1,3-Bis(2-chloroethyl)-1-nitrosourea enhances the inhibitory effect of Resveratrol on 5-fluorouracil sensitive/resistant colon cancer cells

AIM:To study the mechanism of 5-fluorouracil(5-FU)resistance in colon cancer cells and to develop strategies for overcoming such resistance by combination treatment.METHODS:We established and characterized a 5-FU resistance(5-FU-R)cell line derived from continuous exposure(25μmol/L)to 5-FU for 20 wk in 5-FU sensitive HCT-116 cells.The proliferation and expression of different representative apoptosis and anti-apoptosis markers in 5-FU sensitive and 5-FU resistance cells were measured by the MTT assay and by Western blotting,respectively,after treatment with Resveratrol(Res)and/or 1,3-Bis(2-chloroethyl)-1-nitrosourea(BCNU).Apoptosis and cell cycle arrest was measured by 4',6'-diamidino-2-phenylindole hydrochloride staining and fluorescence-activated cell sorting analysis,respectively.The extent of DNA damage was measured by the Comet assay.We measured the visible changes in the DNA damage/repair cascade by Western blotting.RESULTS:The widely used chemotherapeutic agents BCNU and Res decreased the growth of 5-FU sensitive HCT-116 cells in a dose dependent manner.Combined application of BCNU and Res caused more apoptosis in5-FU sensitive cells in comparison to individual treatment.In addition,the combined application of BCNU and Res caused a significant decrease of major DNA base excision repair components in 5-FU sensitive cells.We established a 5-FU resistance cell line(5-FU-R)from 5-FU-sensitive HCT-116(mismatch repair deficient)cells that was not resistant to other chemotherapeutic agents(e.g.,BCNU,Res)except 5-FU.The 5-FU resistance of 5-FU-R cells was assessed by exposure to increasing concentrations of 5-FU followed by the MTT assay.There was no significant cell death noted in5-FU-R cells in comparison to 5-FU sensitive cells after5-FU treatment.This resistant cell line overexpressed anti-apoptotic[e.g.,AKT,nuclear factorκB,FLICE-like inhibitory protein),DNA repair(e.g.,DNA polymerase beta(POL-β),DNA polymerase eta(POLH),protein Flap endonuclease 1(FEN1),DNA damage-binding protein 2(DDB2)]and 5-FU-resistance proteins(thymidylate synthase)but under expressed pro-apoptotic proteins(e.g.,DAB2,CK1)in comparison to the parental cells.Increased genotoxicity and apoptosis were observed in resistant cells after combined application of BCNU and Res in comparison to untreated or parental cells.BCNU increased the sensitivity to Res of 5-FU resistant cells compared with parental cells.Fifty percent cell death were noted in parental cells when 18μmol/L of Res was associated with fixed concentration(20μmol/L)of BCNU,but a much lower concentration of Res(8μmol/L)was needed to achieve the same effect in 5-FU resistant cells.Interestingly,increased levels of adenomatous polyposis coli and decreased levels POL-β,POLH,FEN1 and DDB2 were noted after the same combined treatment in resistant cells.CONCLUSION:BCNU combined with Res exerts a synergistic effect that may prove useful for the treatment of colon cancer and to overcome drug resistance.

Combined Antitumor Effect of Ursolic Acid And 5-Fluorouracil on Human Esophageal Carcinoma Cell Eca-109 In Vitro

Objective： To study the combined antitumor effect and possible mechanisms of ursolic acid with 5-fluorouracil （5-FU） on human esophageal carcinoma cell Eca-109 in vitro. Methods： Eca-109 cells were treated with ursolic acid （10-50 μmol/L） and/or 5-fluorouracil （48.0-768.8 μmol/L） for 48 h in vitro. And then cell proliferation was determined by MTT assay. Cell cycle and apoptosis rate were analyzed by flow cytometry （FCM）. The morphological changes of apoptosis were observed by fluorescent microscopy. At last the expression of P27kipl, bcl-2 and bax were detected by western blot. Results： Results： In comparison with single agent treatment, the combination of ursolic acid and 5-fluorouracil produced greater efficacy in growth inhibition, cell cycle arrest at G0/G1 phase, and apoptosis induction （P〈0.05）. Western blot analysis showed that the combination use of ursolic acid and 5-fluorouracil suppressed the expression of bcl-2 and increased the expressions of bax and P27kip1. Conclusion： Ursolic acid combined with 5-fluorouracil showed adjuvant antiproliferative effects on human esophageal carcinoma cell Eca-109 in vitro, which mainly due to the induction of cell cycle arrest as well as apoptosis.

A novel oral prodrug-targeting transporter MCT 1: 5-fluorouracil-dicarboxylate monoester conjugates

Monocarboxylate transporter 1(MCT1)is responsible for oral absorption of short-chain monocarboxylic acids from small intestine,hence,it’s likely to serve as an ideal design target for the development of oral prodrugs.However,potential application of MCT1 to facilitate the oral delivery is still unclear.Irregular oral absorption,poor permeability and bioavailability greatly limit the oral delivery efficiency of 5-fluorouracil(5-FU).Herein,we design three 5-FU-fatty acid conjugates targeting intestinal MCT1 with different lipophilic linkages.Interestingly,due to high MCT1 affinity and good gastrointestinal stability,5-FUoctanedioic acid monoester prodrug exhibited significant improvement in membrane permeability(13.1-fold)and oral bioavailability(4.1-fold)compared to 5-FU.More surprisingly,stability experiment in intestinal homogenates showed that 5-FU prodrugs could be properly activated to release 5-FU within intestinal cells,which provides an ideal foundation for the improvement of oral bioavailability.In summary,good gastrointestinal stability,high membrane permeability and appropriate intestinal cell bioactivation are the important factors for high-efficiency 5-FU oral prodrugs,and such work provides a good platform for the development of novel oral prodrugs targeting intestinal transporters.

The Synthesis of 1-Aryl-5-Fluorouracil

A series of new type substituted 5-fluorouracil derivatives, 1-aryl-5-fluorouracil (4a-4f), were synthesized via diaryliodonium salts and the structure of the title compound was finally confirmed by IR, UV. H-1-NMR. MS and CHN analysis.

Synthesis and Crystal Structure of N1-(2-(4-Bromo-2-nitrophenoxy)-2-dimethyl acyloxymethyl)-5-fluorouracil

The title compound N1-（2-（4-bromo-2-nitrophenoxy）-2-dimethyl acyloxymethyl）-5-fluorouracil（C15H13BrFN3O7, Mr = 446.19） was synthesized and structurally characterized by ^1H-NMR, -（13）C-NMR, ESI-MS and single-crystal X-ray diffraction. The compound crystallizes in triclinic, space group P1, with a = 8.3325（6）, b = 10.1808（13）, c = 11.8194（16） A, α = 73.194（12）, β= 72.351（9）, γ = 89.509（8）o, V = 911.2（2） A^3, Z = 2, T = 300.79（10） K, μ（Cu Kα） = 3.578 mm^-1, Dc = 1.626 g/cm3, F（000） = 448.0, GOF = 1.060, 5723 reflections measured（8.232≤2θ≤139.592°）, 3339 unique（Rint = 0.0184, Rsigma = 0.0254） which were used in all calculations. The final R = 0.0517（I 〉 2s（I）） and wR = 0.1494（all data）. Hydrogen bonds and π-π interactions together stabilize the structure of the molecule. The preliminary biological test shows that the title compound has good antitumor activity against A549 in vitro and lower toxicity to normal cells.

糖苷合成研究(Ⅹ Ⅸ)1-乙氧甲基-5-氟脲嘧啶糖苷的合成

在相转移条件下,使2,3,4,6-四-O-乙酰基-1-溴-1-脱氧--αD-吡喃葡萄糖(1A)、2,3,4-三-O-乙酰基-1-溴-1-脱氧--αD-吡喃木糖(1B)、2,3,4-三-O-乙酰基-1-溴-1-脱氧--αD-吡喃葡萄糖醛酸甲酯(1C)与1-乙氧甲基-5-氟脲嘧啶2反应,合成了3个新的1-乙氧甲基-5-氟脲嘧啶糖苷3(A^C).3(A^C)经氨解脱乙酰基,得3个新的化合物4(A′~C′),其结构经IR1、HNMR及元素分析证实.

Gli-1 siRNA induced apoptosis in Huh7 cells

AIM： To investigate the effects of Gli-1 small interference RNA （siRNA） on Huh7 cells, and the change of Bcl-2 expression in Huh7 cells. METHODS： Human hepatocellular carcinoma cells Huh7 were used. Cell viability was analyzed by 3-（4, 5-Dimethylthiazol-2-yl）-2, 5-diphenyl tetrazolium bromide （MTT） assay. The expressions of Gli-1 and Bcl-2 family members were detected by RT-PCR and Western blot. Apoptosis was detected by Flow cytometry using propidium iodide, measured by Hoechst 33258 staining using Advanced Fluorescence Microscopy and caspase-3 enzymatic assay. Cell growth was analyzed after treatment with Gli-1 siRNA and 5-fluorouracil （5-Fu）. RESULTS： Inhibition of Gli-1 mRNA in Huh7 cells through Gli-1 siRNA reduced cell viability. Gli-1 siRNA treatment also induced apoptosis by three criteria, increase in the sub-G1 cell cycle fraction, nuclear condensation, a morphologic change typical of apoptosis, and activation of caspase-3. Gli-1 siRNA was also able to down-regulate Bcl-2. However, Gli-1 siRNA resulted in no significant changes in Bcl-xl, Bax, Bad, and Bid. Furthermore, Gli-1 siRNA increased the cytotoxic effect of 5-Fu on Huh7 cell. CONCLUSION： Down-regulation of Bcl-2 plays an important role in apoptosis induced by Gli-1 siRNA in HCC cells. Combination Gli-1 siRNA with chemotherapeutic drug could represent a more promising strategy against HCC. The effects of the strategies need further investigation in vivo and may have potential clinical application.

Bcl-x_L and Myeloid cell leukaemia-1 contribute to apoptosis resistance of colorectal cancer cells

AIM： TO explore the role of Bcl-XL and Myeloid cell leukaemia （Mcl）-I for the apoptosis resistance of colorectal carcinoma （CRC） cells towards current treatment modalities. METHODS： Bcl-XL and Mcl-1 mRNA and protein expression were analyzed in CRC cell lines as well as human CRC tissue by Western blot, quantitative PCRand immunohistochemistry. Bcl-XL and Mcl-1 protein expression was knocked down or increased in CRC cell lines by applying specific siRNAs or expression plasmids, respectively. After modulation of protein expression, CRC cells were treated with chemotherapeutic agents, an antagonistic epidermal growth factor receptor （EGFR1） antibody, an EGFR1 tyrosine kinase inhibitor, or with the death receptor ligand TRAIL. Apoptosis induction and cell viability were analyzed. RESULTS： Here we show that in human CRC tissue and various CRC cell lines both Bcl-xL and Mcl-1 are expressed. Bcl-xL expression was higher in CRC tissue than in surrounding non-malignant tissue, both on protein and mRNA level. Mcl-1 mRNA expression was significantly lower in malignant tissues. However, protein expression was slightly higher. Viability rates of CRC cells were significantly decreased after knock down of Bcl-xL expression, and, to a lower extent, after knock down of Mcl-1 expression. Furthermore, cells with reduced Bcl-xL or Mcl-1 expression was more sensitive towards oxaliplatinand irinotecan-induced apoptosis, and in the case of Bcl-xL also towards 5-FU-induced apoptosis. On the other hand, upregulation of Bcl-xL by transfection of an expression plasmid decreased chemotherapeutic drug-induced apoptosis. EGF treatment clearly induced Bcl-xL and Mcl-1 expression in CRC cells. Apoptosis induction upon EGFR1 blockage by cetuximab or PD168393 was increased by inhibiting Mcl-1 and Bcl-xL expression. More strikingly, CD95- and TRAIL-induced apoptosis was increased by Bcl-xL knock down. CONCLUSION： Our data suggest that Bcl-xL and, to a lower extent, Mcl-1, are important anti-apoptotic factors in CRC. Specific downregulation of Bcl-xL is a promising approach to sensitize CRC cells towards chemotherapy and targeted therapy.

S-1 induced hepatic steatosis in patients with pancreatic cancer:Retrospective analysis

To determine whether S-1 induces hepatic steatosis in patients being treated for pancreatic cancer. METHODSThis retrospective study evaluated 22 patients who received oral S-1 as a first-line treatment for pancreatic cancer between January 2008 and July 2015 at the Ishikawa Prefectural Central Hospital. Patients underwent abdominal computed tomography (CT) scans before chemotherapy and within 3 mo from the start of treatment. CT numbers of the liver and spleen were measured before and after S-1 administration. Steatosis was diagnosed when the ratio of the CT number of the liver to that of the spleen (liver/spleen ratio) was < 0.9. RESULTSMedian patient age was 68 years (range, 48-85 years), and median body mass index was 21 kg/m2 (range, 18-27 kg/m2). Of the 22 patients, six (27%) regularly consumed alcohol, and five (23%) had liver metastases. The mean ratio of CT number of the liver to the spleen was significantly higher before administration of S-1 (1.27 vs 1.09, P = 0.012) compared with after. Of the 22 patients, five (23%) had hepatic steatosis and 17 (77%) did not. The pretreatment demographic and clinical characteristics of these two groups showed no significant differences. The relationship between liver/spleen ratio and alanine transaminase activity in these patients. A statistically significant inverse correlation was observed (r = -0.417, P < 0.027). CONCLUSIONOf the 22 patients with pancreatic cancer, five (23%) experienced S-1 induced hepatic steatosis. Care should be taken during S-1 treatment of patients with pancreatic cancer.

Angelica sinensis polysaccharides ameliorate 5-flourouracil-induced bone marrow stromal cell proliferation inhibition via regulating Wnt/β-catenin signaling

Chemotherapy may cause cellular oxidative stress to bone marrow.Oxidative damage of bone marrow hematopoietic microenvironment is closely related to chronic myelosuppression after chemotherapeutic treatment.Angelica sinensis polysaccharides(ASP)are major effective ingredients of traditional Chinese medicine Angelica with multi-target anti-oxidative stress features.In the current study,we investigated the protective roles and mechanisms of ASP on chemotherapy-induced bone marrow stromal cell(BMSC)damage.The human bone marrow stromal cell line HS-5 cells were divided into control group,5-FU group,5-FU+ASP group,and 5-FU+LiCl group to investigate the mechanism of ASP to alleviate 5-FU-induced BMSC proliferation inhibition.The results showed that 5-FU inhibits the growth of HS-5 cells in a time and dose-dependent manner;however,ASP partially counteracted the 5-FU-induced decrease in cell viability,whereas Wnt signaling inhibitor Dkk1 antagonized the effect of ASP on HS-5 cells.ASP reversed the decrease in total cytoplasmicβ-catenin,p-GSK-3β,and CyclinD1 following 5-FU treatment and modulated nuclear expression ofβ-catenin,Lef-1,and C-myc proteins.Furthermore,ASP also enhanced the antioxidant capacity of cells and reduced 5-FU-induced oxidative stress,attenuated FoxO1 expression,thus weakened its downstream apoptosis-related proteins and G0/G1 checkpoint-associated p27^(Kip1) expression to alleviate 5-FU-induced apoptosis and to promote cell cycle progression.All the results above suggest that the protective role of ASP in 5-FU-treated BMSCs proliferation for the chemotherapy may be related to its activating Wnt/β-catenin signaling and keeping homeostasis betweenβ-catenin and FoxO1 under oxidative stress.The study provides a potential therapeutic strategy for alleviating chemotherapeutic damage on BMSCs.

Influence of 6-shogaol potentiated on 5-fluorouracil treatment of liver cancer by promoting apoptosis and cell cycle arrest by regulating AKT/mTOR/MRP1 signalling

Currently, chemoresistance seriously attenuates the curative outcome of liver cancer. The purpose of our work was to investigate the influence of 6-shogaol on the inhibition of 5-fluorouracil (5-FU) in liver cancer. The cell viability of cancer cells was determined by MTT assay. Liver cancer cell apoptosis and the cell cycle were examined utilizing flow cytometry. Moreover, qRT–PCR and western blotting was used to analyse the mRNA and protein expression levels, respectively. Immunohistochemistry assays were used to examine multidrug resistance protein 1 (MRP1) expression in tumour tissues. In liver cancer cells, we found that 6-shogaol-5-FU combination treatment inhibited cell viability, facilitated G0/G1 cell cycle arrest, and accelerated apoptosis compared with 6-shogaol or 5-FU treatment alone. In cancer cells cotreated with 6-shogaol and 5-FU, AKT/mTOR pathway- and cell cycle-related protein expression levels were inhibited, and MRP1 expression was downregulated. AKT activation or MRP1 increase reversed the influence of combination treatment on liver cancer cell viability, apoptosis and cell cycle arrest. The inhibition of AKT activation to the anticancer effect of 6-shogaol-5-FU could be reversed by MRP1 silencing. Moreover, our results showed that 6-shogaol-5-FU combination treatment notably inhibited tumour growth in vivo. In summary, our data demonstrated that 6-shogaol contributed to the curative outcome of 5-FU in liver cancer by inhibiting the AKT/mTOR/MRP1 signalling pathway.

5-Fluorouracil derivatives ⅩⅩⅢ. Synthesis and antitumor activities of 1-carbamoyl-5-fluorouracils having aromatic ring

In order to get good antitumor agents especially better than 5-fluorouracil,tegafur and l-hexylcarbamoyl-5-fluorouracil (HCFU),fourty nine 1-carbamoyl-5-fluorouracil having aromatic ring were synthesized from 5-fluorouracil and isocyanates or amines.Antitumor activity was tested in the L-1210 tumor system,and 5 compounds gave better value of therapeutic ratio than 5-fluorouracil,tegafur,HCFU.l-(4-Methoxybenzylcarbamoyl)-5-fluorouracil gave the best result.

TRAIL-induced apoptosis of hepatocellular carcinoma cells is augmented by targeted therapies

AIM:To analyze the effect of chemotherapeutic drugs and specific kinase inhibitors,in combination with the death receptor ligand tumor necrosis factor-related apoptosis inducing ligand(TRAIL),on overcoming TRAIL resistance in hepatocellular carcinoma(HCC)and to study the efficacy of agonistic TRAIL antibodies,as well as the commitment of antiapoptotic BCL-2 proteins, in TRAIL-induced apoptosis. METHODS:Surface expression of TRAIL receptors (TRAIL-R1-4)and expression levels of the antiapoptotic BCL-2 proteins MCL-1 and BCL-xL were analyzed by flow cytometry and Western blotting,respectively. Knock-down of MCL-1 and BCL-xL was performed by transfecting specific small interfering RNAs.HCC cellswere treated with kinase inhibitors and chemotherapeutic drugs.Apoptosis induction and cell viability were analyzed via flow cytometry and 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. RESULTS:TRAIL-R1 and-R2 were profoundly expressed on the HCC cell lines Huh7 and Hep-G2. However,treatment of Huh7 and Hep-G2 with TRAIL and agonistic antibodies only induced minor apoptosis rates.Apoptosis resistance towards TRAIL could be considerably reduced by adding the chemotherapeutic drugs 5-fluorouracil and doxorubicin as well as the kinase inhibitors LY294002[inhibition of phosphoinositol- 3-kinase(PI3K)],AG1478(epidermal growth factor receptor kinase),PD98059(MEK1),rapamycin(mam- malian target of rapamycin)and the multi-kinase inhibitor Sorafenib.Furthermore,the antiapoptotic BCL-2 proteins MCL-1 and BCL-xL play a major role in TRAIL resistance:knock-down by RNA interference increased TRAIL-induced apoptosis of HCC cells.Additionally, knock-down of MCL-1 and BCL-xL led to a significant sensitization of HCC cells towards inhibition of both c-Jun N-terminal kinase and PI3K.CONCLUSION:Our data identify the blockage of survival kinases,combination with chemotherapeutic drugs and targeting of antiapoptotic BCL-2 proteins as promising ways to overcome TRAIL resistance in HCC.

Curcumin sensitizes quiescent leukemic cells to antimitotic drug 5-fluorouracil by inducing proliferative responses in them

Long-term quiescence or dormancy is a fundamental feature of cancer stem cells(CSCs)that are genetically identical to the malignant clone but constitute the only cells with tumor propagation potential within the overall tumor population.These quiescent cells show significant resistance to radiation and antiproliferative chemotherapy due to distinctive properties that seem to be related to their stem cell-like character.Hence,successful anticancer therapy must consist of approaches that can target not only the differentiated cancer cells,but also the CSCs.Using serum-starved KG1a cell line as an experimental model system of quiescent leukemic cells(QLCs),the present study demonstrates that QLCs exposed to low concentrations of curcumin show high proliferative potential.Furthermore,when subjected to a combination therapy consisting of low concentrations of curcumin and 5-fluorouracil(5-FU),the QLCs displayed a high kill with an increase in the levels of nitric oxide(NO)and reactive oxygen species.These results were further consolidated with the observation of high caspase-3 activity in cells subjected to the combination therapy.This may suggest that low concentrations of curcumin stimulate the QLCs to become mitotically active,thereby sensitizing them to killing by the antimitotic drug,5-FU.

Chemotherapy suppresses SHH gene expression via a specific enhancer

Sonic hedgehog(SHH)signaling is a key regulator of embryonic development and tissue homeostasis that is involved in gastrointestinal(GI)cancer progression.Regulation of SHH gene expression is a paradigm of long-range enhancer function.Using the classical chemotherapy drug 5-fluorouracil(5FU)as an example,here we show that SHH gene expression is suppressed by chemotherapy.SHH is downstream of immediate early genes(IEGs),including Early growth response 1(Egr1).A specific 139 kb upstream enhancer is responsible for its down-regulation.Knocking down EGR1 expression or blocking its binding to this enhancer renders SHH unresponsive to chemotherapy.We further demonstrate that down-regulation of SHH expression does not depend on 5FU’s impact on nucleotide metabolism or DNA damage;rather,a sustained oxidative stress response mediates this rapid suppression.This enhancer is present in a wide range of tumors and normal tissues,thus providing a target for cancer chemotherapy and its adverse effects on normal tissues.We propose that SHH is a stress-responsive gene downstream of IEGs,and that traditional chemotherapy targets a specific enhancer to suppress its expression.