New piperidine derivative DTPEP acts as dual-acting anti-breast cancer agent by targeting ERα and downregulating PI3K/Akt-PKCα leading to caspase-dependent apoptosis.

OBJECTIVES: In our ongoing studies to develop ER targeting agents, we screened for dual-acting molecules with a hypothesis that a single molecule can also target both ER positive and negative groups of breast cancer. MATERIALS AND METHODS: 1-(2-(4-(Dibenzo[b,f]thiepin-10-yl)phenoxy)ethyl)piperidine (DTPEP) was synthesized and screened in both MCF-7 (ER+ve) and MDA-MB-231 (ER-ve) cells. Assays for analysis of cell cycle, ROS, apoptosis and MMP loss were carried out using flow cytometry. Its target was investigated using western blot, transactivation assay and RT-PCR. In vivo efficacy of DTPEP was validated in LA-7 syngeneic rat mammary tumour model. RESULTS: Here, we report identification of dual-acting molecule DTPEP that downregualtes PI3K/Akt and PKCα expression, induces ROS and ROS-dependent apoptosis, loss of mitochondrial membrane potential, induces expression of caspase indicative of both intrinsic and extrinsic apoptosis in MCF-7 and MDA-MB-231 cells. In MCF-7 cells, DTPEP downregulates ERα expression and activation. In MDA-MB-231 cells, primary cellular target of DTPEP is not clearly known, but it downregualtes PI3K/Akt and PKCα expression. In vivo study showed regression of LA-7 syngeneic mammary tumour in SD rat. CONCLUSIONS: We identified a new dual-acting anti-breast cancer molecules as a proof of concept which is capable of targeting both ER-positive and ER-negative breast cancer.

Synthesis of targeted dibenzo[b,f]thiepines and dibenzo[b,f]oxepines as potential lead molecules with promising anti-breast cancer activity.

A targeted library of substituted dibenzo[b,f]thiepines and dibenzo[b,f]oxepines (prototypes I, II and III), and structurally analogous to tamoxifen have been synthesized as a new class of anti-breast cancer agents. All the prototype molecules exhibited potential antiproliferative activity against ER + ve and ER-ve breast cancer cell lines. Dibenzo[b,f]thiepine prototypes were found to be more active. Of all the compound tested, 14b exhibited potent in-vitro antiproliferative activity at 1.33 µM and 5 µM concentration in MCF-7 and MDA-MB-231 cell lines and was devoid of any cytotoxicity in normal HEK cells even at 50 µM. Cell cycle analysis showed that the compound 14b inhibited cell proliferation due to G0/G1 arrest in MCF-7 cells. Annexin-V FITC and PI staining experiments confirmed that the cell inhibition was primarily due to apoptosis and not by necrosis, which was also supported by LDH release assay experiment. Molecular docking studies showed better binding interaction of the new dibenzo[b,f]thiepine analogue 14b with the estrogen receptor (ER) as compared to 4-hydroxy-tamoxifen and this enhanced binding might be responsible for its estrogen antagonistic activity that induces cell cycle arrest, apoptosis and inhibition of breast cancer cells.

Inhibitory effect of 2-(piperidinoethoxyphenyl)-3-(4-hydroxyphenyl)-2H-benzo(b)pyran (K-1) on human primary endometrial hyperplasial cells mediated via combined suppression of Wnt/ß-catenin signaling and PI3K/Akt survival pathway.

Endometrial hyperplasia is a precursor to the most common gynecologic cancer diagnosed in women. Apart from estrogenic induction, aberrant activation of the Wnt/ß-catenin signal is well known to correlate with endometrial hyperplasia and its carcinoma. The benzopyran compound 2-(piperidinoethoxyphenyl)-3-(4-hydroxyphenyl)-2H-benzo (b) pyran(K-1), a potent antiestrogenic agent, has been shown to have apoptosis-inducing activity in rat uterine hyperplasia. The current study was undertaken to explore the effect of the benzopyran compound K-1 on growth and Wnt signaling in human endometrial hyperplasial cells. Primary culture of atypical endometrial hyperplasial cells was characterized by the epithelial cell marker cytokeratin-7. Results revealed that compound K-1 reduced the viability of primary endometrial hyperplasial cells and expression of ERα, PR, PCNA, Wnt7a, FZD6, pGsk3ß and ß-catenin without affecting the growth of the primary culture of normal endometrial cells. The ß-catenin target genes CyclinD1 and c-myc were also found to be reduced, whereas the expression of axin2 and Wnt/ß-catenin signaling inhibitor Dkk-1 was found to be upregulated, which caused the reduced interaction of Wnt7a and FZD6. Nuclear accumulation of ß-catenin was found to be decreased by compound K-1. K-1 also suppressed the pPI3K/pAkt survival pathway and induced the cleavage of caspases and PARP, thus subsequently causing the apoptosis of endometrial hyperplasial cells. In conclusion, compound K-1 suppressed the growth of human primary endometrial hyperplasial cells through discontinued Wnt/ß-catenin signaling and induced apoptosis via inhibiting the PI3K/Akt survival pathway.

The anti-proliferative effect of 2-[piperidinoethoxyphenyl]-3-[4-hydroxyphenyl]-2H-benzo(b) pyran is potentiated via induction of estrogen receptor beta and p21 in human endometrial adenocarcinoma cells.

In an effort to develop novel therapeutic agents for endometrial cancer, benzopyran derivatives synthesized at our institute display significant inhibitory activity on cellular growth in uterine cancer cells. The current study was undertaken to demonstrate and explore the estrogen receptor (ER) subtype mediated mechanism of action of benzopyran derivative 2-[piperidinoethoxyphenyl]-3-[4-hydroxyphenyl]-2H-benzo(b) pyran (K-1) in human endometrial cancer cells. K-1 competitively inhibited the estradiol binding to human ERα and ERß and showed growth inhibitory activity in human endometrial Ishikawa, HEC1B and primary endometrial adenocarcinoma cells. Transient transactivation assays carried out in COS-1 cells have demonstrated the diminished ERα-ERE mediated- and induced the ERß-ERE mediated-transactivation triggered by compound. It also induced ER-mediated transactivation of the cyclin-dependent kinase inhibitor (CDKI) p21(WAF-1) in both COS-1 cells and in Ishikawa cells. ERß inducing effects of compound were blocked by ICI182,780. In endometrial adenocarcinoma cells, it induced ERß and p21 expression significantly whereas the expression of fos, jun and ERα were significantly reduced. In addition, compound promoted ERα-ß heterodimerization as observed in Ishikawa cells. These results demonstrate that the benzopyran compound suppressed the cellular growth via ERß agonism, induction of p21 and via promoting the ERα-ß heterodimerization, in addition to its antagonistic effects exerted on ERα, in human endometrial cancer cells. The study suggests that the dual action of benzopyran molecule may be of significant therapeutic value in ERα/ß-positive cases of endometrial cancer.

Anti-tumorigenic action of 2-[piperidinoethoxyphenyl]-3-[4-hydroxyphenyl]-2H-benzo(b)pyran: evidence for involvement of GPR30/EGFR signaling pathway.

OBJECTIVE: The aim of the present study was to investigate the effect of non-steroidal, pure antiestrogenic benzopyran derivative i.e., 2-[piperidinoethoxyphenyl]-3-[4-hydroxyphenyl]-2H-benzo(b)pyran (K-1) on the growth of human endometrial cancer cells in vivo and in vitro and to elucidate its mechanism of action. METHODS: Cell proliferation was assayed by measuring the incorporation of 5'-bromo-2'-deoxyuridine in Ishikawa and primary endometrial cancer cells. The expression of proliferation and apoptotic markers was analyzed by immunoblotting. The effect of K-1 on GPR30-regulated proteins was analyzed by ELISA and by immunoblotting. Nude mice bearing subcutaneous implanted-Ishikawa tumors, were treated for 14days with K-1 (200µg/kg body weight/day/orally). The proliferation markers, GPR30-regulated proteins and apoptotic markers were analyzed by immunoblotting in tumor xenograft. The apoptotic effect of compound K-1 was determined by TUNEL assay. RESULTS: Compound K-1 inhibited proliferation of endometrial adenocarcinoma cells and decreased the expression of proliferation markers. It caused apoptosis by increasing the expression of apoptotic markers (NOXA, PUMAα) and reducing the expression of p-CREB and BclxL. Compound interfered with GPR30-regulated-EGFR activation, decreased p-ERK, p-c-jun, c-fos, cyclinD1 and c-myc expression. Treatment of tumor-bearing mice with K-1 resulted in a significant decrease in tumor volume and weight. Decreased expression of p-ERK and its downstream molecules and increased expression of apoptotic markers were observed in tumor in K-1 treated animals. CONCLUSION: Findings suggest the potent inhibitory effect of compound K-1 on endometrial cancer cellular growth (in-vitro) and on tumor size (in-vivo) which is mediated at least, in part, by interference with GPR30-signaling.

2,3-Diaryl-2H-1-benzopyran derivatives interfere with classical and non-classical estrogen receptor signaling pathways, inhibit Akt activation and induce apoptosis in human endometrial cancer cells.

OBJECTIVES: The present study was undertaken to explore the mechanism of anti-proliferative action of benzopyran compound D1 (2-[piperidinoethoxyphenyl]-3-phenyl-2H-benzopyran) and its hydroxy-(D2) and methoxy-(D3) derivatives in Ishikawa and human primary endometrial adenocarcinoma cells. METHODS: Transcriptional activation assays were performed using luciferase reporter system and cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The stage of cell cycle was determined by flow-cytometry and real time analysis of cyclinE1 and cdc2 genes. The apoptotic effects were measured by AnnexinV/PI staining and TUNEL. The expression of PCNA, cyclinD1, pAkt, XIAP, cleaved caspase-9, -3, PARP, Bax and Bcl2 were determined by immunoblotting. The caspase-3 activity and mitochondrial membrane potential were measured by colorimetric assay. RESULTS: All three compounds inhibited E(2)-induced ERE- and AP-1-mediated transactivation and proliferation in endometrial adenocarcinoma cells dose-dependently. Compound D1 caused the arrest of cells in the G(2) phase while D2 and D3 caused arrest in G(1) phase of the cell cycle. All compounds interfered with Akt activation, decreased XIAP expression leading to an increased cleavage of caspase-9, -3, PARP, increased Bax/Bcl2 ratio and caspase-3 activity. CONCLUSION: Findings suggest that benzopyran derivatives inhibit cellular proliferation via modulating ER-dependent classical and non-classical signaling mechanisms, interfere with Akt activation and induce apoptosis via intrinsic pathway in endometrial adenocarcinoma cells.

Synthesis and biological evaluation of 3,4,6-triaryl-2-pyranones as a potential new class of anti-breast cancer agents.

A series of 3,4,6-triaryl-2-pyranones, new class of anti-breast cancer agents, have been synthesized as a structural variants of cyclic triphenylethylenes by replacing the fused benzene ring with pendant phenyl ring to mimic the phenolic A ring of estradiol. Nine of these newly synthesized pyranones exhibited significant anti-proliferative activity in both ER+ve and ER-ve breast cancer cell lines. Four active non-cytotoxic compounds 5c, 5d, 5g and 5h showed specific and selective cytotoxicity and two compounds 5d and 5h induced significant DNA fragmentation in both MCF-7 and MDA-MB-231 cell lines. Based on RBA studies, the molecules probably act in an ER-independent mechanism. The involved pathway was observed as caspase-dependant apoptosis in MCF-7 cells. However, the particular caspases involved and the possible cellular target through which this series of compounds mediate cell death are not known.

Lack of genetic association of promoter and structural variants of mannan-binding lectin (MBL2) gene with susceptibility to generalized vitiligo.

BACKGROUND: Vitiligo is a common depigmenting disorder resulting from the loss of functional melanocytes in the skin. It is hypothesized to be of autoimmune origin. Mannan-binding lectin (MBL) plays an important role in innate immunity. It helps in the clearance of apoptotic cells and in complement activation. Genetic variability due to structural and promoter polymorphisms in the MBL2 gene has been reported to be associated with increased risk for several autoimmune diseases including vitiligo. OBJECTIVES: The aim of this study was to explore whether MBL2 structural and promoter polymorphisms are associated with generalized vitiligo in Gujarat where the prevalence of vitiligo is alarmingly high. MATERIALS AND METHODS: We undertook a case-control study to investigate the association of MBL2 gene exon 1 polymorphisms - codon 52, codon 54 and codon 57 as well as promoter -221 polymorphism in 92 patients with generalized vitiligo and 94 unaffected age-matched controls by polymerase chain reaction-heteroduplex analysis. RESULTS: The genotype and allele frequencies of MBL2 structural and promoter polymorphisms did not differ significantly between the control and patient population (P-values: P < 0.019 for codon 52, P < 0.373 for codon 54, P < 0.855 for codon 57 and P < 0.889 for -221 promoter polymorphisms) after Bonferroni's correction for multiple testing, which suggests that there is no association of MBL2 structural and promoter polymorphisms with generalized vitiligo. CONCLUSIONS: Our results suggest that the well-documented structural and promoter polymorphisms of the MBL2 gene may not be associated with generalized vitiligo in the Gujarat population.

Synthesis and biological evaluation of indolyl bisphosphonates as anti-bone resorptive and anti-leishmanial agents.

A series of indole conjugated bisphosphonate derivatives have been synthesized and evaluated for their in vitro anti-bone resorptive activity using bone marrow osteoclast culture. Two bisphosphonates 23 and 24 significantly inhibited osteoclastogenesis, 23 showed inhibition at 10 and 100 pM which was lower than the concentration of standard drug alendronate, and 24 inhibited osteoclastogenesis at 100 nM which was comparable to alendronate. Two other compounds 13 and 14 also showed inhibition comparable to alendronate, but were cytotoxic in the osteoblast cells. The two active bisphosphonates 23 and 24 induced significant osteoclast apoptosis at concentrations 100 nM for compound 24 and at 10 pM for compound 23 compared to alendronate. In vivo effect of active bisphosphonates 23 and 24 resulted in osteoclastogenesis of bone marrow cells (BMCs) to almost 40-50% (23 showing 8.4% decrease and 24 showing 9.0%) compared to 16.5% of the ovariectomized group. Further, screening of anti-leishmanial activity, four compounds 24-25 and 27-28 showed more than 80% inhibition against both the promastigote and amastigote stages of the Leishmania parasite.

Synthesis and structure guided evaluation of estrogen agonist and antagonist activities of some new tetrazolyl indole derivatives.

Several regioisomeric tetrazolyl indole derivatives with structurally modified alkyl substituents at the tetracyclic indole nitrogen containing N-ethyl amino tetrazole moiety have been synthesized and screened for their ER binding affinity, agonist (estrogenic), antagonist (antiestrogenic) and anti-implantation activities. N-2 regioisomers were found to be moderately antagonists and one compound showed 100% contraceptive efficacy at 10 mg/kg dose. Molecular docking studies carried out in comparison to estradiol and raloxifene showed different binding modes of the two regioisomers to the binding site.

Leishmania donovani: identification of stimulatory soluble antigenic proteins using cured human and hamster lymphocytes for their prophylactic potential against visceral leishmaniasis.

Most of the studies for the identification of prophylactic antigens that elicit T cell responses were concentrated on membrane proteins of Leishmania donovani. This study was taken up to assess L. donovani soluble promastigote antigens for their ability to stimulate proliferation of peripheral blood mononuclear cells (PBMCs) from cured visceral leishmaniasis (VL) patients, endemic and non-endemic controls and lymphocytes/peritoneal macrophages of cured hamsters. The soluble protein was subjected to sequential precipitation with saturated ammonium sulphate (20%, 40%, 60% and 80%), of which largely 80% fractioned protein showed significant cellular responses in cured patients and hamsters. This fraction was further fractionated into five sub fractions by preparative SDS-PAGE and subjected to re-evaluation for their ability to induce cellular responses. Out of these, only F2 sub fraction belonging to the MW of 97.4-68 kDa stimulated remarkable lymphoproliferative and IFN-gamma responses in cured VL patients and in endemic controls. Similarly, significant lymphoproliferative responses and nitric oxide production were also noticed in cured Leishmania infected animals indicating an element of uniformity in responses between hamster and human. F2 sub fraction, when evaluated for its prophylactic efficacy with BCG against L. donovani challenge in hamster exhibited significant parasite inhibition in spleen (71.1%; p<0.001) and liver (68.2%; p<0.001) as compared to their unvaccinated counterpart. The vaccinated animals showed significant lymphoproliferative response and nitric oxide production but leishmania specific IgG level were suppressed. The results indicate the presence of immunostimulatory and protective molecules in F2 sub fraction which may further be exploited for the development of a vaccine against VL, hitherto an unrealized goal.

Development of a fluorescence assay for the detection of L-ficolin-MASP in serum or purified samples.

A fluorescence assay for the detection of L-ficolin-MASP in human serum or purified sample was developed by measuring the cleavage of fluorescent amide substrate by L-ficolin associated MASPs bound to the lipoteichoic acid (LTA). LTA (Staphylococcus aureus DSM 20233) was coated on NuncMaxisorp microtiter plates and serum or purified sample incubated overnight at 4 degrees C to allow the L-ficolin-MASP to bind LTA. Assay conditions for binding and complete cleavage of fluorescent amide substrate were standardized. The optimum temperature, incubation time and molarity of NaCl for LTA-ficolin binding were found to be 4 degrees C for 6 h at 1 M NaCl concentration. The optimum incubation time and pH for complete cleavage of fluorescent amide substrate by LTA bound L-ficolin associated MASP were found to be 2 h at pH 8.5. LTA-ficolin binding was found to be highly specific and was inhibited completely by LTA but not with mannose. A calibration curve was prepared by using the purified ficolin-MASP complex (1 to 12 mug/ml) and could be used to find concentration of ficolin-MASP complex in normal human serum.

Collectins and host defence.

The collectins are a small family of soluble oligomeric proteins containing collagenous regions and C-type lectin domains. They are related in structure and function to complement protein C1q, and to H-, L- and M-ficolins. In humans, the collectins mannose-binding lectin (MBL) and surfactant proteins A and D (SP-A, SP-D) have important roles in innate immunity. MBL occurs mainly in blood plasma and in the upper respiratory tract. It binds to neutral sugar arrays on microorganisms and acts as an opsonin either directly (by binding to cell-surface calreticulin) or indirectly by activating complement. MBL circulates in complex with any of three proteases, named MBL-associated serine proteases (MASPs)-1, -2 and -3. MBL-MASP-2 complexes activate complement, but the role of MBL-MASP-1 and MBL-MASP-3 complexes is not yet known. MBL deficiency occurs at high frequency, and is associated with susceptibility to infection, particularly in infants. SP-A and SP-D are most abundant in the lungs, and also bind to microorganisms and inhaled particulates, mainly by lectin-sugar interactions. They do not activate complement, but act as opsonins and agglutinators, and have additional effects on cellular regulation. Mice deficient in SP-A or SP-D are susceptible to lung infections, and SP-D-deficient mice develop an emphysema-like condition.

Synthesis and biological activities of some new dibenzopyranones and dibenzopyrans: search for potential oestrogen receptor agonists and antagonists.

Continuing our search for newer oestrogen agonists or antagonists and extending our work on the exploration of benzopyran related compounds, some new tricyclic molecules bridged between the active molecules of 3,4-diaryl chroman and 2,3-diaryl benzopyrans have been synthesised. Structural modifications at different positions with elements known to impart agonist or antagonist activities have been carried out to prepare the desired molecules. The target compounds were screened for their anti-osteoporotic (agonist) and anti-uterotrophic (antagonist) activities and were found to be moderately active.

Resolution, molecular structure and biological activities of the D- and L-enantiomers of potent anti-implantation agent, DL-2-[4-(2-piperidinoethoxy)phenyl]-3-phenyl-2H-1-benzopyran.

Compound 1 (DL-2-[4-(2-piperidinoethoxy)phenyl]-3-phenyl-2H-1-benzopyran, CDRI 85/287) a potent anti-estrogen and anti-implantation agent has been successfully resolved into its pure D- and L-enantiomers. Biological studies showed L-enantiomer to be the active form, exhibiting a fivefold higher receptor affinity for the rat uterine cytosolic estrogen receptor, 100% contraceptive efficacy at 1.3 mg/kg dose in single day schedule and 89% inhibition of estradiol induced increase of uterine weight at its contraceptive dose. The absolute stereochemistry determined by X-ray crystallographic analysis showed that the L-enantiomer has 2R configuration at its asymmetric centre.

Changes in membrane fluidity induced by binding of immunoglobulins to sperm Fc receptors.

Receptors for Fc region of immunoglobulins (FcR) are reported to be present on spermatozoa, and also detected in seminal plasma, however their function is still not known. Since various changes in sperm membrane architecture during maturation and passage through female genital tract are reported, experiments were conducted to study the membrane fluidity changes in sperm subsequent to ligation of surface FcR with immunoglobulin and its derivatives. This paper reports that interaction with IgG restricts rotational mobility of cell surface proteins and membrane lipids as studied by EPR spectroscopy using spin probes. Decrease in fluidity was much more pronounced in the presence of aggregated IgG due to crosslinking of sperm FcR by aggregated IgG.

Carbohydrate induced modulation of cell membrane VII. Binding of exogenous lectin increases osmofragility of erythrocytes.

Due to their multivalent binding character, lectins when added exogenously will cross-link membrane surface receptors leading to lateral molecular reorganizations in the plane of the bilayer. This study reports for the first time that agglutination of rabbit erythrocytes by lentil lectin and concanavalin A increases their osmofragility. Increase in osmofragility was detected by measuring the hemolysis of erythrocytes in hypotonic as well as in isotonic solutions. It was also found that agglutination per se does not increase osmofragility but the binding of legume lectin is essential since human Rh+ cells agglutinated by a monoclonal antibody do not exhibit hemolysis.

Carbohydrate induced modulation of cell membrane. VI. Binding of exogenous lectin induces susceptibility of erythrocytes to free radical damage: a spin label study.

The oxidation of erythrocyte membrane has been widely used as a model to study the damage of biomembranes by free radicals. Whether binding of lectin to erythrocytes has any effect on peroxidant injury has never been studied. This study reports for the first time that crosslinking of erythrocyte surface glycoprotein by an exogenous lectin significantly enhances the susceptibility to membrane damage by free radicals, as evidenced by the increase in membrane fluidity measured by EPR using spin label and the increase in the amount of oxyhemoglobin liberated due to cell lysis.

Characterization of a lectin from goat peripheral blood lymphocytes.

A D-glucose specific lectin was isolated from goat peripheral blood lymphocytes by affinity chromatography on N-acetyl D-glucosamine agarose gel. The fluorescence intensity of 4 methyl umbelliferyl D-glucose was quenched to about 62% on addition of the lectin. This lectin gave a single band corresponding to 112 kDa in SDS-PAGE irrespective of treatment with 2-mercaptoethanol. The molecular weight and the Stoke's radius of the lectin in the native conditions were found to be 114 kDa and 4.54 nm, respectively, as determined by gel filtration on Sephacryl S 500 column. The lectin was found to be a glycoprotein with 5.6% of neutral hexose content and 5.5% of sialic acid. The lectin agglutinated trypsinized rabbit erythrocytes and human type A erythrocytes. The hemagglutinating activity was dependent on the presence of divalent cations like Mn2+ and Ca2+. Optimum pH, ionic strength and temperature for rebinding of lectin to acid treated Sephadex G200 were found to be 7.5, 0.16 and 30-37 degrees C, respectively.

Carbohydrate induced modulation of cell membrane. IV: Interaction with mucin and fucoidan totally immobilizes the human platelet membrane.

P selectins and E selectins are cell adhesion molecules that mediate the interaction of platelets and endothelial cells with neutrophils and monocytes. The proposed ligands for these receptors contain the Le(x) core, sialic acid and sulfated fucose. In this paper we report that binding of sialic acid markedly restricts the mobility of membrane proteins and lipids as studied by EPR spectroscopy using spin probes. Binding of mucin and fucoidan totally restricts the mobility probably due to cross-linking of the surface lectins. Binding of these ligands also resulted in an increase in the cytoplasmic viscosity.