Kazuki Sakamoto

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.

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)

Efficacy of combination chemotherapy using a novel oral chemotherapeutic agent, TAS-102, together with bevacizumab, cetuximab, or panitumumab on human colorectal cancer xenografts

TAS-102 is a novel oral nucleoside antitumor agent that consists of trifluridine (FTD) and tipiracil hydrochloride (TPI) at a molecular ratio of 1:0.5, and was approved in Japan in March 2014 for the treatment of patients with unresectable advanced or recurrent colorectal cancer that is refractory to standard therapies. In the present study, we used colorectal cancer xenografts to assess whether the efficacy of TAS-102 could be improved by combining it with bevacizumab, cetuximab or panitumumab. TAS-102 was orally administered twice a day from day 1 to 14, and bevacizumab, cetuximab and panitumumab were administered intraperitoneally twice a week for 2 weeks. Growth inhibitory activity was evaluated based on the relative tumor volume (RTV) after 2 weeks of drug administration and time taken for the relative tumor volume to increase five-fold (RTV5). Tumor growth inhibition and RTV5 with TAS-102 and bevacizumab combination treatment were significantly better than those with TAS-102 or bevacizumab alone in the SW48 and HCT116 tumor models, and the concentration of phosphorylated FTD in tumors determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was higher in the TAS-102 and bevacizumab combination group than in the TAS-102 monotherapy group. The combination of TAS-102 and cetuximab or panitumumab was also significantly more effective than either monotherapy in the SW48 tumor model. There was no significant difference in the body weight between the mice treated with TAS-102 monotherapy and any of the combination therapies on day 29. Our preclinical findings indicate that the combination therapy of TAS-102, bevacizumab and cetuximab or panitumumab is a promising treatment option for colorectal cancer.

Crucial roles of thymidine kinase 1 and deoxyUTPase in incorporating the antineoplastic nucleosides trifluridine and 2'-deoxy-5-fluorouridine into DNA

Trifluridine (FTD) and 2′-deoxy-5-fluorouridine (FdUrd), a derivative of 5-fluorouracil (5-FU), are antitumor agents that inhibit thymidylate synthase activity and their nucleotides are incorporated into DNA. However, it is evident that several differences occur in the underlying antitumor mechanisms associated with these nucleoside analogues. Recently, TAS-102 (composed of FTD and tipiracil hydrochloride, TPI) was shown to prolong the survival of patients with colorectal cancer who received a median of 2 prior therapies, including 5-FU. TAS-102 was recently approved for clinical use in Japan. These data suggest that the antitumor activities of TAS-102 and 5-FU proceed via different mechanisms. Thus, we analyzed their properties in terms of thymidine salvage pathway utilization, involving membrane transporters, a nucleoside kinase, a nucleotide-dephosphorylating enzyme, and DNA polymerase α. FTD incorporated into DNA with higher efficiency than FdUrd did. Both FTD and FdUrd were transported into cells by ENT1 and ENT2 and were phosphorylated by thymidine kinase 1, which showed a higher catalytic activity for FTD than for FdUrd. deoxyUTPase (DUT) did not recognize dTTP and FTD-triphosphate (F3dTTP), whereas deoxyuridine-triphosphate (dUTP) and FdUrd-triphosphate (FdUTP) were efficiently degraded by DUT. DNA polymerase α incorporated both F3dTTP and FdUTP into DNA at sites aligned with adenine on the opposite strand. FTD-treated cells showed differing nuclear morphologies compared to FdUrd-treated cells. These findings indicate that FTD and FdUrd are incorporated into DNA with different efficiencies due to differences in the substrate specificities of TK1 and DUT, causing abundant FTD incorporation into DNA.

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.

Possible antitumor activity of 1‐(3‐C‐ethynyl‐β‐d‐ribo‐pentofuranosyl)cytosine (ECyd, TAS‐106) against an established gemcitabine (dFdCyd)‐resistant human pancreatic cancer cell line

We established a variant of MIAPaCa‐2 human pancreatic cancer cells that is resistant to 2′,2′‐difluorodeoxycytidine (gemcitabine, dFdCyd), MIAPaCa‐2/dFdCyd, and elucidated the biochemical characteristics and mechanism of dFdCyd‐resistance in these cells. We also evaluated 1‐(3‐C‐ethynyl‐β‐d‐ribo‐pentofuranosyl)cytosine (ECyd, TAS‐106, RNA polymerase inhibitor), a new anticancer ribonucleoside, for antitumor activity against the resistant cells in vitro and in vivo. MIAPaCa‐2/dFdCyd cells were 2541‐fold more resistant to dFdCyd than parental MIAPaCa‐2 cells, and the major mechanism of the dFdCyd‐resistance was found to be a decrease in the intracellular pool of dFdCyd and its active metabolites, which would result in a decrease in incorporation of dFdCyd triphosphate into DNA. This finding was confirmed by the discovery of decreased deoxycytidine kinase activity, increased cytidine deaminase and ribonucleotide reductase activity, and increased 5′‐nucleotidase mRNA expression in the MIAPaCa‐2/dFdCyd cells. The cytotoxicity of TAS‐106 as an antitumor nucleoside analog was similar in both parental and dFdCyd‐resistant cells, with IC50 values of 6.25 and 6.27 nM, respectively, and this finding was supported by similar intracellular uptake and metabolism of TAS‐106 in both cell lines. We also evaluated the in vivo antitumor activity of TAS‐106 against MIAPaCa‐2 and dFdCyd‐resistant MIAPaCa‐2/dFdCyd tumors implanted into nude mice. The tumor growth inhibition rate of weekly additions of TAS‐106 (7 mg/kg, iv) against parental and dFdCyd‐resistant tumors was 73% and 76%, respectively, while that of dFdCyd administered twice a week (240 mg/kg, iv) was 84% and 34%, respectively. These results suggest that TAS‐106 would contribute to the treatment of patients with advanced pancreatic carcinomas in whom dFdCyd‐based chemotherapy has failed. (Cancer Sci 2005; 96: 295 –302)

Irinotecan overcomes the resistance to 5-fluorouracil in human colon cancer xenografts by down-regulation of intratumoral thymidylate synthase

To clarify the molecular interaction of irinotecan (CPT-11) and oxaliplatin (l-OHP) in combination with 5-fluorouracil (5-FU), the antitumor effects of CPT-11 and l-OHP combined with the oral 5-FU prodrug, S-1 composed by tegafur, gimeracil and potassium oteracil, were investigated on human colon cancer KM12C xenografts sensitive or resistant to 5-FU in nude mice. In parental KM12C tumor xenografts, combined treatment of CPT-11 with oral S-1 significantly augmented the antitumor activity compared with those of CPT-11 and S-1 alone. Interestingly, combined therapy of CPT-11 with S-1 was markedly effective with almost 90% of inhibition of tumor growth on 5-FU-resistant tumors (KM12C/ 5-FU), and its potency likely corresponded to that in parental tumors. In contrast, combined administration of l-OHP with S-1 did not shown an effect on KM12C/5-FU tumor xenografts. To investigate why only CPT-11 potentiated the anti-tumor activity in combination with 5-FU pro-drugs against 5-FU-resistant colon tumors, the activities or expression levels of thymidylate synthase (TS), ribonucleotide reductase (RNR) and other enzymes in 5-FU-metabolism in both tumors were measured following administration of CPT-11 and/or l-OHP. CPT-11, but not l-OHP, induced a decrease in activities and protein levels of TS and an increase in those of RNR in KM12C/5-FU tumors only, which was likely related to decreased expressions of several proteins in G1/S phase of the cells including CDK4, pRB, and E2F1 in these tumors. These findings suggest that CPT-11, but not l-OHP, would overcome the resistance to 5-FU in combination with 5-FU pro-drugs on 5-FU-resistant colon tumors.

Orotate phosphoribosyltransferase levels measured by a newly established enzyme‐linked immunosorbent assay in gastric carcinoma

A number of enzymes have been shown to be involved in the process of activation and/or degradation of 5‐fluorouracil (5‐FU), and are potential candidates for predicting chemosensitivity to 5‐FU. Among these, orotate phosphoribosyltransferase (OPRT EC 2.4.2.10) is a key enzyme related to the first‐step activation process of 5‐FU and has been shown to be an important enzyme that helps to predict sensitivity to 5‐FU and its related derivatives. We developed a new enzyme‐linked immunosorbent assay (ELISA) to accurately assess intratumoral activity of OPRT. A new sandwich ELISA was established using anti‐OPRT polyclonal antibodies obtained from the rabbit immunized with the recombinant human peptides of the OPRT molecule. OPRT levels were measured in eight human cancer xenografts and in 75 gastric cancer tissues using both a newly established ELISA and a conventional enzyme assay, using radiolabeled 5‐FU as a substrate. There was a significant correlation between OPRT levels measured by this ELISA and OPRT enzyme activity the in eight human cancer xenografts (r2 = 0.782) and gastric carcinoma tissue (r2 = 0.617). The ELISA system for OPRT requires a minimal amount of carcinoma tissue, making it an easy‐to‐use assay system to predict sensitivity to 5‐FU and its derivatives in gastric carcinoma. There was a significant correlation between tumor growth inhibition rates against the oral administration of oral‐uracil/tegafur (UFT) and OPRT enzyme activity in the human cancer xenografts (r2 = 0.574). These results suggest that this newly developed sandwich ELISA system for the quantification of OPRT levels is technically simple, feasible and a useful tool to predict sensitivity to fluoropyrimidine‐based anticancer chemotherapy in patients with gastric carcinoma and other cancers. (Cancer Sci 2006; 97)

Delayed esophageal perforation occurring with endoscopic submucosal dissection: A report of two cases

We report two cases of delayed esophageal perforation occurring with endoscopic submucosal dissection. Our cases involved delayed perforation after 10 d in case 1 and after 6 d in case 2. Both cases were related to solid food. We performed subtotal esophagectomy with gastric tube reconstruction of the esophagus via the subcutaneous route anterior to the thoracic wall without conservative treatment because both cases involved chest pain and major leakage of food into the mediastinum. Postoperative complications were a local factor (including suture failure and esophageal stricture) in case 1, and we performed endoscopic balloon dilatation five times for esophageal stricture. There was no intrathoracic and mediastinal infection in either case. Surgical treatment for delayed esophageal perforation can be performed safely and surely if diagnosis and assessment are not delayed.

Pancreatectomy and splenectomy for a splenic aneurysm associated with segmental arterial mediolysis

Segmental arterial mediolysis (SAM) is characterized by intra-abdominal, retroperitoneal bleeding or bowel ischemia, and the etiology is unknown. A 44-year-old man complaining of abdominal pain was admitted to our hospital. He had been admitted for a left renal infarction three days earlier and had a past medical history of cerebral aneurysm with spontaneous remission. The ruptured site of the splenic arterial aneurysm was clear via a celiac angiography, and we treated it using trans-arterial embolization. Unfortunately, the aneurysm reruptured after two weeks, and we successfully treated it with distal pancreatomy and splenectomy. We recommended a close follow-up and prompt radiological or surgical intervention because SAM can enlarge rapidly and rupture.