Downregulation Of Thymidylate Synthase Research Articles

Phase I study of the HDAC inhibitor vorinostat in combination with capecitabine in a biweekly schedule in advanced breast cancer.

2587 Background: Synergy between histone deacetylase inhibitors and 5-fluorouracil is thought to be due to down regulation of thymidylate synthase. Study objectives are to assess maximum tolerated dose (MTD), dose limiting toxicities (DLT) and objective response rate of vorinostat (V) in combination with capecitabine (C). Secondary aims are pharmacodynamics of V. Methods: Cohorts of 3-6 patients (pts) were enrolled on a 3+3 dose-escalation phase to assess MTD of escalating doses of oral C (1500/1800/2000mg) with V 200 mg BID on days 1-7 and 15-21 of a 28 d cycle. 10 pts enrolled on dose expansion phase and treated at MTD. Pts received a “run in” treatment of V for 5 d and pre/post V biopsy (bx) collected when feasible. DLT was defined as ≥ grade 3 non hematological toxicity, grade 4 thrombocytopenia, grade 4 neutropenia > 5 d, grade 4 febrile neutropenia requiring hospitalization, QTc >500ms,or treatment delay > 2 weeks from toxicity. Microarray analysis was performed using Illumina HT-12 gene arrays on pre/post bx from 4 pts. Results: 23 ptswith median age 51 (range 33-69) were treated: 8 pts at 1500/200mg, 5 pts at 1800/200mg, and 10 pts treated on dose expansion phase at 1500/200mg. Median # of prior lines of chemotherapy was 2 (range 0-7). 3 pts are still on study. Median # of cycles on study were 2 (range 1-42). Cycle 1 DLT was grade 3 fatigue in 2 pts treated on 1800/200mg, and in 1 pt treated on 1500/200mg. Other grade 3/4 toxicities are summarized in the Table. 14 pts are evaluable for response (12 pts at 1500/200mg and 2 pts at 1800/200mg). No objective responses were seen, 3 pts had stable disease > 6 months (1 pt completed 42 cycles). Changes in pathways involved in extracellular matrix and TGFb pathway were noted. Conclusions: DLT of C concomitant with V was fatigue. MTD was 1500 mg C combined with 200 mg V BID. Combination has modest clinical activity. Consistent modulation of pathways involved in extracellular matrix and TGFb pathway, suggesting biomarkers of response to V. Clinical trial information: NCT00719875. [Table: see text]

Phase I dose escalation of the oral histone deacetylase inhibitor (HDACi) resminostat in combination with FOLFIRI in colorectal cancer (CRC) patients: The SHORE trial.

3625 Background: Resminostat is a novel oral HDAC inhibitor with broad activity in various cancer models. In CRC models, resminostat revealed synergistic effects with 5-FU and irinotecan/SN-38, indicating its (re-)sensitization potential when applied in combination therapy. Furthermore, resminostat downregulates thymidylate synthase, involved in drug resistance to 5-FU and effectively inhibits HDAC2, one of the target enzymes believed to critically support development of CRC. The phase I/II SHORE trial investigates resminostat in combination with FOLFIRI in patients previously treated with 5-FU. Methods: Patients (pts) with advanced CRC having previously received 5-FU alone or in combination with other agents who were scheduled for FOLFIRI in second or later treatment lines were included. The phase I comprised an open-label, inter-patient, ‘3+3’ dose escalation design with increasing doses of resminostat combined with standard FOLFIRI. Pts received resminostat on 5 consecutive days, followed by a 9-day drug free period (‘5+9’ scheme, i.e. 14-day cycles). On days 3 and 4 of each cycle (C), FOLFIRI was administered. Primary objective of the Phase I part was to determine safety and tolerability, the maximum tolerated dose and pharmacokinetics of the combination. Results: 17 pts (median age 61 yrs; 12 males; 11 ECOG 0; 6 ECOG 1; median therapy line 2 [2-6]) were enrolled in 4 dose levels of resminostat 200, 400, 600 mg QD (3 pts each) and 400 mg BID (6 pts) plus FOLFIRI. Two pts discontinued in C1 and were replaced. No DLT occurred. AEs consisted mainly of GI symptoms of mild and moderate intensity (nausea, vomiting and diarrhea) leading to decreased electrolyte plasma levels in some pts. In the highest dose level tested (400 mg BID) hematological toxicity, mainly neutropenia up to grade 4, was observed leading to dose reductions in 3 pts in C3 and C7. No objective responses were reported, however some pts showed SD for prolonged time (up to 32 w). Results of the completed phase I part will be reported. Conclusions: The combination of resminostat with standard FOLFIRI was safe and well tolerated warranting continuation into the Phase II part of the study. Clinical trial information: NCT01277406.

Sustained inhibition of deacetylases is required for the antitumor activity of the histone deactylase inhibitors panobinostat and vorinostat in models of colorectal cancer

Despite compelling preclinical data in colorectal cancer (CRC), the efficacy of HDACIs has been disappointing in the clinic. The goal of this study was to evaluate the effectiveness of vorinostat and panobinostat in a dose- and exposure-dependent manner in order to better understand the dynamics of drug action and antitumor efficacy. In a standard 72 h drug exposure MTS assay, notable concentration-dependent antiproliferative effects were observed in the IC50 range of 1.2-2.8 μmol/L for vorinostat and 5.1-17.5 nmol/L for panobinostat. However, shorter clinically relevant exposures of 3 or 6 h failed to elicit any significant growth inhibition and in most cases a >24 h exposure to vorinostat or panobinostat was required to induce a sigmoidal dose-response. Similar results were observed in colony formation assays where ≥ 24 h of exposure was required to effectively reduce colony formation. Induction of acetyl-H3, acetyl-H4 and p21 by vorinostat were transient and rapidly reversed within 12 h of drug removal. In contrast, panobinostat-induced acetyl-H3, acetyl-H4, and p21 persisted for 48 h after an initial 3 h exposure. Treatment of HCT116 xenografts with panobinostat induced significant increases in acetyl-H3 and downregulation of thymidylate synthase after treatment. Although HDACIs exert both potent growth inhibition and cytotoxic effects when CRC cells were exposed to drug for ≥ 24 h, these cells demonstrate an inherent ability to survive HDACI concentrations and exposure times that exceed those clinically achievable. Continued efforts to develop novel HDACIs with improved pharmacokinetics/phamacodynamics, enhanced intratumoral delivery and class/isoform-specificity are needed to improve the therapeutic potential of HDACIs and HDACI-based combination regimens in solid tumors.

Abstract A24: Enhancing chemotherapy efficacy and antitumor immune activity by combined antisense downregulation of TS and IDO.

Abstract Chemotherapy plays a major role in treatment of many common cancers. However, it is limited by intrinsic and/or acquired tumor cell resistance to anticancer drugs, often mediated by increased expression of genes mediating that resistance. An approach to increase chemotherapy effectiveness is to reduce expression of such genes using small molecules or RNAi to silence specific targets that cancer cells rely on for escape from death due to exogenous treatment or immune cell attack. We have previously shown that siRNA downregulation of thymidylate synthase (TS), the enzyme responsible for the de novo synthesis of thymidylate and a common target for some anti-cancer drugs (5FUdR, pemetrexed, and others) enhances the activity of these drugs in vitro. Using lower concentrations of anticancer drugs has the potential to decrease deleterious side effects, including therapy-induced toxicity to T cells capable of recognizing and killing tumor cells. In addition, certain tumor proteins including indoleamine 2,3-dioxygenase [IDO] can regulate immunosurveillance by inhibiting antitumor T and NK cell activity. IDO can also mediate tumor cell resistance to some anticancer drugs, such as imatinib. To assess the capacity of combined siRNA knockdown of TS and IDO in human tumor cells to increase sensitivity to anticancer drugs, we report that: a) TS siRNA downregulates TS by 90% in HCT-15 colon carcinoma cells, over 90% percent in A549 lung carcinoma cells, and by 80% in H441 lung carcinoma cells, b) shRNA expression vectors stably expressing IDO siRNA in multiple clonally-derived populations of A549 reduces IFNγ-induced IDO protein expression by over 95%. Transiently-transfected TS siRNA is being used, alone and in combination with stable shRNA-mediated reduction in IDO, to assess the utility of targeting both mRNAs to enhance sensitivity to TS-targeting drugs (5-FU, 5-FUdR, pemetrexed), DNA-damaging anticancer drugs (cisplatin, melphalan) and both, both in vitro and against human A549-derived tumour xenografts in immunocompromised mice without T cells but competent with respect to NK cell activity. Combined antisense targeting of both TS and IDO in human tumor cells has the potential to increase the effectiveness of anti-TS and other drugs, both by increasing tumor cell drug sensitivity and by combined enhancement of sensitivity to drug treatment and reduced inhibition of NK cell activity. Citation Format: Saman Maleki Vareki, James Koropatnick. Enhancing chemotherapy efficacy and antitumor immune activity by combined antisense downregulation of TS and IDO. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A24.

385 Dovitinib, a Multitargeted Kinase Inhibitor, Actively Inhibits Human Gastric Cancer Cells Via On-target Effects, and Acts Synergistically with 5-fluorouracil Via Off-target Effects with Down-regulation of Thymidylate Synthase

385 Dovitinib, a Multitargeted Kinase Inhibitor, Actively Inhibits Human Gastric Cancer Cells Via On-target Effects, and Acts Synergistically with 5-fluorouracil Via Off-target Effects with Down-regulation of Thymidylate Synthase

Changes in histone deacetylase (HDAC) expression patterns and activity of HDAC inhibitors in urothelial cancers

ObjectiveTo determine histone deacetylase (HDAC) isoenzyme expression patterns in urothelial cancer tissues and cell lines and investigate their potential to predict the efficacy of the HDAC inhibitor vorinostat. Materials and methodsExpression of HDAC mRNAs was determined by quantitative RT-PCR in 18 urothelial cancer cell lines (UCC), normal uroepithelial controls (NUC), 24 urothelial cancer tissues, and 12 benign controls. Results were compared with published microarray data. Effects of pan-HDAC inhibitor vorinostat and on UCCs were determined by viability and apoptosis assays, cell cycle analysis, and measurements of p21CIP1, thymidylate synthase (TS), and EZH2. In addition, protein expression levels of HDACs were investigated in UCCs. ResultsProminent changes in UCCs were HDAC2 and/or HDAC8 up-regulation in 11 of 18 cell lines and decreased expression of HDAC4, HDAC5, and/or HDAC7 mRNA in 15 of 18 cell lines. In cancer tissues, HDAC8 was likewise significantly up-regulated (P = 0.002), whereas HDAC2 up-regulation was detected only in a subset of tumors (9/24, P = 0.085). Overexpression of HDAC2 and HDAC8 mRNA did not correspond with the protein level. Vorinostat induced G2/M arrest, an increase in the sub-G1 fraction, up-regulation of p21, and down-regulation of TS in all UCC. Effects on EZH2 and PARP cleavage as well as activation of caspase 3/7 differed between cell lines. Associations between the overall sensitivity to the pan-HDACi vorinostat and overexpression of HDAC2 and HDAC8 mRNA were not observed. ConclusionsIn urothelial cancer, up-regulation of HDAC2 and HDAC8 and down-regulation of HDAC4, HDAC5, and HDAC7 mRNA are common findings. The treatment effect of the pan-HDAC inhibitor vorinostat was variable in UCCs and up-regulation of HDAC2 and HDAC8 was not predictive for treatment response. Whether selective targeting of HDAC2, HDAC8, or other HDACs deregulated in urothelial cancer (e.g., HDAC4, HDAC5, and HDAC7) result in a more consistent treatment response needs further investigation.

Antitumor efficacy of sequential treatment with docetaxel and 5-fluorouracil against human oral cancer cells

Docetaxel (DOC) and 5-fluorouracil (5-FU) are important anticancer agents widely used in the treatment of a variety of cancers including oral squamous cell carcinoma (OSCC). The purpose of this study was to determine the antitumor efficacy of the sequential administration of DOC and 5-FU against OSCC cells (B88 and CAL27 cells) in vitro and in vivo. In in vitro growth inhibition assays, sequential treatment with DOC followed by 5-FU was more effective in inhibiting cancer cell growth than 5-FU followed by DOC, single treatment with DOC or 5-FU, or combined treatment with DOC and 5-FU. Furthermore, DOC followed by 5-FU significantly inhibited tumor growth in vivo compared to 5-FU followed by DOC. To understand the mechanisms underlying the enhanced growth inhibitory effect of the administration sequence, DOC followed by 5-FU, we examined the expression of 5-FU metabolic enzymes such as thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and orotate phosphoribosyl transferase (OPRT), which were known to regulate the antitumor effect of 5-FU, by real-time RT-PCR and western blot analysis. Downregulation of TS and DPD expression and upregulation of OPRT expression were induced by DOC treatment, suggesting that DOC enhanced the efficacy of 5-FU by altering the expression of its metabolic enzymes. These results indicate that sequential treatment with DOC followed by 5-FU could be a promising therapeutic strategy for oral cancer.

Sequential irinotecan hydrochloride/S-1 for S-1-resistant inoperable gastric cancer: A feasibility study

Irinotecan hydrochloride (CPT-11) is reported to be involved in the downregulation of thymidylate synthase (TS), a target molecule of 5-fluorouracil (5-FU) and oral fluoropyrimidine S-1. Therefore, we hypothesized that a preceding administration of CPT-11 against S-1-resistant tumors may recover sensitivity to S-1. To this end, we planned a S-1/CPT-11 sequential therapy as a feasibility study in S-1-refractory gastric cancer patients. In the first course, CPT-11 was administered intravenously at 150 mg/m(2) on days 1 and 15. Subsequently, S-1 was administered orally for 4 weeks from day 29 to 57, followed by a 2-week interval (sequential S-1/CPT-11). When the tumor showed a complete response (CR) or partial response (PR), the same dose of S-1 monotherapy was continued unless progressive disease (PD) was observed. When the response was stable disease (SD), S-1 was administered at the same dose for just 2 weeks (days 1-15), no drug was administered for the following 2 weeks (4-week cycle) and CPT-11 was administered intravenously at 100 mg/m(2) on days 1 and 15 (concurrent S-1/CPT-11) unless PD was observed. In the case of PD, the study was terminated. The primary endpoint was an antitumor effect and secondary endpoints were median survival time (MST), progression-free survival (PFS), time-to-treatment failure (TTF) and safety. The response rate (RR) following the first course was only 5.9% and the most positive RR was 11.8%. The MST, median TTF and PFS were 381, 69 and 71 days, respectively. Leukocytopenia was observed in more than half of the patients. Since the RR was lower than estimated in an interim analysis, the trial was terminated and the protocol was concluded to be unfeasible.

A phase I study of biweekly capecitabine and the histone deacetylase inhibitors (HDACi) vorinostat in for advanced breast cancer (BC).

e13609 Background: The HDACi vorinostat (V) inhibits growth of BC cell lines and downregulates thymidylate synthase (TS), an enzyme thought to be involved in resistance to 5-fluoruracil. Methods: We designed a phase I/II clinical trial of V + capecitabine (C) for advanced BC. The primary aims are to determine the maximum tolerated dose (MTD), dose-limiting toxicities (DLT) and response by RECIST criteria. Secondary aims are pharmacodynamic (PD) measures of gene signatures and candidate transcription factors in pre/post V biopsies using RNA-seq. Cohorts (Co) of 3-6 patients (pts) were enrolled and treated with escalating doses of C (1,500/1,800/2,000 mg) + V 200 mg BID on days 1-7 and 15-21 of a 28 day cycle. Pts received V on days -10, -9, -8, -7, -6 and C on day -3 to facilitate PD studies. A DLT is defined as ≥ grade 3 non-hematologic toxicity, grade 4 thrombocytopenia, grade 4 neutropenia > 5 days, grade 4 febrile neutropenia requiring hospitalization, or treatment delay of > 2 weeks due to unresolved toxicity. Results: 13 pts enrolled on phase I study: (Co1 1,500/200 mg, n= 8), (Co2 1,800/200 mg, n=5). Median age was 59 yrs (range 46-68); median number of prior therapy was 5 (range 2-9); 10 pts had ER/PR+ BC; 1 pt had HER-2+ BC; 3 pts had prior C therapy. Nine of 13 pts were evaluable for toxicity; 3 DLTS (grade 3 fatigue) occurred in Co 1 (2) and Co 2 (1). Other grade 3/4 toxicities included: 1 pt had grade 3 neutropenia, anemia, fatigue, hyponatremia, hypoalbuminemia, ataxia, infection, muscle weakness, headache and grade 4 thrombocytopenia; 1 pt had grade 3 hand/foot syndrome; 1 pt had grade 3 syncope and grade 4 hypokalemia; 1 pt had grade 3 thrombocytopenia; 1 pt had grade 3 anemia;1 pt had grade 3 neutropenia and infection; 1 pt had grade 3 neutropenia and lymphopenia; and 1 pt had grade 3 headache. Seven pts completed > 2 cycles and were evaluable for response; 3 pt had stable disease, notably 1 pt is on treatment for 27 cycles and 1 pt completed 6 cycles. Extraction/amplification/ sequencing from core biopsies shows acceptable read-mapping (45-50 % of RefSeq genes) on Illumina GA II platform. Conclusions: The MTD and recommended dose for phase II study is C1,500 mg BID and V 200 mg BID in a biweekly schedule. PD results will be presented.

Low-dose docetaxel enhances the sensitivity of S-1 in a xenograft model of human castration resistant prostate cancer

S-1 is a recently developed dihydropyrimidine dehydrogenase inhibitor fluoropyrimidine and has demonstrated high maximum plasma 5-Fluorouracil (5-FU) levels with mild toxicity, and an oral formulation has resulted in an improvement in patient quality of life. The aims of the present study were to determine the efficacy of S-1 or S-1 combined with docetaxel (DOC) using castration resistant prostate cancer (CRPC) cells and to explore their clinical potential for treating CRPC patients. LNCaP cells, androgen dependent prostate cancer (ADPC) cells and C4-2 cells, which are a CRPC subline of LNCaP cells, were used. Specimens obtained from ADPC and CRPC patients were also evaluated. The CRPC specimens and C4-2 cells exhibited significantly lower thymidylate synthase (TS) expression, a target of 5-FU, than the ADPC specimens and LNCaP cells. In vitro, C4-2 cells exhibited higher sensitivity to 5-FU than LNCaP cells. In C4-2 xenograft model, S-1 monotherapy suppressed tumor growth and low-dose DOC enhanced the anti-tumor effect of S-1. In vitro, low-dose DOC, which did not induce G2/M arrest, increased p53 and p21 and resulted in down-regulation of TS in C4-2 cells, and down-regulation of TS is considered to be responsible for the synergistic effect of S-1 in vivo. The present findings indicate that CRPC patients with androgen ablation may be good candidates for 5-FU based chemotherapy, and these regimens have attractive therapeutic potential for clinical practice, and they may have a significant impact on therapeutic options.

1284 LOW DOSE DOCETAXEL ENHANCES THE SENSITIVITY OF S-1 IN A XENOGRAFT MODEL OF HUMAN CASTRATION RESISTANT PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 20111284 LOW DOSE DOCETAXEL ENHANCES THE SENSITIVITY OF S-1 IN A XENOGRAFT MODEL OF HUMAN CASTRATION RESISTANT PROSTATE CANCER Masanori Hasegawa, Akira Miyajima, Takeo Kosaka, Yota Yasumizu, Nobuyuki Tanaka, Takahiro Maeda, Suguru Shirotake, Hiroki Ide, Eiji Kikuchi, and Mototsugu Oya Masanori HasegawaMasanori Hasegawa Tokyo, Japan More articles by this author , Akira MiyajimaAkira Miyajima Tokyo, Japan More articles by this author , Takeo KosakaTakeo Kosaka Tokyo, Japan More articles by this author , Yota YasumizuYota Yasumizu Tokyo, Japan More articles by this author , Nobuyuki TanakaNobuyuki Tanaka Tokyo, Japan More articles by this author , Takahiro MaedaTakahiro Maeda Tokyo, Japan More articles by this author , Suguru ShirotakeSuguru Shirotake Tokyo, Japan More articles by this author , Hiroki IdeHiroki Ide Tokyo, Japan More articles by this author , Eiji KikuchiEiji Kikuchi Tokyo, Japan More articles by this author , and Mototsugu OyaMototsugu Oya Tokyo, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.970AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES No standard therapy is currently available for castration resistant prostate cancer (CRPC) patients who have disease progression during docetaxel (DOC) treatment. This study focused on S-1, oral 5-fluorouracil (5-FU)-based antitumor drug which demonstrated high maximum plasma 5-FU levels with mild gastrointestinal toxicity. In addition, an oral formulation has resulted in an improvement in patient quality of life. S-1 is now used as a front-line chemotherapeutic agent for gastric cancer in Japan. The aims of the present study were to determine the efficacy of S-1 or S-1 combined with low dose DOC against CRPC cell line and to explore their clinical potential for treating CRPC patients. METHODS The androgen dependent prostate cancer (ADPC) cell line LNCaP and its hormone refractory sub-line C4-2, and a subcutaneous tumor xenograft model prepared with C4-2 in SCID mice were used. Specimens obtained from ADPC and CRPC patients were also evaluated. To investigate mechanisms potentially underlying the synergistic inhibition of cancer growth, we examined thymidylate synthase (TS), a target of 5-FU. A high level of TS is thought to be related to resistance against 5-FU chemotherapy. RESULTS The CRPC specimens and C4-2 showed significantly lower TS expression than the ADPC specimens and LNCaP. In vivo, daily oral administration of S-1 (3 mg/kg, adjusted for human dose) significantly suppressed tumor growth. Although a single intraperitoneal injection of low dose DOC (2 mg/kg) did not show tumor suppression, low dose DOC enhanced anti-tumor effect of S-1. In vitro, low dose DOC, which did not induce G2/M arrest, increased p53 and p21, suppressed CDK4 activity, and resulted in down regulation of TS in C4-2. 5-FU enhanced TS expression, and combined with low dose DOC suppressed the TS which was enhanced by 5-FU. In addition, we also confirmed in vivo using immunostaining that S-1 enhanced TS expression, low dose DOC suppressed the expression of TS, and combined S-1/DOC suppressed the TS, which was enhanced by S-1. CONCLUSIONS The present findings indicate that CRPC patients with androgen ablation may be good candidates for 5-FU based chemotherapy, and these regimens have attractive therapeutic potential for clinical practice, and they may have a significant impact on therapeutic options. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e514 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Masanori Hasegawa Tokyo, Japan More articles by this author Akira Miyajima Tokyo, Japan More articles by this author Takeo Kosaka Tokyo, Japan More articles by this author Yota Yasumizu Tokyo, Japan More articles by this author Nobuyuki Tanaka Tokyo, Japan More articles by this author Takahiro Maeda Tokyo, Japan More articles by this author Suguru Shirotake Tokyo, Japan More articles by this author Hiroki Ide Tokyo, Japan More articles by this author Eiji Kikuchi Tokyo, Japan More articles by this author Mototsugu Oya Tokyo, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Vorinostat synergises with capecitabine through upregulation of thymidine phosphorylase

Background:Potentiation of anticancer activity of capecitabine is required to improve its therapeutic index. In colorectal cancer (CRC) cells, we evaluated whether the histone deacetylase-inhibitor vorinostat may induce synergistic antitumour effects in combination with capecitabine by modulating the expression of thymidine phosphorylase (TP), a key enzyme in the conversion of capecitabine to 5-florouracil (5-FU), and thymidylate synthase (TS), the target of 5-FU.Methods:Expression of TP and TS was measured by real-time PCR, western blotting and immunohistochemistry. Knockdown of TP was performed by specific small interfering RNA. Antitumour activity of vorinostat was assessed in vitro in combination with the capecitabine active metabolite deoxy-5-fluorouridine (5′-DFUR) according to the Chou and Talay method and by evaluating apoptosis as well as in xenografts-bearing nude mice in combination with capecitabine.Results:Vorinostat induced both in vitro and in vivo upregulation of TP as well as downregulation of TS in cancer cells, but not in ex vivo treated peripheral blood lymphocytes. Combined treatment with vorinostat and 5′-DFUR resulted in a synergistic antiproliferative effect and increased apoptotic cell death in vitro. This latter effect was impaired in cells where TP was knocked. In vivo, vorinostat plus capecitabine potently inhibited tumour growth, increased apoptosis and prolonged survival compared with control or single-agent treatments.Conclusions:Overall, this study suggests that the combination of vorinostat and capecitabine is an innovative antitumour strategy and warrants further clinical evaluation for the treatment of CRC.

RAD001 shows activity against gastric cancer cells and overcomes 5-FU resistance by downregulating thymidylate synthase

We evaluated RAD001, an inhibitor of the mammalian target of rapamycin (mTOR) in human gastric cancer cell lines and determined the molecular mechanisms. RAD001 has marked growth inhibitory activity against the SNU-1 and SNU-216 cells. It inhibited phosphorylation of mTOR and S6 K, and induced G1 cell cycle arrest. Synergistic growth-inhibitory effects in combination with 5-fluorouracil (5-FU) was identified. Furthermore, RAD001 conferred sensitivity to 5-FU-resistant cell lines by downregulating thymidylate synthase (TS). In conclusion, RAD001 showed growth inhibitory activity against gastric cancer cells and acted synergistically with cytotoxic agents such as 5-FU by downregulating TS.

Genetic Determinants of Pemetrexed Responsiveness and Nonresponsiveness in Non-small Cell Lung Cancer Cells

Pemetrexed disodium (Alimta), LY231514, is an antifolate that is able to simultaneously inhibit the synthesis of purines and pyrimidines. Pemetrexed has been approved for first- and second-line treatment in patients with non-small cell lung cancer (NSCLC). However, there is still a lack of clinical biomarkers for predicting the therapeutic response to pemetrexed. The aim of this study is to establish new biomarkers for pemetrexed treatment in NSCLC. Human NSCLC cell lines were exposed to pemetrexed. The antitumor effect was measured by growth inhibition with MTT assay and expression of cell cycle mediators with immunoblots. Using the Superarray cancer pathway gene array, 482 genes were screened for differential expression in A549 cells that were untreated or treated with pemetrexed. A549 cells exhibited sensitivity but H1355 cells showed resistance to pemetrexed. To investigate the mechanisms of responsiveness and nonresponsiveness to pemetrexed in these cell lines, we measured the expression levels of thymidylate synthase (TS), dihydrofolate reductase (DHFR), reduced folate carrier, and folylpoly-gamma-glutamate synthetase genes. TS, DHFR, and reduced folate carrier gene expressions were significantly reduced in A549 and H1355 cells. Pemetrexed caused cell cycle arrest in the G1 phase and S phase in H1355 and A549 cells, respectively. Significantly higher expressions of many genes, especially lipocalin-2 (Lcn-2) and nm23-H1 proteins, were noted in A549 cells treated with pemetrexed in comparison with untreated cells. Furthermore, reverse transcriptase polymerase chain reaction and Western blot showed that Lcn-2 and nm23-H1 expressions increase in response to pemetrexed treatment in a dose-responsive manner in pemetrexed-sensitive A549 cells but not in resistant H1355 cells. Our results indicated that downregulation of TS and DHFR genes and upregulation of p21, p27, Lcn-2, and nm23-H1 genes may serve as new biomarkers for predicting responsiveness to pemetrexed.

A Phase I, Pharmacokinetic, and Pharmacodynamic Study of Two Schedules of Vorinostat in Combination with 5-Fluorouracil and Leucovorin in Patients with Refractory Solid Tumors

We conducted a phase I clinical trial to determine the maximum tolerated dose (MTD) of daily or twice daily vorinostat x 3 days when combined with fixed doses of 5-fluorouracil (FU) and leucovorin every 2 weeks. Vorinostat doses were escalated in a standard 3 x 3 phase I design. FU/leucovorin was started on day 2 of vorinostat and consisted of leucovorin 400 mg/m(2) i.v. over 2 hours followed by FU 400 mg/m(2) i.v. bolus and 2,400 mg/m(2) over 46 hours (sLV5FU2). Forty-three patients were enrolled. Grade 3 fatigue, and hand and foot syndrome were the dose-limiting toxicities (DLT) at the 2,000 mg vorinostat once-daily dose level. Grade 3 fatigue and mucositis were DLTs at the 800 mg vorinostat twice-daily dose level. None of six patients at the 1,700 mg once daily or six patients at the 600 mg twice daily dose levels had a DLT; those dose levels represent the MTD. Twenty-one of 38 patients with FU-refractory colorectal cancer had stable disease, and one had a partial response. Vorinostat maximum serum concentrations at the MTD exceeded concentrations associated with thymidylate synthase downregulation in vitro. No pharmacokinetic interactions were noted between vorinostat and FU. The MTD of vorinostat in combination with sLV5FU2 is 1,700 mg orally once daily x 3 or 600 mg orally twice daily x 3 days every 2 weeks. Clinical activity in refractory colorectal cancer supports further clinical development of this combination.

Enhanced Anticancer Effect of the Combination of BIBW2992 and Thymidylate Synthase–Targeted Agents in Non–Small Cell Lung Cancer with the T790M Mutation of Epidermal Growth Factor Receptor

Most non-small cell lung cancer (NSCLC) tumors with activating mutations of the epidermal growth factor receptor (EGFR) are initially responsive to first-generation, reversible EGFR tyrosine kinase inhibitors (TKI) such as gefitinib, but they subsequently develop resistance to these drugs through either acquisition of an additional T790M mutation of EGFR or amplification of the proto-oncogene MET. We have now investigated the effects of combination treatment with thymidylate synthase (TS)-targeting drugs and the second-generation, irreversible EGFR-TKI BIBW2992 on the growth of NSCLC cells with the T790M mutation. The effects of BIBW2992 on EGFR signaling and TS expression in gefitinib-resistant NSCLC cells were examined by immunoblot analysis. The effects of BIBW2992 and the TS-targeting agents S-1 (or 5-fluorouracil) or pemetrexed on the growth of gefitinib-resistant NSCLC cells were examined both in vitro and in vivo. The combination of BIBW2992 with 5-fluorouracil or pemetrexed synergistically inhibited the proliferation of NSCLC cells with the T790M mutation in vitro, whereas an antagonistic interaction was apparent in this regard between gefitinib and either of these TS-targeting agents. BIBW2992 induced downregulation of TS in the gefitinib-resistant NSCLC cells, implicating depletion of TS in the enhanced antitumor effect of the combination therapy. The combination of BIBW2992 and either the oral fluoropyrimidine S-1 or pemetrexed also inhibited the growth of NSCLC xenografts with the T790M mutation to an extent greater than that apparent with either agent alone. The addition of TS-targeting drugs to BIBW2992 is a promising strategy to overcome EGFR-TKI resistance in NSCLC with the T790M mutation of EGFR.

A Phase I Study of 5-Fluorouracil/Leucovorin and Arsenic Trioxide for Patients with Refractory/Relapsed Colorectal Carcinoma

This Phase I study was designed to determine a safe combination dose of 5-fluorouracil (5-FU) and arsenic trioxide (ATO) to treat 5-FU-resistant relapsed/refractory colorectal cancer patients. We studied the effect of ATO in the downregulation of thymidylate synthase (TS) in peripheral blood mononuclear cells and in tumor biopsies. ATO was administered for 5 consecutive days during the first week and twice during weeks 2 to 3 and once on week 4. 5-FU/leucovorin (LV) was administered on days 8, 15, and 22. A modified accelerated titration design was used. 5-FU was dose escalated first followed by a planned dose increase for ATO. No dose-limiting toxicities were seen in seven patients who received 0.15 mg/kg ATO; grade 3 toxicities were as follows: neutropenia 1, diarrhea 1, and bowel obstruction 1. In patients receiving 0.20 mg/kg ATO, grade 3 toxicities were QTc prolongation 1, fatigue 4, alkaline phosphatase elevation 2, diarrhea 2, and peripheral edema 1. TS gene expression in peripheral blood mononuclear cell decreased in all patients. Eight tumors were biopsied, four showed TS downregulation, three showed upregulations, and one did not change. Estimated median progression-free survival and overall survival were 3.1 and 13.9 months, respectively. In patients who showed TS increase or no change versus TS reduction, estimated median progression-free survival was 2.6 versus 7.9 months (P = 0.188) and overall survival was 8.6 versus 11.7 months (P = 0.44), respectively. Thus, we determined 0.20 mg/kg ATO, 2,600 mg/m(2) 5-FU, and 500 mg/m(2) leucovorin (LV) to be the recommended phase II dose.

Synergistic Antitumor Effect of S-1 and HER2-Targeting Agents in Gastric Cancer with HER2 Amplification

Amplification of human epidermal growth factor receptor 2 (HER2) has been detected in 20% to 30% of gastric cancers and is associated with a poor outcome. Combination therapies with HER2-targeting agents and cytotoxic agents are considered a potential therapeutic option for gastric cancer with HER2 amplification. We have now investigated the effects of combination treatment with the oral fluoropyrimidine S-1 and the HER2-targeting agents lapatinib or trastuzumab in gastric cancer cells with or without HER2 amplification. We used 5-fluorouracil (5FU) instead of S-1 for in vitro experiments, given that tegafur, a component of S-1, is metabolized to 5FU in the liver. The combination of 5FU and HER2-targeting agents synergistically inhibited cell proliferation and exhibited an enhanced proapoptotic effect in gastric cancer cells with HER2 amplification, but not in those without it. Lapatinib or trastuzumab also induced downregulation of thymidylate synthase (TS) expression and activity only in cells with HER2 amplification. The combination of 5FU and TS depletion by RNA interference also exhibited an enhanced proapoptotic effect in cells with HER2 amplification. These observations thus suggest that lapatinib-induced or trastuzumab-induced downregulation of TS is responsible, at least in part, for the synergistic antitumor effect of combined treatment with 5FU and HER2-targeting agents. The antitumor effect of the combination of S-1 and HER2-targeting agents in vivo was also greater than that of either drug alone. Our preclinical findings thus indicate that the combination of S-1 and HER2-targeting agents is a promising treatment option for gastric cancer with HER2 amplification.

Abstract 3640: Increased anti-tumor effects of sequential docetaxel followed by 5-FU treatment against human oral cancer cells

Abstract [Background] Docetaxel (TXT), a novel member of the taxoid family, and 5-fluorouracil (5-FU) are key drugs against oral cancer chemotherapy. The promising clinical activity of TXT and 5-FU has promoted considerable interest in combining these drugs. Although the combination treatment has been reported to improve the response rates, sequential treatment with TXT and 5-FU was unknown. [Purpose] The aim of this study was to evaluate the anti-tumor effects of sequential treatment with TXT and 5-FU against oral squamous cell carcinoma cells. [Methods and Results] Two human oral squamous cell carcinoma cell lines, B88 and CAL27 cells were used in this study. The effects of sequential treatment on the inhibition of tumor growth with TXT and 5-FU were investigated by 3-(4, 5-dimethylthiaol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometric analysis, and the xenograft on nude mice. In vivo study, B88 or CAL cells (5×106) were subcutaneously inoculated into the backs of nude mice. When the tumor reached 0.5 cm in diameter, tumor-bearing nude mice received either the intraperitoneal administration of TXT (10 mg/kg) alone, 5-FU (15 mg/kg) alone, TXT followed by 5-FU or 5-FU followed by TXT treatment. TXT followed by 5-FU treatment more effectively inhibited the tumor growth in vitro and in vivo, and induced apoptosis than 5-FU followed by TXT treatment. To elucidate the mechanisms underlying growth inhibitory effect of TXT followed by 5-FU, we examined the expression of 5-FU metabolic enzymes, thymidylate synthase (TS), dehydropyrimidine dehydrogenase (DPD), and ortate phosphorybosyl transferase (OPRT) by real-time PCR and western blot analysis. Down-regulation of TS and DPD expression, and up-regulation of OPRT expression were induced by TXT treatment compared with 5-FU, suggesting that they enhanced the efficacy of 5-FU. [Conclusions]These findings demonstrated that sequential treatment with TXT followed by 5-FU can be effective for the patients with oral squamous cell carcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3640.

Abstract 3858: Synergistic antitumor effects of combination therapy with S-1 and HER2 targeting agents in gastric cancer with HER2 amplification

Abstract HER2 amplification is observed in 20-30% of gastric cancer and it has been correlated to poor outcomes and more aggressive disease. Combination therapies with HER2 targeting agents and cytotoxic agents are considered a therapeutic option for patients with HER2 amplified gastric cancer. We now investigated the effects of the combination treatment with S-1(or 5-fluorouracil (5FU)) and either lapatinib or trastuzumab on the growth of gastric cancer cells with or without HER2 amplification in vitro and in vivo. The combination of 5FU and either lapatinib or trastuzumab showed a synergistic antiproliferative effect in HER2 amplified gastric cancer cells. Combined treatment with 5FU and lapatinib or trastuzumab induced synergistic increases in the number of apoptotic cells and in the activity of caspase-3 in HER2 amplified cells, wheareas, such synergistic antiproliferative effect nor the enhancement of apoptosis was observed in cells without HER2 amplification. Thymidylate synthase (TS) is an important target enzyme for 5FU, with a reduced level of TS expression having been associated with a higher rate of response to 5FU-based chemotherapy. To elucidate the molecular mechanism of the synergistic effect by the combination of 5FU and lapatinib or trastuzumab, we examined the effects of lapatinib or trastuzumab on the expression and activity of TS in gastric cancer cells. Both lapatinib and trastuzumab induced down-regulation of TS expression and inhibition of TS activity in HER2 amplified cells, whereas neither lapatinib nor trastuzumab has such effects on TS expression and activity in cells without HER2 amplification. Similar to the effects of lapatinib or trastuzumab, TS depletion by siRNA resulted in the enhancement of 5FU-induced apoptosis, suggesting that TS down-regulation attributes, at least in part, to the synergistic proapoptotic interaction with 5FU. Furthermore, the combination of oral fluoropyrimidine S-1 and lapatinib or trastuzumab synergistically inhibited the growth of gastric cancer cell xenografts with HER2 amplification. Our results thus suggest that the combination of S-1 and lapatinib or trastuzumab has synergistic antitumor effects in HER2 amplified gastric cancer cells and that effect is likely attributable to the down-regulation of TS expression and inhibition of TS activity by lapatinib or trastuzumab. In conclusion, these observations provide a basis for clinical evaluation of combination chemotherapy with S-1 and HER2 targeting agents, lapatinib or trastuzumab, in gastric cancer patients with HER2 amplification. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3858.