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Our previous study demonstrated that gemcitabine (GEM), S‑1, and a combination of GEM and S‑1 (GS) induced S‑phase arrest and increased the phosphorylation of checkpoint kinase 1 (Chk1), which is a critical mediator of cell survival under impaired DNA replication, in pancreatic cancer cell lines. The aim of the present study was to investigate the combined effect of the Chk1 inhibitor prexasertib and antitumor drugs (GEM and S‑1) on pancreatic cancer cell line SUIT‑2. Furthermore, we conducted mechanistic analysis of the combined effect. The MTT assay revealed that a combination of prexasertib and GS showed a strong effect. Mechanistic analysis of the combined effect showed effective induction of apoptosis. Furthermore, a combination of prexasertib and GS downregulated the expression of antiapoptotic protein Bcl‑2. Chk1 knockdown with small interfering RNA and GS treatment resulted in strong induction of apoptosis. Our results provide evidence to show that the combination of prexasertib and GS has a strong antitumor effect and effectively induces apoptosis in pancreatic cancer cells through downregulation of the antiapoptotic protein Bcl‑2. Prexasertib could possibly enhance the effects of standard drugs, including GEM, S‑1, and GS, against pancreatic cancer.

Chemoradiotherapy is a useful treatment strategy in patients with locally advanced cancers. In particular, combination of 5-fluorouracil (5-FU) with X-ray irradiation is effective for the treatment of some types of gastrointestinal cancers. We investigated the antitumor effects of combination treatment with X-ray and S-1, a unique formulation of 5-FU, on human cancer xenografts in nude mice and compared the efficacy of this treatment to that of radiotherapy combined with cisplatin, UFT, another oral 5-FU prodrug, and intravenous 5-FU. Tumors implanted into the left hind legs of mice were treated with a dose of 2 or 5 Gy X-ray irradiation on days 1 and 8, and S-1, UFT and 5-FU were administered for 14 days. The efficacy of combined treatment with 8.3 mg/kg S-1 and 2 Gy X-ray irradiation in treating non-small cell lung cancer xenografts (Lu-99 and LC-11) was significantly higher than that of treatment with S-1 alone or 2 Gy X-ray irradiation alone, and the antitumor activity of combined treatment was similar to that of 5 Gy X-ray irradiation alone. Although 8.3 mg/kg S-1 and 17.5 mg/kg UFT had equivalent antitumor activity; the antitumor efficacy of combination treatment with S-1 and 2 Gy X-ray irradiation on LC-11 tumors was significantly higher than that of combination treatment with UFT and 2 Gy X-ray irradiation. Combination treatment with S-1 and X-ray irradiation was also more effective against pancreatic tumors than combination treatment with intravenous 5-FU and X-ray irradiation. To elucidate the reason for the increased antitumor efficacy of combination treatment with S-1 and X-ray irradiation, the antitumor effect of gimeracil, one of the components of S-1, was tested in combination with 2 Gy X-ray irradiation. These experiments demonstrated that gimeracil enhanced the efficacy of X-ray irradiation against lung as well as head and neck cancer xenografts in a dose-dependent manner. Furthermore, we observed decreased expression of γ-H2AX protein, a marker of DNA repair, in LC-11 tumors treated with X-ray irradiation and gimeracil compared to that observed in tumors treated with X-ray irradiation alone, suggesting that gimeracil may inhibit rapid repair of X-ray-induced DNA damage in tumors. The present study suggests that chemoradiotherapy using S-1 acts through a novel mechanism and may prove useful in treating patients with locally advanced cancers whose disease progression is difficult to control using chemotherapy alone.

Neoadjuvant chemotherapy (NAC) by 5-fluorouracil including S-1 is administered to advanced gastric cancer patients. However, the therapeutic benefit from this pre-operative treatment remains uncertain. The present study analyzed the expression of 5-fluorouracil related enzymes, TS, DPD and OPRT in 47 gastric cancer biopsy specimens using quantitative double-fluorescence immunohistochemistry (qDFIHC), which is a newly developed system to quantify protein expression. The study first determined whether the cancer heterogeneity within the sample influences evaluation by qDFIHC system. Thereafter, the expression values of the TS, DPD, OPRT and OPRT/TS, OPRT/DPD, OPRT/(TS+DPD) ratios were retrospectively correlated with the clinical or pathological response in the patients. The expression values of TS, DPD and OPRT at a single field were significantly correlated with mean of the values evaluated at three fields. Among the 6 candidate factors analyzed, OPRT, OPRT/TS, OPRT/DPD and OPRT/(TS+DPD) showed significant correlations with the clinical response in 47 patients. Cut-off values to differentiate the clinical response were determined in the four factors. OPRT/TS showed the strongest correlation with the clinical response. qDFIHC was able to quantify the TS, DPD and OPRT expressions in cancer biopsy specimens without being affected by the heterogeneity. Effective therapy using tailored S-1 NAC according to the OPRT/TS ratio is therefore expected in advanced gastric cancer patients.

S-1, an oral fluorouracil antitumor drug, is composed of three agents: tegafur (FT), 5-chloro-2,4-dihydroxypyridine (CDHP), and potassium oxonate (Oxo). Approximately 50% of oral squamous cell carcinomas (OSCC) exhibit cervical lymph node metastasis. The extent of lymph node involvement is a major determinant in both staging and prognosis of the majority of OSCC. The purpose of this study was to examine the effect of S-1 on the metastatic potential of OSCC cells. We used orthotopic green fluorescence protein (GFP) SAS-L1, in BALB/c nu/nu mice. Mice received oral doses of either 5% hydroxypropylmethylcellulose (HPMC) for control or S-1 (20 mg/kg) and were autopsied at 2 weeks. We also performed in vitro experiments using concomitant 5-fluorouracil (5-FU) and CDHP as a drug model of S-1 to determine the effect of S-1 on OSCC invasion and metastasis. Although 100% (11 of 11) of mice not treated with S-1 showed cervical lymph node metastasis, only 54.4% (6 of 11) of S-1 treated mice demonstrated metastasis. In in vitro experiments, OSCC cells treated with 5-FU and CDHP showed a marked reduction in invasiveness and in adhesion to laminin coated plates. Western blot analysis revealed that treatment with 5-FU and CDHP suppressed expression of integrins alphav, alpha3, alpha6, beta1, beta3, beta4, beta5, and beta6. These results suggest that S-1 inhibits tumor proliferation and lymph node metastasis in OSCC cells. Moreover, expression of integrin subunits and the integrin signal transduction pathway may be closely related to metastasis suppression.

It has been reported that S-1 can exert antitumor effects on various human cancers including oral squamous cell carcinoma (OSCC). However, little is known about the detailed mechanisms of the antitumor activity of S-1. In the present study, we determined whether S-1 could suppress the angiogenesis and growth of human OSCC cells in vitro and in vivo. The S-1 component (5-FU plus CDHP) significantly suppressed the growth and migration of OSCC cells and BAEC, which inhibited tubule formation in HUVECs in vitro. Also, S-1 inhibited the nuclear factor-kappaB (NF-kappaB) activity in human OSCC cells in vitro. Moreover, S-1 inhibited the expression of survival signal, phosphorylated Akt (p-Akt), and of two major proangiogenic molecules, vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2), in cells implanted into the subcutaneous tissue of nude mice. The decreased expression of p-Akt, VEGF and FGF-2 correlated with decreased tumorigenicity and decreased vascularization of lesions in vivo. These findings suggest that S-1 can suppress the angiogenesis and growth of OSCC cells by inhibiting the expression of p-Akt, VEGF and FGF-2 involved in the blockade of Akt/NF-kappaB pathway.

The present study assesses the effects of neo-adjuvant chemotherapy (NAC) with uracil and tegafur (UFT) alone vs UFT plus cyclophosphamide (CPA), on the activity of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) in breast cancer tissues. Breast cancer patients were randomly assigned to 3 groups; the control (no-treatment) group (n=13), the UFT (5-8 mg/kg/day) alone group (n=10) and the UFT plus CPA (1 mg/kg/one day interval) (UC) group (n=9), and they received NAC for 2-4 weeks. A total of 32 invasive ductal breast carcinomas were used to assay for TS and DPD activity. There were no statistically significant differences in tumor size or stage classification between the 3 groups. The DPD activity was inversely and significantly correlated with the tumor size and pT, but the TS activity was not correlated with these clinicopathological factors. The TS activity was decreased by NAC with UFT, and the addition of CPA resulted in an increased inhibition of TS activity. In contrast, DPD activity was increased by NAC with UFT administration, but its increased activity was significantly inhibited by the addition of CPA. Multiple regression analyses demonstrated that the total dose of UFT was a significant variable for inhibiting TS activity, and that CPA was a significant variable for inhibiting DPD activity. The DPD activity increased by UFT can be inhibited by CPA, and this may represent one of the possible mechanisms responsible for the anti-tumor activity of 5-FU or its derivatives as enhanced by CPA.

To evaluate the antitumor and antimetastatic efficacy of oral fluoropyrimidines, alone and combined with taxane on human breast cancer xenografts model, we developed a breast cancer model that spontaneously metastasizes to the lung by orthotopic implantation of MDA-MB-435S-HM tumors into the mammary fat pad (mfp) of SCID mice. The activity of the 5-fluorouracil (5-FU)-degrading enzyme dihydropyrimidine dehydrogenase (DPD) was significantly higher in the metastatic tumors than in the primary tumors. Based on this enzymatic characteristic of pulmonary metastases of breast cancer in regard to 5-FU metabolism, we investigated the antitumor activity of two types of oral 5-FU prodrugs, with and without paclitaxel, on both orthotopically implanted breast tumors and metastatic lung tumors in mice. The drugs and doses used were: S-1, a new oral DPD-inhibiting fluoropyrimidine (DIF) 8.3 mg/kg/day, capecitabine 360 mg/kg/day as a non-DIF, and paclitaxel 50 mg/kg, all of which display minimal toxicity in mice. In the primary tumors, paclitaxel and S-1 displayed a significant antitumor activity, with 57 and 41%, respectively inhibition of tumor growth (p < 0.01), but capecitabine had no effect. When S-1 and paclitaxel were combined, they synergistically caused tumor regression (tumor growth inhibition ratio 94%, p < 0.01) in mice compared to capecitabine plus paclitaxel, without any toxicity. In the pulmonary metastasis model, paclitaxel, and both S-1 alone and combined with paclitaxel, but not capecitabine alone or combined with paclitaxel, diaplayed almost complete antimetastatic activity. These results strongly suggest that combination of S-1, as a DIF with taxanes will show a potent high antitumor and antimetastatic effect on refractory human breast cancers, especially those expressing strong DPD activity.

S-1 is a new oral antineoplastic agent which can inhibit cell growth and induces apoptosis in certain types of cancer cells including gastric carcinomas, colorectal cancers and salivary gland cancers, but its effect on response of tumor cells to radiation has not been clarified yet. We have reported that S-1 can sensitize human oral cancer cells to radiation, and that S-1 in combination with radiation can exert remarkable effects on decreasing clonogenic survival and in vivo tumor growth. Here, we demonstrate the mechanism of apoptosis enhancing activity by the combination treatment of S-1 and radiation in salivary gland cancer cells. S-1 in combination with radiation have a remarkable effect on decreasing in vitro cell growth. In addition, the combined treatment of S-1 and radiation resulted in an increased DNA fragmentation by detecting Hoechst 33258 staining. Moreover, apoptosis of the cells by combined treatment of S-1 and radiation was associated with reactive oxygen/nitrogen species generation and the activation of caspase-8, -9 and -3. These results indicate that S-1 in combination with radiation can markedly enhance apoptosis of salivary gland cancer cells, and the combined therapy of S-1 and radiation are currently leading to the design of clinical studies.