Tohru Obata

Evaluation of antitumour effects of docetaxel (Taxotere®) on human gastric cancers In vitro and In vivo

In vitro antitumour effects of docetaxel (Taxotere) were examined in nine cultured human gastric cancer cell lines and 18 clinical gastric cancer specimens. In vivo antitumour effects were examined in human gastric cancer xenografts in nude mice. The activity was compared with paclitaxel (Taxol). Docetaxel was more effective than paclitaxel in six of the nine cell lines and the effectiveness rates of docetaxel and paclitaxel were 56% (10/18) and 6% (1/17), respectively, in the clinical gastric cancer specimens. In vivo docetaxel showed superior antitumour effect on well differentiated (MKN-28), poorly differentiated (MKN-45) and undifferentiated (KKLS) gastric cancer xenografts. We conclude that docetaxel promises to be clinically active against gastric carcinomas.

A CRUCIAL ROLE OF URIDINE/CYTIDINE KINASE 2 IN ANTITUMOR ACTIVITY OF 3-ETHYNYL NUCLEOSIDES

The antitumor 3′-ethynyl nucleosides, 1-(3-C-ethynyl-β-d-ribopentofuranosyl)cytosine (ECyd) and 1-(3-C-ethynyl-β-d-ribopentofuranosyl)uridine (EUrd), are potent inhibitors of RNA polymerases and show excellent antitumor activity against various human solid tumors in xenograft models. ECyd is being investigated in phase I clinical trials as a novel anticancer drug possessing a unique antitumor action. ECyd and EUrd require the activity of uridine/cytidine kinase (UCK) to produce the corresponding active metabolite. The UCK family consists of two members, UCK1 and UCK2, and both UCKs are expressed in many tumor cells. It was unclear, however, whether UCK1 or UCK2 is responsible for the phosphorylation of the 3′-ethynyl nucleosides. We therefore established cell lines that are highly resistant to the 3′-ethynyl nucleosides from human fibrosarcoma HT-1080 and gastric carcinoma NUGC-3. All the resistant cell lines showed a high cross-resistance to ECyd and EUrd. As a result of cDNA sequence analysis, we found that UCK2 mRNA expressed in EUrd-resistant HT-1080 cells has a 98-base pair deletion of exon 5, whereas EUrd-resistant NUGC-3 cells were harboring the point mutation at nucleotide position 484 (C to T) within exon 4 of UCK2 mRNA. This mutation was confirmed by genome sequence analysis of the UCK2 gene. Moreover, the expression of UCK2 protein was decreased in these resistant cells. In contrast, no mutation in the mRNA or differences in protein expression levels of UCK1 were shown in the EUrd-resistant HT-1080 and NUGC-3 cells. These results suggest that UCK2 is responsible for the phosphorylation and activation of the antitumor 3′-ethynyl nucleosides.

Recent advances in the treatment of peritoneal dissemination of gastrointestinal cancers by nucleoside antimetabolites

Peritoneal dissemination is the most common cause of metastasis from malignancies in the abdominal cavity. There are no standard treatments for peritoneal dissemination and the results are poor. The reasons for this are as follows: (1) no effective chemotherapeutic agents have been identified or developed; (2) surgical cytoreduction has little effect on survival improvement; and (3) the molecular mechanisms of peritoneal dissemination have not been clarified and no therapy against the target molecules has been developed. However, studies on the molecular mechanisms of peritoneal dissemination have elucidated some of the target molecules and the development of new multimodal therapies has also improved survival. Early postoperative intraperitoneal chemotherapy, hyperthermic intraperitoneal perfusion chemotherapy and neoadjuvant intraperitoneal‐systemic chemotherapy have been newly developed, and a novel surgical therapy named peritonectomy has been proposed to perform complete cytoreduction of peritoneal dissemination. At present, these approaches appear to be effective therapeutic modalities for peritoneal dissemination. However, TS‐1 and capecitabine have shown worthwhile results in recent clinical trials for patients with advanced gastric cancer. We recently found that newly developed antitumor cytosine nucleoside analogs show a survival advantage in peritoneal dissemination models using human cancer cells. These non‐fluoropyrimidine nucleosides may potentially help to improve the poor prognosis observed in patients with advanced cancers involving peritoneal dissemination. (Cancer Sci 2007; 98: 11–18)

Cellular localization and functional characterization of the equilibrative nucleoside transporters of antitumor nucleosides

Nucleoside transporters play an important role in the disposition of nucleosides and their analogs. To elucidate the relationship between chemosensitivity to antitumor nucleosides and the functional expression of equilibrative nucleoside transporters (ENT), we established stable cell lines of human fibrosarcoma HT‐1080 and gastric carcinoma TMK‐1 that constitutively overexpressed green fluorescent protein‐tagged hENT1, hENT2, hENT3 and hENT4. Both hENT1 and hENT2 were predictably localized to the plasma membrane, whereas hENT3 and hENT4 were localized to the intracellular organelles. The chemosensitivity of TMK‐1 cells expressing hENT1 and hENT2 to cytarabine and 1‐(3‐C‐ethynyl‐β‐d‐ribopentofuranosyl) cytosine increased markedly in comparison to that of mock cells. However, no remarkable changes in sensitivity to antitumor nucleosides were observed in cell lines that expressed both hENT3 and hENT4. These data suggest that hENT3 and hENT4, which are mainly located in the intracellular organelles, are not prominent nucleoside transporters like hENT1 and hENT2, which are responsible for antitumor nucleoside uptake. (Cancer Sci 2007; 98: 1633–1637)

The yeast eIF4E-associated protein Eap1p attenuates GCN4 translation upon TOR-inactivation.

Amino acid‐starved yeast activates the eIF2α kinase Gcn2p to suppress general translation and to selectively derepress the transcription factor Gcn4p, which induces various biosynthetic genes to elicit general amino acid control (GAAC). Well‐fed yeast activates the target of rapamycin (TOR) to stimulate translation via the eIF4F complex. A crosstalk was demonstrated between the pathways for GAAC and TOR signaling: the TOR‐specific inhibitor rapamycin activates Gcn2p. Here we demonstrate that, upon TOR‐inactivation, the putative TOR‐regulated eIF4E‐associated protein Eap1p likely functions downstream of Gcn2p to attenuate GCN4 translation via a mechanism independent of eIF4E‐binding, thereby constituting another interface between the two pathways.

Influence of Cytidine Deaminase on Antitumor Activity of 2′-Deoxycytidine Analogs In Vitro and In Vivo

Antitumor 2′-deoxycytidine (dCyd) analogs such as gemcitabine (dFdC), cytarabine (Ara-C), and 2′-C-cyano-2′-deoxy-1-β-d-arabinofuranosylcytosine (CNDAC) are activated by dCyd kinase, whereas cytidine deaminase (CDA) inactivates them by conversion to their uracil forms. To elucidate the relationship between the chemosensitivity to antitumor dCyd nucleosides and CDA expression, we established a stable line of human gastric carcinoma TMK-1 cells constitutively overexpressing CDA (TMK-1/CDA) and examined its chemosensitivity to antitumor dCyd analogs in vitro and in vivo. We observed comparable reactivity for dFdC and Ara-C, and the substrate reactivity of CNDAC to recombinant human CDA was more than 10 times less efficient than those of Ara-C and dFdC. Next, we examined the in vitro chemosensitivity of TMK-1/CDA and observed a marked decrease in the sensitivity of TMK-1/CDA to Ara-C, dFdC, and CNDAC compared with mock-transfected cells. In addition, we transplanted TMK-1/CDA cells into a nude mouse xenograft model and examined their in vivo chemosensitivity to CNDAC. The in vivo antitumor effect of CNDAC on TMK-1/CDA cells was substantially reduced compared with that of mice transplanted with mock-transfected cells. These results indicate that CDA could play an important role in regulating susceptibility to antitumor dCyd analogs in vitro and in vivo. In addition, the expression level of CDA was found to affect the antitumor activity of CNDAC, even though the substrate reactivity of CNDAC to CDA is relatively low.

Deletion mutants of human deoxycytidine kinase mRNA in cells resistant to antitumor cytosine nucleosides

We studied mutational events in deoxycytidine (dCyd) kinase mRNA expression, focusing on aberrant dCyd kinase mRNA, which has been frequently observed in established cell lines resistant to antitumor dCyd nucleoside analogues such as 1‐β‐D‐arabinofuranosyl cytosine (Ara‐C), gemcita‐bine (dFdC) and 2′‐C‐cyano‐2′‐deoxy‐l‐β‐D‐arabinofuranosylcytosine (CNDAC). We describe here the expression of aberrant dCyd kinase mRNAs identified as splicing mutants. These mutants included deletions of the fifth exon in CNDAC‐resistant cells (originating from HT‐1080 cells), of the third exon in Ara‐C‐resistant cells (originating from SK‐MEL‐28 cells) and of the fourth exon in 2′‐deoxy‐2′‐methylidenecytidine (DMDC)‐resistant cells (originating from SK‐MEL‐28 cells). Various nucleoside‐resistant cells originating from the same parental HT‐1080 cells were established. The resulting cells expressed the same mRNA with deletion of the fifth exon, and the location of splicing was independent of the type of nucleosides used for the establishment of resistant cells. The deletion of the fifth exon in dCyd kinase seems to be a target for acquisition of resistance to antitumor cytosine nucleosides. However, distinct mutations in the dCyd kinase gene seem to be associated with acquisition of resistance to different antitumor cytosine nucleosides.

Synthesis, configurational stability and stereochemical biological evaluations of (S)- and (R)-5-hydroxythalidomides

The first asymmetric synthesis of (S)- and (R)-5-hydroxythalidomides, one of thalidomides major metabolites, was achieved using HMDS/ZnBr(2)-induced imidation as a key reaction. 5-Hydroxythalidomide was found to be configurationally more stable than thalidomide at physiological pH. Stereochemical biological effects of thalidomide and 5-hydroxythalidomide on anti-angiogenesis and antitumor activities were also investigated using racemic and pure enantiomers.

Indomethacin Analogues that Enhance Doxorubicin Cytotoxicity in Multidrug Resistant Cells without Cox Inhibitory Activity

Conformationally restricted indomethacin analogues were designed and prepared from the corresponding 2-substituted indoles, which were synthesized by a one-pot isomerization/enamide-ene metathesis as the key reaction. Conformational analysis by calculations, NMR studies, and X-ray crystallography suggested that these analogues were conformationally restricted in the s-cis or the s-trans form due to the 2-substituent as expected. Their biological activities on cyclooxygenase-1 (COX-1) inhibition, cyclooxygenase-2 (COX-2) inhibition, and modulation of MRP-1-mediated multidrug resistance (MDR) are described. Some of these indomethacin analogues enhanced doxorubicin cytotoxicity, although they do not have any COX inhibitory activity, which suggests that the MDR-modulating effect of an NSAID can be unassociated with its COX-inhibitory activity. This may be an entry into the combination chemotherapy of doxorubicin with a MDR modulator.

Photodynamic therapy of photofrin II and excimer dye laser on experimental tumors.

Photodynamic therapy (PDT) by combination of photofrin II and excimer dye laser was evaluated for its usefulness in experimental tumors. High antitumor effects of PDT on sarcoma 180 solid type were obtained when PDT was performed with laser irradiation at an energy of 50 or 100 J/cm2 (40 or 80 Hz, 4 mJ/pulse) 48 h after i.v. administration of photofrin II at a dose of 25 mg/kg. Under the same conditions, the antitumor effects of PDT on murine Lewis lung carcinoma, human fibrosarcoma HT-1080 and human bladder transitional cell carcinoma KK-47 were also observed. These results suggest that clinical application of PDT with photofrin II and excimer dye laser might be useful.