Synthesis and anti breast cancer activity of biphenyl based chalcones

A series of (2E,2′E)-1,1′-(3-hydroxy-5-methylbiphenyl-2,6-diyl)-bis(3-pheylprop-2-ene-1-ones (5–33) were prepared by the reaction of 1,3-diacetyl biphenyls (1–4) with different aldehydes in presence of catalytic amount of solid KOH in ethanol in excellent yields. The compounds were evaluated for anticancer activity against human breast cancer MCF-7 (estrogen responsive proliferative breast cancer model) and MDA-MB-231 (estrogen independent aggressive breast cancer model) cell lines, HeLa (cervical cancer) cell line, and human embryonic kidney (HEK-293) cells. Most of the compounds preferentially inhibited the growth of the aggressive human breast cancer cell lines, MDA-MB-231 in the range of 4.4–30 μM. The two compounds 9 and 29 proved to be better anticancer agents than the standard drug tamoxifen against the MDA-MB-231 cell lines. Mode of action of these compounds was established to be apoptosis, cell cycle arrest and loss of mitochondrial membrane potential.     

Interaction of eosinophil granule major basic protein with synthetic lipid bilayers: A mechanism for toxicity

Summary Eosinophil granule major basic protein (MBP) is a potent toxin for mammalian cells and helminths, but the mechaism of its toxicity is not known. Here we tested whether MBP toxicity is exerted through its effect on the lipid bilayer of its targets. Liposomes prepared from synthetic phospholipids were used as targets for MBP and their properties examined by fluorescence and circular dichroism (CD) spectroscopy. MBP caused a change in the temperature transition profiles of acidic liposomes (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl serine or an equimolar mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid) and induced their aggregation as shown by fluorescence resonance energy transfer experiments. The CD spectra and fluorescence characteristics of MBP itself were altered by its interaction with acidic lipids. Blue shifts in the emission maxima of the Trp, and of the dimethylaminonaphthyl moiety in acrylodan-labeled MBP, and a reduction in the effectiveness of quenching of Trp fluorescence by acrylamide were observed in the presence of acidic lipids. None of these effects were noted with zwitterionic lipids. This MBP : lipid bilayer interaction resulted in fusion and lysis of liposomes as indicated by the fluorescent indicator calcein. The results demonstrate that MBP associates with acidic lipids and that it disrupts, aggregates, fuses, and lyses liposomes prepared from such lipids. Such interaction might account for its wide range of toxicity.Abbreviations used Acrylodan 6-acryloyl-2-dimethylam-inonaphthalene - CD circular dichroism - DMPA 1,2-dimyrist-oyl-sn-glycero-3-phosphatidic acid - DMPC 1,2-dimyristoyl-sn-glycero-3-phosphocholine - DPH 1,6-diphenyl-1,3,5-hexatriene - DTT dithiothreitol - FRET fluorescence resonance energy transfer - HEPES N-2-hydroxyethyl piperazine-N-2-ethane sulfonic acid - K _sv Stern-Volmer constant - K _q bimolecular quenching coefficient - _em emission wavelength - _ex excitation wavelength - MBP major basic protein - MOPC 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine - NBD-PE N-(7-nitro-2,1,3-benzo-xadiazol-4-yl)-phosphatidylethanolamine - nMBP native major basic protein - PBS phosphate-buffered saline - POPC 1-palmit-oyl-2-oleoyl-sn-glycero-3-phosphocholine - POPS 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl serine - raMBP reduced and alkylated major basic protein - RHO-PE rhodamine-phosphatidylethanolamine - Tes N-tris[hydroxymethyl]-methyl-2-amino-ethane-sulfonic acid - Tris tris[hydroxymethyl]-amino-methane We would like to thank Dr. Predrag J.K. Ilich for assistance with initial data analysis, Dr. Salah S. Sedarous for the lifetime data and for helpful discussions, Dr. S. Yu. Venyaminov for helpful discussions, Mr. Kenneth D. Peters and Mr. Peter J. Callahan for assistance with some of the illustrations, and Ms. Jill Wagner for performing the radioimmunoassays. We would also like to thank Ms. Jill Kappers for excellent secretarial work. This work was supported in part by a Fellowship grant from the American Heart Association, Minnesota Affiliate, and by grants from the National Institutes of Health AI 09728 and from the Mayo Foundation. RIA-G is a Fellow of the American Heart Association.     

Neo-tanshinlactone inspired synthesis, in vitro evaluation of novel substituted benzocoumarin derivatives as potent anti-breast cancer agents

A small library of novel benzocoumarin derivatives based on naturally occurring neo-tanshinlactone scaffold was constructed and their antiproliferative activities against breast cancer cells MCF-7 and MDA-MB-231 were evaluated. A number of derivatives showed good anti-breast cancer activity, in some cases higher to that of the reference compound tamoxifen. In particular, benzocoumarins Bc-5, Bc-8 and Bc-9 strongly inhibited the proliferation of MCF-7 cancer cell line with the IC(50) values of 3.8, 7.9 and 6.5 μM, respectively. The compounds were capable of inducing nuclear fragmentation, cell cycle arrest and caspase dependent apoptosis in MCF-7 cell lines. In addition, these derivatives were devoid of cytotoxic effect against normal osteoblast cells. These synthetic benzocoumarins hold promises for developing safer alternative to the existing anti-breast cancer agents.     

Unlocking Synergistic Potential: Agomelatine Enhances the Chemotherapeutic Effect of Paclitaxel in Breast Cancer Cell Through MT1 Melatonin Receptors and ER-alpha Axis

This study investigates the potential of agomelatine (AGO), a synthetic melatoninergic drug, in combination with paclitaxel (PTX) for the treatment of breast cancer. The effects of AGO, PTX and melatonin (MTN) on breast cancer cell viability were investigated, focusing on the role of MT1 receptors. Cell viability and gene expression were analyzed in MCF-7 and MDA-MB-231 breast cancer cell experiments. The results show that AGO has cytotoxic effects on breast cancer cells similar to MTN. Combining AGO and MTN with PTX showed synergistic effects in MCF-7 cells. The study also reveals differences in the molecular mechanisms of breast cancer between estrogen-positive MCF-7 cells and estrogen-negative MDA-MB-231 cells. Combination with AGO and PTX affects apoptosis-associated proteins in both cell types. The findings suggest that AGO, combined with PTX, may be a promising adjuvant therapy for breast cancer and highlight the importance of MTN receptors in its mechanism of action.     

Design,Synthesis, and Biological Evaluation of Novel Amyl Ester Tethered Dihydroartemisinin-Isatin Hybrids as Potent Anti-Breast Cancer Agents

A series of novel amyl ester tethered dihydroartemisinin-isatin hybrids 4a–d and 5a–h were designed, synthesized, and evaluated as anti-breast cancer agents. The synthesized hybrids were preliminarily screened against estrogen receptor-positive (MCF-7 and MCF-7/ADR) and triple-negative (MDA-MB-231 and) breast cancer cell lines. Three hybrids 4a , d and 5e not only were more potent than artemisinin and adriamycin against drug-resistant MCF-7/ADR and MDA-MB-231/ADR breast cancer cell lines, but also displayed non-cytotoxicity towards normal MCF-10 A breast cells, and the SI values were >4.15, indicating their excellent selectivity and safety profiles. Thus, hybrids 4a , d and 5e could act as potential anti-breast cancer candidates and were worthy of further preclinical evaluations. Moreover, the structure–activity relationships which may facilitate further rational design of more effective candidates were also enriched.     

Amphotericin B-induced ion flux is markedly attenuated in phosphatidylglycerol membrane as evidenced by a newly devised fluorometric method

The effect of phospholipid head group on the membrane-permeabilizing activity of amphotericin B (AmB) was examined using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) liposomes. The activity of AmB was evaluated as K⁺ influx measured as pH change inside liposomes by fluorescent measurements of 2′,7′-bis(carboxyethyl)-4 or 5-carboxyfluorescein (BCECF). AmB showed prominent permeability in POPC liposomes, whereas hardly inducing ion flux in POPG membrane. POPC added to POPG liposomes as a minor constituent markedly enhanced membrane permeability, indicating the importance of a phosphonocholine group of PC for the drug’s activity.     

Comparative study between synthetic and phospholipids of natural origin: effect of phospholipid selection on the behavior of a topical liposomal dosage form incorporating terbinafine

Selection of excipients used is a critical step in the design of a pharmaceutical dosage form as it affects its behavior upon application, as during storage. The purpose of the present study is to evaluate and compare the behavior of six liposomal formulations intended for topical application composed of two widely used phospholipids 1,2-diacyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine with and without incorporation of cholesterol. Liposomal hydrogels made of hydroxyethylcellulose 3% and incorporating the anti-fungal agent terbinafine hydrochloride (E)-N-(6,6-dimethyl-2-hepten-4-inyl)-N-methyl-1-naphthalene-methanamine (-hydrochloride) were prepared, their viscosity was measured and in vitro drug release was studied. Moreover, physical stability and drug retention during storage at two different temperatures (2–8?°C and RT) were examined over time. The results showed differences in the behavior between the two phospholipids while incorporation of cholesterol at the studied concentrations was found to be of minor importance. Drug release was found to be favorable from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomal hydrogels and drug retention was found to be higher at lower storage temperature for all batches. Original physicochemical properties of all batches were found to be retained at least for a week.     

Synergistic enhancement of apoptosis by coralyne and paclitaxel in combination on MDA-MB-231 a triple-negative breast cancer cell line

Triple-negative breast cancer (TNBC) is the most outrageous subtype of breast cancer. Emphasizing the urge of new approach in cancer therapy, combinational drug therapy may be proven as an effective strategy. In our previous study, we reported that coralyne (COR) with paclitaxel (PTX) efficiently decreases the proliferation of MDA-MB-231 compared with MCF-7 cell line. Thus, we studied the effect of COR and PTX in combination on apoptosis of MDA-MB-231 cell line. In silico results demonstrated that COR intercalates DNA at a minor groove. In vitro approaches revealed that in combination (COR and PTX) increases the efficacy of apoptosis in MDA-MB-231 cell line by a significant increase in G1/S phase arrest, DNA fragmentation, and change in mitochondria membrane potential. The expression of ATM and ATR a serine/threonine-protein kinase, ataxia telangiectasia and Rad3-related protein were depleted with an increase in time from 24 to 48 hours in concurrent with increased levels of γH2AX indicating that DNA damage routes cells to enter apoptosis. This was confirmed by high levels of caspase-3 and cytochrome c. Also, the decrease in the expression levels of matrix metalloproteinase-9 confirmed the antimetastatic efficacy of COR + PTX. The present study indicates that the synergistic effect of COR and PTX can enhance apoptosis in MDA-MB-231 cell line and may be proven as a potential anticancer therapy against TNBC.     

Label-free,chronological and selective detection of aggregation and fibrillization of amyloid β protein in serum by microcantilever sensor immobilizing cholesterol-incorporated liposome

To facilitate the early diagnosis of Alzheimer's disease and mild cognitive impairment patients, we developed a cantilever-based microsensor that immobilized liposomes of various phospholipids to detect a trace amount of amyloid β (Aβ) protein, and investigated its aggregation and fibrillization on model cell membranes in human serum. Three species of liposomes composed of different phospholipids of 1,2-dipalmtoyl-sn-glycero-3-phosphocholine (DPPC), DPPC/phosphatidyl ethanolamine and 1,2-dipalmitoyl-sn-glycero-3-phosphorylglycerol having varied hydrophilic groups were applied, which showed different chronological interactions with Aβ(1–40) protein and varied sensitivities of the cantilever sensor, depending on their specific electrostatic charged conditions, hydrophilicity, and membrane fluidity. 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) having short hydrophobic carbon chains confirmed to show a large interaction with Aβ(1–40) and a high sensitivity. Furthermore, the incorporation of cholesterol into DMPC was effective to selectively detect Aβ(1–40) in human serum, which effect was also checked by quartz crystal microbalance. Finally, Aβ detection of 100-pM order was expected selectively in the serum by using the developed biosensor.     

Carboxylated phytosterol derivative-introduced liposomes for skin environment-responsive transdermal drug delivery system

Abstract

Transdermal drug delivery systems are a key technology for skin-related diseases and for cosmetics development. The delivery of active ingredients to an appropriate site or target cells can greatly improve the efficacy of medical and cosmetic agents. For this study, liposome-based transdermal delivery systems were developed using pH-responsive phytosterol derivatives as liposome components. Succinylated phytosterol (Suc-PS) and 2-carboxy-cyclohexane-1-carboxylated phytosterol (CHex-PS) were synthesized by esterification of hydroxy groups of phytosterol. Modification of phytosterol derivatives on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes was confirmed by negatively zeta potentials at alkaline pH and the change of zeta potentials with decreasing pH. In response to acidic pH and temperatures higher than body temperature, Suc-PS-containing and CHex-PS-containing liposomes exhibited content release at intracellular acidic compartments of the melanocytes at the basement membrane of the skin. Phytosterol-derivative-containing liposomes were taken up by murine melanoma-derived B16-F10 cells. These liposomes delivered their contents into endosomes and cytosol of B16-F10 cells. Furthermore, phytosterol-derivative-containing liposomes penetrated the 3?D skin models and reached the basement membrane. Results show that pH-responsive phytosterol-derivative-containing DMPC liposomes are promising for use in transdermal medical or cosmetic agent delivery to melanocytes.     

Pattern of serum immunoreactivity against breast cancer cell lysates may predict severity of disease in breast cancer patients

Humoral tumor-specific immunity has been investigated as a potential tool to identify tumor-associated antigens and evaluate cancer diagnosis and prognosis. Using SDS-PAGE and western blotting techniques we investigated the humoral immune response against tumor cell antigens in 36 breast cancer patients, 17 node-positive (NP) and 19 node-negative (NN). As a source of antigens, we prepared protein lysates from four breast cancer cell lines (AU565, BT474, MCF-7 and MDA-MB-231) which in vitro exhibit different features of invasion, estrogen receptor/progesterone receptor status and HER2/neu expression thereby potentially representing mild to aggressive forms of clinical disease. A higher number of immunocomplexes Ag–Ab were formed when serum from NN patients was immunoreacted against lysates from AU565 and MCF-7 in comparison to serum from NP patients (P < 0.01). BT474 cells were not a good antigenic source. MDA-MB-231 cells could not significantly discriminate between NN and NP patients since both groups showed higher amounts of reactivity against the lysate. However, comparative analysis of protein preparations purified from MCF-7 and MDA-MB-231 cells and immunodetected concomitantly with the same serum samples showed that serum from patients with cancers with worse prognosis (stage, nodality, HER2/neu and hormonal status) reacted more intensely to proteins purified from the relatively more invasive cell line MDA-MB-231 compared to MCF-7. These findings suggest that the study of serum antibody reactivity to antigens purified from breast cancer cell lines with different invasive properties should be further investigated for its potential in providing beneficial prognostic information in breast cancer. Supported by the United States Military Cancer Institute and the Department of Clinical Investigation at Walter Reed Army Medical Center. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of the Army or the Department of Defense.     

Formation of transition metal-doxorubicin complexes inside liposomes

Doxorubicin complexation with the transition metal manganese (Mn²⁺) has been characterized, differentiating between the formation of a doxorubicin-metal complex and doxorubicin fibrous-bundle aggregates typically generated following ion gradient-based loading procedures that rely on liposome encapsulated citrate or sulfate salts. The physical and chemical characteristics of the encapsulated drug were assessed using cryo-electron microscopy, circular dichroism (CD) and absorbance spectrophotometric analysis. In addition, in vitro and in vivo drug loading and release characteristics of the liposomal formulations were investigated. Finally, the internal pH after drug loading was measured with the aim of linking formation of the Mn²⁺ complex to the presence or absence of a transmembrane pH gradient. Doxorubicin was encapsulated into either 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/cholesterol (Chol) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/Chol liposomes, where the entrapped salts were citrate, MnSO₄ or MnCl₂. In response to a pH gradient or a Mn²⁺ ion gradient, doxorubicin accumulated inside to achieve a drug-to-lipid ratio of approximately 0.2:1 (wt/wt). Absorbance and CD spectra of doxorubicin in the presence of Mn²⁺ suggested that there are two distinct structures captured within the liposomes. In the absence of added ionophore A23187, drug loading is initiated on the basis of an established pH gradient; however, efficient drug uptake is not dependent on maintenance of the pH gradient. Drug release from DMPC/Chol is comparable regardless of whether doxorubicin is entrapped as a citrate-based aggregate or a Mn²⁺ complex. However, in vivo drug release from DSPC/Chol liposomes indicate less than 5% or greater than 50% drug loss over a 24-h time course when the drug was encapsulated as an aggregate or a Mn²⁺ complex, respectively. These studies define a method for entrapping drugs possessing coordination sites capable of complexing transition metals and suggest that drug release is dependent on lipid composition, internal pH, as well as the nature of the crystalline precipitate, which forms following encapsulation.     

l-Leucine transport in human breast cancer cells (MCF-7 and MDA-MB-231): kinetics, regulation by estrogen and molecular identity of the transporter

The transport of l-leucine by two human breast cancer cell lines has been examined. l-Leucine uptake by MDA-MB-231 and MCF-7 cells was via a BCH-sensitive, Na⁺-independent pathway. l-Leucine uptake by both cell lines was inhibited by l-alanine, d-leucine and to a lesser extent by l-lysine but not by l-proline. Estrogen (17β-estradiol) stimulated l-leucine uptake by MCF-7 but not by MDA-MB-231 cells. l-Leucine efflux from MDA-MB-231 and MCF-7 cells was trans-stimulated by BCH in a dose-dependent fashion. The effect of external BCH on l-leucine efflux from both cell types was almost abolished by reducing the temperature from 37 to 4 °C. There was, however, a significant efflux of l-leucine under zero-trans conditions which was also temperature-sensitive. l-Glutamine, l-leucine, d-leucine, l-alanine, AIB and l-lysine all trans-stimulated l-leucine release from MDA-MB-231 and MCF-7 cells. In contrast, d-alanine and l-proline had little or no effect. The anti-cancer agent melphalan inhibited l-leucine uptake by MDA-MB-231 cells but had no effect on l-leucine efflux. Quantitative real-time PCR revealed that LAT1 mRNA was approximately 200 times more abundant than LAT2 mRNA in MCF-7 cells and confirmed that MDA-MB-231 cells express LAT1 but not LAT2 mRNA. LAT1 mRNA levels were higher in MCF-7 cells than in MDA-MB-231 cells. Furthermore, LAT1 mRNA was more abundant than CD98hc mRNA in both MDA-MB-231 and MCF-7 cells. The results suggest that system L is the major transporter for l-leucine in both MDA-MB-231 and MCF-7 cells. It is possible that LAT1 may be the major molecular correlate of system L in both cell types. However, not all of the properties of system L reflected those of LAT1/LAT2/CD98hc.     

Interaction of exogenous hypochlorite or hypochlorite produced by myeloperoxidase + H2O2 + Cl- system with unsaturated phosphatidylcholines

The interaction between unsaturated phosphatidylcholines and either exogenous or endogenous (produced by the enzyme system involving myeloperoxidase (MPO), H₂ O₂ ,and Cl^–) hypochlorite was studied in multilayer liposomes containing oleic, linoleic, and arachidonic acid residues using MALDI TOF mass spectrometry. At pH 7.4, hypochlorite reacts with the double bond of the oleic acid residue in 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine producing oleic acid chlorohydrin as the main product. Minor amounts of glycols and epoxides were also detected. The main products of the reaction of hypochlorite with 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine were mono and di chlorohydrins of linoleic acid. The signals of monoglycol, epoxide, and glycol or epoxide containing monochlorohydrin derivatives were also present in the mass spectrum. The main products of the reaction of hypochlorite with 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine were lysophosphatidylcholine (1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine) and mono-, di-, and trichlorohydrin. Monoglycol and its derivatives containing one or two chlorohydrin groups were also detected. Along with those, carbonyl compounds (aldehyde and acid) formed as a result of double bond breakage in fifth position of arachidonate were detected. Monochlorohydrin was also found when liposomes comprising 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine were incubated in the presence of enzymatic mixture, MPO +H₂ O₂ +Cl^–,at pH 6.0. In the absence of the enzyme or either of its substrates (H₂ O₂ or Cl^–) or in the presence of the MPO inhibitor (sodium azide) or hypochlorite scavengers (taurine or methionine), monochlorohydrin formation was not observed. These data confirm the suggestion that just the hypochlorite generated in MPO catalysis provides for chlorohydrin formation. Thus, the use of MALDI TOF mass spectrometry has shown, along with chlorohydrins, glycols and epoxides as the products of hypochlorite interaction with unsaturated phosphatidylcholines at physiological pH. It was first determined that hypochlorite breaks double bonds in polyunsaturated phosphatidylcholine and also causes lysophosphatidylcholine formation.     

L-leucine transport in human breast cancer cells (MCF-7 and MDA-MB-231): kinetics, regulation by estrogen and molecular identity of the transporter

The transport of L-leucine by two human breast cancer cell lines has been examined. L-leucine uptake by MDA-MB-231 and MCF-7 cells was via a BCH-sensitive, Na+ -independent pathway. L-leucine uptake by both cell lines was inhibited by L-alanine, D-leucine and to a lesser extent by L-lysine but not by L-proline. Estrogen (17beta-estradiol) stimulated L-leucine uptake by MCF-7 but not by MDA-MB-231 cells. L-leucine efflux from MDA-MB-231 and MCF-7 cells was trans-stimulated by BCH in a dose-dependent fashion. The effect of external BCH on L-leucine efflux from both cell types was almost abolished by reducing the temperature from 37 to 4 degrees C. There was, however, a significant efflux of L-leucine under zero-trans conditions which was also temperature-sensitive. L-glutamine, L-leucine, D-leucine, L-alanine, AIB and L-lysine all trans-stimulated L-leucine release from MDA-MB-231 and MCF-7 cells. In contrast, D-alanine and L-proline had little or no effect. The anti-cancer agent melphalan inhibited L-leucine uptake by MDA-MB-231 cells but had no effect on L-leucine efflux. Quantitative real-time PCR revealed that LAT1 mRNA was approximately 200 times more abundant than LAT2 mRNA in MCF-7 cells and confirmed that MDA-MB-231 cells express LAT1 but not LAT2 mRNA. LAT1 mRNA levels were higher in MCF-7 cells than in MDA-MB-231 cells. Furthermore, LAT1 mRNA was more abundant than CD98hc mRNA in both MDA-MB-231 and MCF-7 cells. The results suggest that system L is the major transporter for L-leucine in both MDA-MB-231 and MCF-7 cells. It is possible that LAT1 may be the major molecular correlate of system L in both cell types. However, not all of the properties of system L reflected those of LAT1/LAT2/CD98hc.     

Molecular Modification of Metadherin/MTDH Impacts the Sensitivity of Breast Cancer to Doxorubicin

BackgroundBreast cancer is a leading cause of death in women and with an increasing worldwide incidence. Doxorubicin, as a first-line anthracycline-based drug is conventional used on breast cancer clinical chemotherapy. However, the drug resistances limited the curative effect of the doxorubicin therapy in breast cancer patients, but the molecular mechanism determinants of breast cancer resistance to doxorubicin chemotherapy are not fully understood. In order to explore the association between metadherin (MTDH) and doxorubicin sensitivity, the differential expressions of MTDH in breast cancer cell lines and the sensitivity to doxorubicin of breast cancer cell lines were investigated.MethodsThe mRNA and protein expression of MTDH were determined by real-time PCR and Western blot in breast cancer cells such as MDA-MB-231, MCF-7, MDA-MB-435S, MCF-7/ADR cells. Once MTDH gene was knocked down by siRNA in MCF-7/ADR cells and overexpressed by MTDH plasmid transfection in MDA-MB-231 cells, the cell growth and therapeutic sensitivity of doxorubicin were evaluated using MTT and the Cell cycle assay and apoptosis rate was determined by flow cytometry.ResultsMCF-7/ADR cells revealed highly expressed MTDH and MDA-MB-231 cells had the lowest expression of MTDH. After MTDH gene was knocked down, the cell proliferation was inhibited, and the inhibitory rate of cell growth and apoptosis rate were enhanced, and the cell cycle arrest during the G0/G1 phase in the presence of doxorubicin treatment. On the other hand, the opposite results were observed in MDA-MB-231 cells with overexpressed MTDH gene.ConclusionMTDH gene plays a promoting role in the proliferation of breast cancer cells and its high expression may be associated with doxorubicin sensitivity of breast cancer.     

Heparan Sulfate Proteoglycans Play a Dual Role in Regulating Fibroblast Growth Factor-2 Mitogenic Activity in Human Breast Cancer Cells

The human breast cancer cell lines MCF-7 and MDA-MB-231 differ in their responsiveness to fibroblast growth factor-2 (FGF-2). This growth factor stimulates proliferation in well-differentiated MCF-7 cells, whereas the less well-differentiated MDA-MB-231 cells are insensitive to this molecule. To investigate the potential regulation of FGF-2 mitogenic activity by heparan sulfate proteoglycans (HSPG), we have treated human breast cancer cells by glycosaminoglycan degrading enzymes or a metabolic inhibitor of proteoglycan sulfation: sodium chlorate. The interaction between FGF-2 and proteoglycans was assayed by examining the binding of¹²⁵I-FGF-2 to breast cancer cell cultures as well as to cationic membranes loaded with HSPG. Using MCF-7 cells, we showed that heparinase treatment inhibited FGF-2 binding to HSPG and completely abolished FGF-2 induced growth; chlorate treatment of MCF-7 cells decreased FGF-2 binding to HSPG and cell responsiveness in a dose-dependent manner. This demonstrates a requirement of adequately sulfated HSPG for FGF-2 growth-promoting activity on MCF-7 cells. In highly invasive MDA-MB-231 cells which produce twice as much HSPG as MCF-7 cells and which are not normally responsive to exogenously added FGF-2, chlorate treatment decreased FGF-2 binding to HSPG and induced FGF-2 mitogenic effect. This chlorate effect was dose dependent and observed at concentrations of 10–30 mM;higher chlorate concentrations completely abolished the FGF-2 effect. This shows that the HSPG level of sulfation can also negatively regulate the biological activity of FGF-2. Taken together, these results demonstrate a crucial role for HSPG in both positive and negative control of FGF-2 mitogenic activity in breast cancer cell proliferation.     

Cellular effect of styrene substituted biscoumarin caused cellular apoptosis and cell cycle arrest in human breast cancer cells

BackgroundCoumarins occurs naturally across plant kingdoms exhibits significant pharmacological properties and pharmacokinetic activity. The conventional, therapeutic agents are often associated with poor stability, absorption and increased side effects. Therefore, identification of a drug that has little or no-side effect on humans is consequential. Here, we investigated the antiproliferative activity of styrene substituted biscoumarin against various human breast cancer cell lines, such as MCF-7, (ER-) MDA-MB-231 and (AR+) MDA-MB-453. Styrene substituted biscoumarin induced cell death by apoptosis in MDA-MB-231 cell line was analyzed.MethodsAntiproliferative activity of Styrene substituted biscoumarin was performed by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Styrene substituted biscoumarin induced apoptosis was assessed by Hoechst staining, Annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) staining and flow cytometric analysis. Migratory and proliferating characteristic of breast cancer cell line MDA-MB-231 was also analyzed by wound healing and colony formation assay. Furthermore, mRNA expression of BAX and BCL-2 were quantified using qRT-PCR and protein expression level analyzed by Western blot.ResultsThe inhibition concentration (IC₅₀) of styrene substituted biscoumarin was assayed against three breast cancer cell lines. The inhibition concentration (IC₅₀) value of styrene substituted biscoumarin toward MDA-MB-231, MDA-MB-453 and MCF-7 cell lines was 5.63, 7.30 and 10.84 μg/ml respectively. Styrene substituted biscoumarin induced apoptosis was detected by Hoechst staining, DAPI/PI analysis and flow-cytometric analysis. The migration and proliferative efficiency of MDA-MB-231 cells were completely arrested upon styrene substituted biscoumarin treatment. Also, mRNA gene expression and protein expression of pro-apoptotic (BAX) and anti-apoptotic (BCL-2) genes were analyzed by qRT-PCR and western blot analysis upon styrene substituted biscoumarin treatment to MDA-MB-231 cells. Our results showed that styrene substituted biscoumarin downregulated BCL-2 gene expression and upregulated BAX gene expression to trigger apoptotic process.ConclusionStyrene substituted biscoumarin could induce apoptosis through intrinsic mitochondrial pathway in breast cancer cell lines, particularly in MDA-MB-231. Our data suggest that styrene substituted biscoumarin may act as a potential chemotherapeutic agent against breast cancer.     

Specific RNA binding to ordered phospholipid bilayers

We have studied RNA binding to vesicles bounded by ordered and disordered phospholipid membranes. A positive correlation exists between bilayer order and RNA affinity. In particular, structure-dependent RNA binding appears for rafted (liquid-ordered) domains in sphingomyelin-cholesterol-1,2-dioleoyl-sn-glycero-3-phosphocholine vesicles. Binding to more highly ordered gel phase membranes is stronger, but much less RNA structure-dependent. All modes of RNA-membrane association seem to be electrostatic and headgroup directed. Fluorometry on 1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomes indicates that bound RNA broadens the gel-fluid melting transition, and reduces lipid headgroup order, as detected via fluorometric measurement of intramembrane electric fields. RNA preference for rafted lipid was visualized and confirmed using multiple fluorophores that allow fluorescence and fluorescence resonance energy transfer microscopy on RNA molecules closely associated with ordered lipid patches within giant vesicles. Accordingly, both RNA structure and membrane order could modulate biological RNA–membrane interactions.     

Development of methotrexate proline prodrug to overcome resistance by MDA-MB-231 cells

The resistance to methotrexate by a number of cancer cells such as breast cancer cell-line MDA-MB-231 due to poor permeability renders it less effective as an anticancer agent for these cells. Proline prodrug of methotrexate (Pro-MTX) was designed as a substrate of prolidase which is specific for imido bond of dipeptide containing proline and expected to penetrate MDA-MB-231 cells more efficiently. The prodrug was synthesized by solid-phase peptide synthesis method and examined as a substrate of pure prolidase as well as cell homogenate. The cytotoxicity against MDA-MB-231 and non-methotrexate resistant breast cancer cell line, MCF-7 was also examined by XTT assay. The results showed that Pro-MTX was a substrate of prolidase. It was also shown that the prodrug could be converted to parent drug methotrexate in Caco-2 and HeLa cell homogenate. When tested with Caco-2 and MCF-7 cells, Pro-MTX showed weaker cytotoxicity compared with methotrexate. But for methotrexate resistant MDA-MB-231 cells, Pro-MTX showed stronger activity than methotrexate. The results indicated that the proline prodrug of methotrexate may overcome the resistance of human breast cancer cells in culture.