of IL-6 in the serum of mice bearing clone 5, which does not induce weight loss, clearly indicates that IL-6 is not solely responsible for the induction of cachexia. © 1995 Wiley-Liss, Inc.

The objective of this study was to examine the association between the immunohistochemical Ki67 labeling index (IHC Ki67), Ki67 mRNA expression level, and first‐generation gene signatures in a cohort of breast cancer patients. We assessed associations between IHC Ki67 and first‐generation gene signatures in a panel of 39 tumor samples, using an oligonucleotide microarray. Gene expression analyses included Ki67 alone (MKi67), 21‐gene signature, mitosis kinome score signature, and genomic grade index. Correlation coefficients were calculated by Spearman's rank correlation test. In all cases, IHC Ki67, MKi67, and three genetic markers were highly correlated (ρ, 0.71–0.97). Estrogen receptor (ER)‐positive cases showed strong correlations between IHC Ki67 and other signatures (ρ, 0.79–0.83). The ER‐negative cases showed slightly lower correlations (ρ, 0.58–0.73). In ER‐positive cases, the low IHC Ki67 group showed significantly longer relapse‐free survival than the high IHC Ki67 group (P = 0.007). This difference was confirmed by multivariate analysis. Our data indicate that IHC Ki67 shows similar predictive power for proliferation in ER‐positive cancers as genomic markers. Further study of IHC Ki67 is needed to define prognostic factors and predictive factors for chemotherapy using central laboratory assessment. (Cancer Sci, doi: 10.1111/j.1349‐7006.2012.02319.x, 2012)

We examined the efficacy of chemoendocrine therapy using capecitabine as a chemotherapeutic agent in premenopausal and postmenopausal models with estrogen receptor (ER)-positive human breast cancer xenografts. Tamoxifen and letrozole were used as endocrine therapeutic agents for premenopausal and postmenopausal models, respectively. The antitumor activity of capecitabine in combination was significantly superior to either monotherapy treatment in both premenopausal (p

Recombinant human erythropoietin (rhEPO) has been used in the EU and the United States for the treatment of anemia in cancer patients after myelosuppressive chemotherapy or radiotherapy. However, several conflicting results have been reported concerning the detrimental effect of rhEPO on survival benefit in cancer patients. In experimental studies, contradictory results were also reported in in vitro tumor cell proliferation studies and in vivo tumor growth studies using tumor cells expressing EPO-receptor (EPO-R). Therefore, we tried to clarify the effect of epoetin β, a product of rhEPO, on tumor growth in xenograft models using five EPO-R-positive human cancer cell lines, namely the MCF7 breast, 786-O renal, SCH gastric, A549 lung and SK-OV-3 ovarian cancer cell lines. Epoetin β was administered once a week for 3 weeks at doses of 1,000, 3,000 and 10,000 IU/kg in accordance with the clinical administration schedule and dosages. As a result, no enhancement of tumor growth from the administration of epoetin β was observed in any of the xenograft models throughout the experiment duration. The effect of epoetin β on the antitumor activity of bevacizumab, an anti-angiogenic agent, was additionally examined using A549 and MCF7 xenograft models, since rhEPO reportedly stimulates tumor neovascularization. Epoetin β showed no significant effect on the antitumor activity of bevacizumab in either xenograft model. These findings suggest that epoetin β is not involved in in vivo tumor growth promotion.

Erlotinib is used as a standard treatment for recurrent advanced non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) mutations in NSCLC have been shown to be a predictive factor of erlotinib, although the relationship between K-ras oncogene (KRAS) mutations and erlotinib resistance is controversial. Recently, in vitro sequence-dependent interactions of erlotinib and docetaxel have been studied on as a novel therapeutic approach against NSCLC. The purpose of the present study was to determine the optimum novel regimen of erlotinib and docetaxel against NSCLC cells which have EGFR mutation (HCC827 cells), KRAS mutation (A549 cells) or both wild-type (NCI-H292 cells). First, we analyzed the effects of in vitro combination for cell proliferation-inhibition using a combination index. In all cell lines, docetaxel followed by erlotinib treatment showed nearly additive effects. On the other hand, erlotinib followed by docetaxel treatment showed remarkable antagonistic interactions. Second, we examined the effect of combinations on the in vitro apoptosis induction. Erlotinib followed by docetaxel treatment reduced apoptosis induction compared with docetaxel alone; in contrast, docetaxel followed by erlotinib treatment had no inhibitory effects on docetaxel-induced apoptosis in any of the cell lines. Finally, an in vivo tumor growth inhibition test was performed using xenograft models. Docetaxel followed by erlotinib administration resulted in significant tumor growth inhibition compared with erlotinib or docetaxel monotherapy in all models. In conclusion, we demonstrated that docetaxel followed by erlotinib therapy was a potentially optimum regimen against NSCLC regardless of the mutation status of EGFR and KRAS.

We carried out gene expression profiling of forty human tumor cells for research choice method of the most fitting anticancer drug, using unsupervised hierarchal clustering analysis. This clustering analysis is based on a tumor growth inhibition panel of nine antitumor drugs (MMC, CDDP, ACNU, CPT-11, CPA, FT-207, UFT, 5'-DFUR and ADM) for forty human cancers. These cancers(eleven stomach, seven colon, six breast, three pancreas, five lung, two esophageal carcinomas, one liver, one renal cell carcinoma, one uterus, two ovarian, and one melanoma) have been maintained by serial s. c. passages in nude mice of the same sex of donor patients. Nine antitumor drugs were divided into two groups, a 5-FU-related drug group (5'-DFUR, FT-207 and UFT) and another group. On the other hands, forty cells were clustered into four groups. By using GeneChip (Hu95Av2, Affymetrix), we investigated gene expression profiling of the matched tumor cells and selected specific genes in each group. Interestingly, a pathway analysis revealed that expressions of p53-related genes were up-regulated in the 5-FU-sensitive groups. This result suggested that chemosensitivity was predicted by gene expression profiling of tumor cells. We considered that microarray analysis would be a good tool for further tailor-made medications.

The measurement of glycated hemoglobin A1c (HbA1c) has important implications for diagnosis of diabetes and assessment of treatment effectiveness. We proposed specific sequence motifs to identify enzymes that oxidize glycated compounds from genome database searches. The gene encoding a putative fructosyl amino acid oxidase was found in the Phaeosphaeria nodorum SN15 genome and successfully expressed in Escherichia coli. The recombinant protein (XP_001798711) was confirmed to be a novel fructosyl peptide oxidase (FPOX) with high specificity for α-glycated compounds, such as HbA1c model compounds fructosyl-αN-valine (f-αVal) and fructosyl-αN-valyl-histidine (f-αVal-His). Unlike previously reported FPOXs, the P. nodorum FPOX has a Km value for f-αVal-His (0.185 mM) that is considerably lower than that for f-αVal (0.458 mM). Based on amino acid sequence alignment, three dimensional structural modeling, and site-directed mutagenesis, Gly60 was found to be a determining residue for the activity towards f-αVal-His. A flexible surface loop region was also found to likely play an important role in accepting f-αVal-His. Biotechnol. Bioeng. 2010; 106: 358–366. © 2010 Wiley Periodicals, Inc.

We have evaluated the antitumor activity of XELOX (a combination therapy of capecitabine (Xeloda) and oxaliplatin) in human colorectal and gastric cancer xenograft models. In human colorectal cancer model CXF280, antitumor activity of the combination at two-thirds of the maximum tolerated dose (MTD) was superior to that of each monotherapy at MTD. Furthermore, in human colorectal cancer model COL-05-JCK and human gastric cancer xenograft model GXF 97, the combination also showed at least additive antitumor activity. In addition, toxicity was not augmented with the combination therapy in these three models. As demonstrated using ELISA or immunohistochemistry, oxaliplatin in xenograft model tumors up-regulated the level of thymidine phosphorylase (dThdPase), a key enzyme for the metabolism of capecitabine to 5-fluorouracil. These results suggest that oxaliplatin might potentiate the antitumor activity of capecitabine by up-regulating the tumor level of dThdPase. Based on these results, clinical trials of XELOX against colorectal and gastric cancers are warranted.

To clarify the antitumor activity of trastuzumab and its potential as an effective treatment for gastric cancer patients.Levels of HER2 expression in tumor tissues of gastric cancer cell lines were examined using immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and mRNA quantification. Efficacy of trastuzumab was examined as a single agent or in combination with chemotherapeutic agents widely used clinically for gastric cancers in HER2-overexpressing human gastric cancer xenograft models.Two of nine human gastric cancer xenograft models, NCI-N87 and 4-1ST, showed overexpression of HER2 mRNA and protein by IHC (HercepTest) and HER2 gene amplification by FISH (Pathvysion). HER2 protein showed potent staining in peripheral membranes, similar to the staining pattern of breast cancer. FISH scores were also comparable to those of breast cancer models. Trastuzumab as a single agent inhibited the tumor growth in both of the HER2-overexpressing models but not in the HER2-negative models, GXF97 and MKN-45. In any combination with capecitabine, cisplatin, irinotecan, docetaxel, or paclitaxel, trastuzumab showed more potent antitumor activity than the anticancer agents alone. A three-drug combination of capecitabine, cisplatin, and trastuzumab showed remarkable tumor growth inhibition. In NCI-N87 in vitro, trastuzumab showed direct antiproliferative activity according to cell count or crystal violet dying, and showed indirect antitumor activity such as antibody-dependent cellular cytotoxicity.The antitumor activity of trastuzumab observed in human gastric cancer models warrants consideration of its use in clinical treatment regimens for human gastric cancer as a single agent or a combination drug with various chemotherapeutic agents.

It has been reported that one of the hyperthermostable aminopeptidases from Pyrococcus horikoshii exhibits hydrolytic activity toward short peptides and acyl-peptides (deblocking activity). In the genome database of P. horikoshii, two new open reading frames homologous to the hyperthermostable aminopeptidase of P. horikoshii were found. The two new genes for the proteins were cloned, expressed using E. coli, and characterized. The purified proteins gave a single band on SDS-PAGE corresponding to molecular masses of 42 kDa and 41 kDa respectively, and exhibited aminopeptidase activity, including deblocking activity. These enzymes are likely to exist as oligomeric structures at neutral pH. The optimum pHs of the two enzyme activities were in the range of 7.0 to 7.5, and the optimum temperatures for the activities were around 100 degrees C. The enzymes exhibited low hydrolytic activity for peptide substrates longer than 10 residues. They were activated by cobalt and zinc ions. Their substrate specificities and activation factors are different. It was confirmed that P. horikoshii has three similar aminopeptidases with deblocking activity and that these enzymes appear to play important roles in hydrolyzing small peptides in P. horikoshii cells.

The combination of irinotecan and a fluoropyrimidine has been widely accepted as a treatment for advanced colorectal carcinoma. However, there have been no evaluable data on the feasibility of these combinations. To assess the significance of such combinations, we attempted to identify gene expression patterns in response to irinotecan and two different types of fluoropyrimidines. In 12 patients dispositioned to receive preoperative chemotherapy for colorectal carcinoma, pre-therapy tumor biopsies and final resected specimens were available for analysis. Patients were randomly assigned to receive one of the following four regimens: (I), oral doxifluridine; (II), intravenous infusion of 5-FU; (III), intravenous infusion of irinotecan; (IV), combination of doxifluridine and irinotecan (I+III). To identify genes whose expressions changed, we analyzed the gene expression profiles prior to and after these therapies using an oligonucleotide microarray consisting of 12,000 genes. Next, we focused on the genes that demonstrated similar kinetics of altered expression in all patients in each of the regimens. We identified two proto-oncogenes, nuclear receptor of T-cells (NOT) and c-fos, that were up-regulated in doxifluridine- and irinotecan-related regimens but unchanged in the 5-FU-related regimen. Moreover, group IV tumors showed the highest apoptotic rate and lowest proliferation activity following the combined chemotherapy. These results suggest that doxifluridine has a synergistic impact on the therapeutic effect of irinotecan by up-regulating proto-oncogenes such as NOT and c-fos, and thus justify the use of one of the irinotecan and fluoropyrimidine combinations.

The deblocking aminopeptidase (DAP) of Pyrococcus horikoshii is a hyperthermophilic exoprotease that cleaves the N-terminal amino acid of peptide substrates with a putative deblocking activity for acylated peptides. DAP has been found to be homologous to a tetrahedral aminopeptidase from the halophilic Haloarcula marismortui. The latter enzyme is a dodecameric complex and has been revealed to be a self-compartmentalized protease whose central cavity harbouring the catalytic site is accessible through several channels of different size, unlike all other known proteolytic complexes. Three paralogues of DAP have been identified in P. horikoshii, with about 40% identity between them. Each of them has been overexpressed in Escherichia coli, purified and crystallized in the native and selenomethionine-substituted states. The results indicate that they form two kinds of assemblies, of 12 and of 24 subunits, with a molecular weight of approximately 400 and approximately 800 kDa, respectively. Crystals of the different variants of DAP and in their different oligomeric states diffract up to a resolution of 3 A.

Capecitabine (N4-pentyloxycarbonyl-5′-deoxy-5-fluorocytidine) is a novel oral fluoropyrimidine carbamate, which is converted to 5-fluorouracil (5-FU) selectively in tumours through a cascade of three enzymes. The present study investigated tissue localisation of the three enzymes in humans, which was helpful for us to design the compound. Carboxylesterase was almost exclusively located in the liver and hepatoma, but not in other tumours and normal tissue adjacent to the tumours. Cytidine (Cyd) deaminase was located in high concentrations in the liver and various types of solid tumours. Finally, thymidine phosphorylase (dThdPase) was also more concentrated in various types of tumour tissues than in normal tissues. These unique tissue localisation patterns enabled us to design capecitabine. Oral capecitabine would pass intact through the intestinal tract, but would be converted first by carboxylesterase to 5′-deoxy-5-fluorocytidine (5′-dFCyd) in the liver, then by Cyd deaminase to 5′-deoxy-5-fluorouridine (5′-dFUrd) in the liver and tumour tissues and finally by dThdPase to 5-FU in tumours. In cultures of human cancer cell lines, the highest level of cytotoxicity was shown by 5-FU itself, followed by 5′-dFUrd. Capecitabine and 5′-dFCyd had weak cytotoxic activity only at high concentrations. The cytotoxicity of the intermediate metabolites 5′-dFCyd and 5′-dFUrd was suppressed by inhibitors of Cyd deaminase and dThdPase, respectively, indicating that these metabolites become effective only after their conversion to 5-FU. Capecitabine, which is finally converted to 5-FU by dThdPase in tumours, should be much safer and more effective than 5-FU, and this was indeed the case in the HCT116 human colon cancer and the MX-1 breast cancer xenograft models.

Murine colon 26 carcinoma causes cachexia even when the tumor burden is small. In this tumor model, murine IL-12 suppressed the induction of cancer cachexia and also inhibited tumor growth. IL-12 reduced the serum levels of IL-6, a cachexia mediator in this model, and alleviated the body weight loss and other abnormalities associated with cachexia, such as adipose tissue wasting and hypoglycemia. The anticachectic activity was observed even at low doses of IL-12, insufficient to inhibit tumor growth. IL-12 greatly increased levels of IFN-gamma in the tumor tissue and, to a lesser extent, in the circulation. IFN-gamma given intraperitoneally also prevented cancer cachexia, although it did not reduce IL-6 levels either in the tumor or in the circulation. In athymic mice bearing the same colon 26 tumor, IL-12 was no longer anticachectic and did not induce IFN-gamma. These results indicate that the anticachectic activity of IL-12 is T-cell-dependent and results from at least 2 mechanisms, the down-regulation of IL-6 and the up-regulation of IFN-gamma.

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Background Bevacizumab is a humanized monoclonal antibody to human vascular endothelial cell growth factor (VEGF) and has been used for many types of cancers such as colorectal cancer, non-small cell lung cancer, breast cancer, and glioblastoma. Bevacizumab might be effective against gastric cancer, because VEGF has been reported to be involved in the development of gastric cancer as well as other cancers. On the other hand, there are no established biomarkers to predict the bevacizumab efficacy in spite of clinical needs. Therefore, we tried to identify the predictive markers for efficacy of bevacizumab in gastric cancer patients by using bevacizumab-sensitive and insensitive tumor models. Methods Nine human gastric and two colorectal cancer mouse xenografts were examined for their sensitivity to bevacizumab. We examined expression levels of angiogenic factors by ELISA, bioactivity of VEGF by phosphorylation of VEGFR2 in HUVEC after addition of tumor homogenate, tumor microvessel density by CD31-immunostaining, and polymorphisms of the VEGF gene by HybriProbe™ assay. Results Of the 9 human gastric cancer xenograft models used, GXF97, MKN-45, MKN-28, 4-1ST, SC-08-JCK, and SC-09-JCK were bevacizumab-sensitive, whereas SCH, SC-10-JCK, and NCI-N87 were insensitive. The sensitivity of the gastric cancer model to bevacizumab was not related to histological type or HER2 status. All tumors with high levels of VEGF were bevacizumab-sensitive except for one, SC-10-JCK, which had high levels of VEGF. The reason for the refractoriness was non-bioactivity on the phosphorylation of VEGFR2 and micro-vessel formation of VEGF, but was not explained by the VEGF allele or VEGF165b. We also examined the expression levels of other angiogenic factors in the 11 gastrointestinal tumor tissues. In the refractory models including SC-10-JCK, tumor levels of another angiogenic factor, bFGF, were relatively high. The VEGF/bFGF ratio correlated more closely with sensitivity to bevacizumab than with the VEGF level. Conclusions VEGF levels and VEGF/bFGF ratios in tumors were related to bevacizumab sensitivity of the xenografts tested. Further clinical investigation into useful predictive markers for bevacizumab sensitivity is warranted.