Synthesis and biological evaluation of imidazol-2-one derivatives as potential antitumor agents

A new series of aryl substituted imidazol-2-one derivatives structurally related to combretastatin A-4 (CA-4) were synthesized and evaluated for their cytotoxic activities in vitro against various human cancer cell lines including MDR cell line. The cytotoxic effects of compounds 7b and 7i proved to be similar to or greater than that of docetaxel. The highly active compound 7b also exhibited excellent inhibitory activity on tumor growth in vivo.     

Reversal of multidrug resistance in vitro and in vivo by 5-N-formylardeemin,a new ardeemin derivative

Because multidrug resistance (MDR) is a serious impediment to the use of chemotherapy in treating cancer patients, great efforts have been made to search for effective MDR-reversing agents. We have developed a brand new synthetic ardeemin derivative, 5-N-formylardeemin, and investigated the activity of which in reversing MDR in MDR cancer cell lines derived from human breast cancer (MCF-7-R) or lung cancer (A549-R). 5-N-formylardeemin strongly enhanced the anti-cancer efficacy of doxorubicin, vincristine through potentiation of apoptosis in both MCF-7-R and A549-R at relatively noncytotoxic concentrations in vitro. Mechanistic studies showed that 5-N-formylardeemin inhibited the expression of MDR-1 (P-gp) and increased the intracellular accumulation of cytotoxic drugs in the MDR cells, suggesting that 5-N-formylardeemin reverses MDR activities through inhibiting MDR-1 expression. Interestingly, 5-N-formylardeemin also sensitized the parent wild-type cancer cells toward these chemotherapeutic agents to various extents. Importantly, in vivo studies demonstrated that 5-N-formylardeemin significantly improved the therapeutic effects of doxorubicin in nude mice bearing A549-R xenografts, which was associated with reduced expression of MDR-1 protein level and increased apoptosis in tumor tissues. These results underscore 5-N-formylardeemin as a potential sensitizer for chemotherapy against multidrug resistant cancers.     

Molecular docking studies and in vitro screening of new dihydropyridine derivatives as human MRP1 inhibitors

The overexpression of multidrug resistance protein 1 (MRP1) by tumor cells results in multidrug resistance (MDR) to structurally unrelated anticancer drugs. Circumvention of MDR by combination of chemosensitizers with antitumor compounds is a new field of investigation in cancer chemotherapy. Much effort has been put-in recently to identify the modulators/inhibitors of MRP1 to overcome the MDR. 1,4-Dihydropyridine (DHP) derivatives are indicated to be a new class of MRP1 inhibitors in cancer treatment. Molecular docking studies were carried out on 48 newly synthesized DHP derivatives with the crystal structure of MRP1 to gain some structural insights on the binding mode and possible interactions with the active site of MRP1 (NBD1). The 10 top-ranked molecules were selectively evaluated, experimentally for their MRP1 inhibitory effect using the insect cell membrane MRP1 ATPase assay. The inhibitory capacity (IC(50) concentrations) of the test compounds was compared with the reported IC(50)- or the K(i)-concentrations for benzbromarone, a standard MRP1 inhibitor. Amongst the compounds tested, compounds IA(1) and IIA(5) were found to exhibit a potent MRP1 inhibitory action with IC(50) values of 20±4 and 14±2 μM (mean±SD), respectively as compared to benzbromarone (IC(50)=4 μM). The compound IIA(5), in particular was found to be more potent than IA(1) in accordance with the docking results. These new DHP derivatives possess promising characteristics for their development as MDR reversal agents.     

FUT family mediates the multidrug resistance of human hepatocellular carcinoma via the PI3K/Akt signaling pathway

The fucosyltransferase (FUT) family is the key enzymes in cell-surface antigen synthesis during various biological processes such as tumor multidrug resistance (MDR). The aim of this work was to analyze the alteration of FUTs involved in MDR in human hepatocellular carcinoma (HCC) cell lines. Using mass spectrometry (MS) analysis, the composition profiling of fucosylated N-glycans differed between drug-resistant BEL7402/5-FU (BEL/FU) cells and the sensitive line BEL7402. Further analysis of the expressional profiles of the FUT family in three pairs of parental and chemoresistant human HCC cell lines showed that FUT4, FUT6 and FUT8 were predominant expressed in MDR cell lines. The altered levels of FUT4, FUT6 and FUT8 were responsible for changed drug-resistant phenotypes of BEL7402 and BEL/FU cells both in vitro and in vivo. In addition, regulating FUT4, FUT6 or FUT8 expression markedly modulated the activity of the phosphoinositide 3 kinase (PI3K)/Akt signaling pathway and MDR-related protein 1 (MRP1) expression. Inhibition of the PI3K/Akt pathway by its specific inhibitor wortmannin, or by Akt small interfering RNA (siRNA), resulted in decreased MDR of BEL/FU cells, partly through the downregulation of MRP1. Taken together, our results suggest that FUT4-, FUT6- or FUT8-mediated MDR in human HCC is associated with the activation of the PI3K/Akt pathway and the expression of MRP1, but not of P-gp, indicating a possible novel mechanism by which the FUT family regulates MDR in human HCC.     

Tomoeones A-H, cytotoxic phloroglucinol derivatives from Hypericum ascyron

Phloroglucinol derivatives tomoeones A-H (1-8) and three known compounds were isolated from leaves of Hypericum ascyron. Their structures were established based on spectroscopic analyses. They are all acylphloroglucinol derivatives possessing a spiro skeleton with geminal isoprenyl groups and a monoterpene moiety, and they are stereoisomers to each other at C-4 and C-13. They appear to be a class of phloroglucinol derivatives. Cytotoxicities of the isolated phloroglucinol derivatives against human tumor cell lines, including multidrug-resistant (MDR) cancer cell lines, were evaluated. Tomoeone F (6) demonstrated significant cytotoxicity against KB cells with an IC50 value of 6.2 microM. Compound 6 was also cytotoxic against MDR cancer cell lines (KB-C2 and K562/Adr), which was more potent than doxorubicin.     

Regulation of [Ca(2+)](i) homeostasis in MRP1 overexpressing cells

Regulation of capacitative Ca(2+) entry was studied in two different multidrug resistance (MDR) protein (MRP1) overexpressing cell lines, HT29(col) and GLC4/ADR. MRP1 overexpression was accompanied by a decreased response to thapsigargin. Moreover, inhibition of capacitative Ca(2+) entry by D, L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) was abolished in MRP1 overexpressing cells. Both PDMP and the MRP1 inhibitor MK571 greatly reduced InsP(3)-mediated (45)Ca(2+) release from intracellular stores in HT29 cells. Again, these effects were virtually abolished in HT29(col) cells. Our results point to a modulatory role of MRP1 on intracellular calcium concentration ([Ca(2+)](i)) homeostasis which may contribute to the MDR phenotype.     

Preferential use of CXCR4 by R5X4 human immunodeficiency virus type 1 isolates for infection of primary lymphocytes

Coreceptor specificity of human immunodeficiency virus type 1 (HIV-1) strains is generally defined in vitro in cell lines expressing CCR5 or CXCR4, but lymphocytes and macrophages are the principal targets in vivo. CCR5-using (R5) variants dominate early in infection, but strains that use CXCR4 emerge later in a substantial minority of subjects. Many or most CXCR4-using variants can use both CXCR4 and CCR5 (R5X4), but the pathways that are actually used to cause infection in primary cells and in vivo are unknown. We examined several R5X4 prototype and primary isolates and found that they all were largely or completely restricted to CXCR4-mediated entry in primary lymphocytes, even though lymphocytes are permissive for CCR5-mediated entry by R5 strains. In contrast, in primary macrophages R5X4 isolates used both CCR5 and CXCR4. The R5X4 strains were also more sensitive than R5 strains to CCR5 blocking, suggesting that interactions between the R5X4 strains and CCR5 are less efficient. These results indicate that coreceptor phenotyping in transformed cells does not necessarily predict utilization in primary cells, that variability exists among HIV-1 isolates in the ability to use CCR5 expressed on lymphocytes, and that many or most strains characterized as R5X4 are functionally X4 in primary lymphocytes. Less efficient interactions between R5X4 strains and CCR5 may be responsible for the inability to use CCR5 on lymphocytes, which express relatively low CCR5 levels. Since isolates that acquire CXCR4 utilization retain the capacity to use CCR5 on macrophages despite their inability to use it on lymphocytes, these results also raise the possibility that a CCR5-mediated macrophage reservoir is required for sustained infection in vivo.     

2-(3-Aryl-2-propenoyl)-3-methylquinoxaline-1,4-dioxides: A novel cluster of tumor-specific cytotoxins which reverse multidrug resistance

A series of 2-(3-aryl-2-propenoyl)-3-methylquinoxaline-1,4-dioxides 3a–l were prepared by condensation of various aryl aldehydes with 2-acetyl-3-methylquinoxaline-1,4-dioxide 2. These compounds inhibit the growth of human Molt 4/C8 and CEM T-lymphocytes and the IC₅₀ values are mainly in the 5–30 μM range. The quinoxaline 1,4-dioxide 3j inhibited the growth of 58 human tumor cell lines, particularly leukemic and breast cancer neoplasms. All of the compounds 3a–l reversed the multidrug resistance (MDR) properties of murine L-5178Y leukemic cells which were transfected with the human MDR1 gene. The MDR-reversing effect may be due to the conjugated π-electron system forming a weak electron charge transfer complex with the P-glycoprotein-mediated efflux pump. The compounds in series 2 and 3 were assessed against HL-60, HSC-2, HSC-3 and HSC-4 malignant cells as well as HGF, HPC and HPLF normal cell lines which revealed that the majority of the compounds displayed a greater toxicity to neoplastic than normal cells. Various ways in which the project may be expanded are presented.     

Functional comparison between YCF1 and MRP1 expressed in Sf21 insect cells

YCF1 is a yeast vacuole membrane transporter involved in resistance to Cd(2+) and to exogenous glutathione S-conjugate precursors. MRP1 contributes to multidrug resistance (MDR) in tumor cells. MRP1 and YCF1 have extensive amino acid sequence homology (63% amino acid similarity). We expressed MRP1 or YCF1 in insect cell membranes and compared their functions to know more about their structure-function relationships. YCF1 and MRP1 with His epitopes were expressed in Sf21 insect cells; both of them in the plasma membrane. The ATP-dependent transport of [(3)H]LTC(4) in Sf/YCF1-His vesicles was osmotically sensitive and showed saturable kinetics with an apparent K(m) of 758 nM for LTC(4) and 94 microM for ATP which were similar to those in yeast cells. The K(m) of YCF1 for LTC(4) (758 nM) was sevenfold higher than that of MRP1 (108 nM). MK-571 and ONO-1078, reversing agents for MRP1-mediated MDR, considerably inhibited the transport of LTC(4) by both YCF1 and MRP1. However, PAK-104P, a pyridine analog that reverses MDR associated with P-gp and MRP1, inhibited the transporting activity of MRP1 stronger than that of YCF1. KE1, another MDR reversing agent, moderately inhibited the transport of LTC(4) by MRP1 but not that of YCF1. In conclusion, we successfully expressed yeast YCF1 in Sf21 insect cells and found that the localization of the protein was different from that in yeast. The function of YCF1 in Sf21 insect cells was similar but not identical to that of MRP1.     

Glutathione-dependent binding of a photoaffinity analog of agosterol A to the C-terminal half of human multidrug resistance protein

MRP1 is a 190-kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells. MRP1 is characterized by an N-terminal transmembrane domain (TMD(0)), which is connected to a P-glycoprotein-like core region (DeltaMRP) by a cytoplasmic linker domain zero (L(0)). It has been demonstrated that GSH plays an important role in MRP1-mediated MDR. However, the mechanism by which GSH mediates MDR and the precise roles of TMD(0) and L(0) are not known. We synthesized [(125)I]11-azidophenyl agosterol A ([(125)I]azidoAG-A), a photoaffinity analog of the MDR-reversing agent, agosterol A (AG-A), to photolabel MRP1, and found that the analog photolabeled the C-proximal molecule of MRP1 (C(932-1531)) in a manner that was GSH-dependent. The photolabeling was inhibited by anticancer agents, reversing agents and leukotriene C(4). Based on photolabeling studies in the presence and absence of GSH using membrane vesicles expressing various truncated, co-expressed, and mutated MRP1s, we found that L(0) is the site on MRP1 that interacts with GSH. This study demonstrated that GSH is required for the binding of an unconjugated agent to MRP1 and suggested that GSH interacts with L(0) of MRP1. The photoanalog of AG-A will be useful for identifying the drug binding site within MRP1, and the role of GSH in transporting substrates by MRP1.     

Anti-leukemic activity of Lintuzumab (SGN-33) in preclinical models of acute myeloid leukemia

Despite therapeutic advances, the long-term survival rates for acute myeloid leukemia (AML) are estimated to be 10% or less, pointing to the need for better treatment options. AML cells express the myeloid marker CD33, making it amenable to CD33-targeted therapy. Thus, the in vitro and in vivo anti-tumor activities of lintuzumab (SGN-33), a humanized monoclonal anti-CD33 antibody undergoing clinical evaluation, were investigated. In vitro assays were used to assess the ability of lintuzumab to mediate effector functions and to decrease the production of growth factors from AML cells. SCID mice models of disseminated AML with the multi-drug resistance (MDR)-negative HL60 and the MDR+, HEL9217 and TF1-α, cell lines were developed and applied to examine the in vivo antitumor activity. In vitro, lintuzumab significantly reduced the production of TNF-α-induced pro-inflammatory cytokines and chemokines by AML cells. Lintuzumab promoted tumor cell killing through antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) activities against MDR- and MDR+ AML cell lines and primary AML patient samples. At doses from 3 to 30 mg/kg, lintuzumab significantly enhanced survival and reduced tumor burden in vivo, regardless of MDR status. Survival of the mice was dependent upon the activity of resident macrophages and neutrophils. The results suggest that lintuzumab may exert its therapeutic effects by modulating the cytokine milieu in the tumor microenvironment and through effector mediated cell killing. Given that lintuzumab induced meaningful responses in a phase 1 clinical trial, the preclinical antitumor activities defined in this study may underlie its observed therapeutic efficacy in AML patients.     

Four Ardeemin Analogs from Endophytic Aspergillus fumigatus SPS‐02 and Their Reversal Effects on Multidrug‐Resistant Tumor Cells

Four ardeemin derivatives, 5‐N‐acetylardeemin ( 1 ), 5‐N‐acetyl‐15bβ‐hydroxyardeemin ( 2 ), 5‐N‐acetyl‐15b‐didehydroardeemin ( 3 ), and 5‐N‐acetyl‐16α‐hydroxyardeemin ( 4 ), were isolated from the fermentation broth of an endophytic Aspergillus fumigatus SPS‐02 associated with Artemisia annua L . The structures of these metabolites were elucidated by a combination of spectroscopic data, including 1D‐, 2D‐NMR and MS. In vitro chemosensitization assay indicated that these ardeemins had different activities of reversing the multidrug‐resistant (MDR) phenotype in three cancer cell lines, leukemia doxorubicin resistant cell K562/DOX, human lung adenocarcinoma cis‐platin‐resistant cell A549/DDP, and ovarian cancer cisplatin‐resistant cell SK‐OV‐S/DDP. Compound 4 exhibited the strongest MDR reversing effect at 5 μM concentration in K562/DOX and A549/DDP cell lines 5.2±0.18‐fold, 8.2±0.23‐fold, respectively, while compound 2 had the highest reversal capacity in SK‐OV‐S/DDP cell line with 10.8±0.28 fold. Preliminary investigation of their structure? activity relationship suggested that a OH group at C(15b) or C(16) in ardeemin plays a key role in reversing the MDR effect. It is the first report on ardeemin analogs from endophytic A. fumigatus with reversal effects on MDR cancer cell lines K562/DOX, A549/DDP and SK‐OV‐S/DDP.     

Substrate specificities of rat oatp1 and ntcp: implications for hepatic organic anion uptake

Transport of a series of 3H-radiolabeled C23, C24, and C27 bile acid derivatives was compared and contrasted in HeLa cell lines stably transfected with rat Na+/taurocholate cotransporting polypeptide (ntcp) or organic anion transporting polypeptide 1 (oatp1) in which expression was under regulation of a zinc-inducible promoter. Similar uptake patterns were observed for both ntcp and oatp1, except that unconjugated hyodeoxycholate was a substrate of oatp1 but not ntcp. Conjugated bile acids were transported better than nonconjugated bile acids, and the configuration of the hydroxyl groups (alpha or beta) had little influence on uptake. Although cholic and 23 norcholic acids were transported by ntcp and oatp1, other unconjugated bile acids (chenodeoxycholic, ursodeoxycholic) were not. In contrast to ntcp, oatp1-mediated uptake of the trihydroxy bile acids taurocholate and glycocholate was four- to eightfold below that of the corresponding dihydroxy conjugates. Ntcp mediated high affinity, sodium-dependent transport of [35S]sulfobromophthalein with a Km similar to that of oatp1-mediated transport of [35S]sulfobromophthalein (Km = 3.7 vs. 3.3 muM, respectively). In addition, for both transporters, uptake of sulfobromophthalein and taurocholic acid showed mutual competitive inhibition. These results indicate that the substrate specificity of ntcp is considerably broader than previously suspected and caution the extrapolation of transport data obtained in vitro to physiological function in vivo.     

Gliadin-specific type 1 regulatory T cells from the intestinal mucosa of treated celiac patients inhibit pathogenic T cells

Celiac disease (CD) results from a permanent intolerance to dietary gluten and is due to a massive T cell-mediated immune response to gliadin, the main component of gluten. In this disease, the regulation of immune responses to dietary gliadin is altered. Herein, we investigated whether IL-10 could modulate anti-gliadin immune responses and whether gliadin-specific type 1 regulatory T (Tr1) cells could be isolated from the intestinal mucosa of CD patients in remission. Short-term T cell lines were generated from jejunal biopsies, either freshly processed or cultured ex vivo with gliadin in the presence or absence of IL-10. Ex vivo stimulation of CD biopsies with gliadin in the presence of IL-10 resulted in suppression of Ag-specific proliferation and cytokine production, indicating that pathogenic T cells are susceptible to IL-10-mediated immune regulation. T cell clones generated from intestinal T cell lines were tested for gliadin specificity by cytokine production and proliferative responses. The majority of gliadin-specific T cell clones had a Th0 cytokine production profile with secretion of IL-2, IL-4, IFN-gamma, and IL-10 and proliferated in response to gliadin. Tr1 cell clones were also isolated. These Tr1 cells were anergic, restricted by DQ2 (a CD-associated HLA), and produced IL-10 and IFN-gamma, but little or no IL-2 or IL-4 upon activation with gliadin or polyclonal stimuli. Importantly, gliadin-specific Tr1 cell clones suppressed proliferation of pathogenic Th0 cells. In conclusion, dietary Ag-specific Tr1 cells are present in the human intestinal mucosa, and strategies to boost their numbers and/or function may offer new therapeutic opportunities to restore gut homeostasis.     

Modulation of human mammary cell sensitivity to paclitaxel by new quinoline sulfonamides.

Sulfonamide derivatives of chloroquine and primaquine were synthesised and evaluated against both paclitaxel-sensitive and paclitaxel-resistant mammarian cancer cell lines. All derivatives exhibited at least 96% MDR reversal activity when co-administered with paclitaxel at 5 microM. The best compound, a chloroquine derivative, exhibited 99% MDR reversal activity when co-administered with paclitaxel at 1 microM. Molecular modelling studies reveal that these derivatives share a common pharmacophore with taxane MDR reversal agents.