Rapid decrease of intracellular pH associated with inhibition of Na+/H+ exchanger precedes apoptotic events in the MNK45 and MNK74 gastric cancer cell lines treated with 2-aminophenoxazine-3-one.

The effects of Phx-3 on changes in intracellular pH (pHi) in the MKN45 and MKN74 human gastric cancer cell lines were evaluated in order to determine the mechanism for the proapoptotic effects of 2-aminophenoxazine-3-one (Phx-3) on these cells. Phx-3 (100 µM) reduced pHi in MKN45 from 7.45 to 5.8, and in MKN74 from 7.5 to 6.2 within 1 min of engagement with these cells. Such a decrease of pHi was closely correlated with the dose of this phenoxazine and continued for 4 h. The activity of Na+/H+ exchanger isoform l (NHE1), which is involved in H+ extrusion from the cells, was dose-dependently suppressed by Phx-3 in these cells, and was greatly suppressed in the presence of 100 µM Phx-3. This result indicates that the decrease of pHi in MKN45 and MKN74 cells is closely associated with the inhibition of NHE1 in these cells. The morphology of these cells at 24 h after treatment with Phx-3 indicated shrinkage of the cells and condensation of the nuclear chromatin structure, which are characteristic of the apoptotic events in these gastric cancer cells. Cytotoxicity of Phx-3 against MKN45 and MKN74 cells was extensive because almost all MKN45 cells lost viability at 24 h in the presence of 20 µM Phx-3, and nearly 50% of the MKN74 cells lost viability in the presence of 50 µM Phx-3. These results suggest that rapid and extensive decrease of pHi in human gastric cancer MKN45 and MKN74 cells caused by Phx-3 might disturb intracellular homeostasis, leading to apoptotic and cytotoxic events in these cells. Phx-3 is a good candidate for therapeutics of gastric cancer that is intractable to conventional chemopreventive therapies.

[Review of our home nutrition therapy cases].

We provided a home care service to 118 patients from December 1996 to December 2007. One hundred three of 118 patients (87.3%) suffered from malignant disease. We had 90 patients on HPN and 4 patients on HEN using PEG. Home anti-cancer chemotherapy was performed for 29 patients. Most of the patients in our home care suffered from malignant disease, and they were maintained to protein-calorie malnutrition (PCM), so it is important to continue a home care nutrition therapy for survival and QOL advantages. Also, it is important for patient undergoing anti-cancer chemotherapy to maintain a good nutrition for avoiding a complication and to have a continuous chemotherapy. In our home nutrition therapy cases, HPN consisted of large number because of many digestive cancer patients existed, so HEN could not be used. In the case of HEN practicable that is first choice in home care nutrition, the other HPN was suitable for a pain control of terminal state patients. It is important for HPN to educate and to teach how to manage a patient cleanly and to teach a caregiver how to use a portable pump. We intended to simplify the content of infusion for patients, and to change the volume according to the patient status. HEN cases could be performed safely and for a long-term practice by a caregiver.

Characteristics of an aminopeptidase from Japanese cedar (Cryptomeria japonica) pollen.

A peptidase from Japanese cedar pollen, Jc-peptidase, was clarified to preferentially hydrolyze an MCA substrate of Phe-MCA (L-phenylalanyl-4-methylcoumaryl-7-amide). This study examined substrate specificities of Jc-peptidase using oligopeptides. Jc-peptidase hydrolyzed Phe-Phe and Tyr-Phe effectively and hydrolyzed Leu-Phe, Met-Phe, and Arg-Phe moderately. Other substrates such as Ala-Phe, Asp-Phe, and Pro-Phe were not hydrolyzed with the peptidase. Results obtained with a series of aminoacyl-Phe peptides were compatible with the facts obtained for MCA substrates except for Arg-MCA. Effects of amino acid residues in the P1' position were also examined using Phe-amino acids. An N-terminal phenylalanine residue was actually released from bioactive peptides such as molluscan cardioexcitatory neuropeptide (FMRF-NH(2)). Because the activity was inhibited with Zn(2+) and EDTA, Jc-peptidase was inferred to belong to the metalloproteases. The N-terminal amino acid sequence was determined to be APIGVQLEIEENYVHMYNGF and an internal sequence to be EIFAATFNVDEETEA, but no homology with other proteins was found.

Isolation and characterization of aminopeptidase (Jc-peptidase) from Japanese cedar pollen (Cryptomeria japonica).

An aminopeptidase, Jc-peptidase, was purified from Japanese cedar pollen by seven steps, including precipitation with ammonium sulfate, ion-exchange chromatography, gel filtration, hydrophobic interaction chromatography on phenyl-agarose, and high-performance liquid chromatography. Purified Jc-peptidease has a molecular weight of 42 kDa and hydrolyzes the synthetic substrates of L-phenylalanyl-4-methylcoumaryl-7-amide (Phe-MCA) with Km = 5 x 10(-5) M, Tyr-MCA with Km = 7 x 10(-4) M, Leu-MCA with Km = 1 x 10(-3) M, and Met-MCA with Km = 1 x 10(-3) M. Other MCA analogues such as Arg-MCA or Glu-MCA failed to serve as its substrates. The activity was inhibited in the presence of phebestin, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-valyl]-L-phenylalanine, with Ki = 4.7 x 10(-5) M, or bestatin, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-leucine, with Ki = 1.1 x 10(-4) M. According to amino acid sequence analysis, the N-terminal amino group seems to be blocked. The physiological function of the aminopeptidase (Jc-peptidase) has not been clarified in vivo.