Design,synthesis and anticancer activity of novel nopinone-based thiosemicarbazone derivatives

A series of new nopinone-based thiosemicarbazone derivatives were designed and synthesized as potent anticancer agents. All these compounds were identified by ¹H NMR, ¹³C NMR, HR-MS spectra analyses. In the in vitro anticancer activity, most derivatives showed considerable cytotoxic activity against three human cancer cell lines (MDA-MB-231, SMMC-7721 and Hela). Among them, compound 4i exhibited most potent antitumor activity against three cancer cell lines with the IC₅₀ values of 2.79 ± 0.38, 2.64 ± 0.17 and 3.64 ± 0.13 μM, respectively. Furthermore, the cell cycle analysis indicated that compound 4i caused cell cycle arrest of MDA-MB-231 cells at G2/M phase. The Annexin V-FITC/7-AAD dual staining assay also revealed that compound 4i induced the early apoptosis of MDA-MB-231 cells.     

Design,synthesis and in vitro anticancer activity of novel quinoline and oxadiazole derivatives of ursolic acid

A series of new quinoline derivatives of ursolic acid were designed and synthesized in an attempt to develop potential anticancer agents. The structures of these compounds were identified by ¹H NMR, ¹³C NMR, IR and ESI-MS spectra analysis. The target compounds were evaluated for their in vitro cytotoxicity against three human cancer cell lines (MDA-MB-231, Hela and SMMC-7721). From the results, compounds 3a–d displayed significant antitumor activity against three cancer cell lines. Especially, compound 3b was found to be the most potent derivative with IC₅₀ values of 0.61 ± 0.07, 0.36 ± 0.05, 12.49 ± 0.08 μM against MDA-MB-231, HeLa and SMMC-7721 cells, respectively, stronger than positive control etoposide. Furthermore, the Annexin V-FITC/PI dual staining assay revealed that compound 3b could significantly induce the apoptosis of MDA-MB-231 cells in a dose-dependent manner. The cell cycle analysis also indicated that compound 3b could cause cell cycle arrest of MDA-MB-231 cells at G0/G1 phase.     

三阴性乳腺癌干细胞的富集及CD44+CD24-/low、CXCR4表达测定

目的:探讨MDA-MB-231细胞经无血清培养富集三阴性乳腺癌干细胞,观察再成球、集落形成及CD44+CD24-/low、CXCR4表达。方法:将MDA-MB-231乳腺癌细胞进行微球体培养,取培养第7-9天的微球体,判断干细胞富集的程度;比较不同细胞浓度对癌球细胞成球率影响;流式细胞仪测定CD44+CD24-/low含量;Western blot法分析CXCR4蛋白表达;单个癌球细胞再成球能力;观察癌球与贴壁细胞集落形成。结果:1).在含20 ng/m L EGF,10 ng/m L b FGF,2%b27无血清培养基中可培养三阴性乳腺癌癌球,1×104/m L、2×104/m L、3×104/m L、4×104/m L、5×104/m L细胞浓度癌球细胞成球率分别为(5.61±0.02)%、(3.23±0.54)%、(2.28±0.48)%、(1.05±0.13)%、(0.91±0.01)%,组间比较差异有统计学意义P值均0.05。2).贴壁MDA-MB-231细胞与癌球细胞CD44+CD24-/low含量分别为(38.54±2.00)%VS(66.35±2.06)%,差异有统计学意义P=0.003。3).癌球细胞CXCR4蛋白表达高于贴壁MDA-MB-231细胞,灰度扫描分析差异有统计学意义,P=0.03。4).单个癌球细胞具有再成球能力。5).软琼脂糖集落形成能力癌球需200个细胞即可见集落形成,而贴壁细胞需1 000个MDA-MB-231细胞。结论:1.通过无血清培养可以富集三阴性乳腺癌干细胞,低细胞密度有利于癌球形成。2.癌球中CD44+CD24-/low含量高于贴壁MDA-MB-231细胞。3.CXCR4在癌球中表达高于贴壁MDA-MB-231细胞。     

Synthesis and Evaluation of Piperazine-Tethered Derivatives of Alepterolic Acid as Anticancer Agents

Alepterolic acid is a natural diterpenoid isolated from Aleuritopteris argentea with potential anti-cancer activity. In this study, alepterolic acid was modified to construct a series of arylformyl piperazinyl derivatives ( 3a – 3p ). The synthesized derivatives were fully characterized with HRMS, NMR, and IR. Four compounds with inhibition rate higher than 30 % at 10 μM ( 3f , 3n , 3g and 3k ) were further measured to obtain the IC₅₀ values against four cancer cell lines, including hepatoma cell lines HepG2, lung cancer cell lines A549, estrogen receptor-positive cell lines MCF7, and triple-negative breast cancer (TNBC) cell lines MDA-MB-231 by MTT assay. It was found that these compounds were more effective to HepG2 and MDA-MB-231 cells, while less toxic to A549 and MCF7 cells, and compound 3n as the most toxic derivatve against MDA-MB-231 cell lines, with IC₅₀ value of 5.55±0.56 μM. Trypan blue staining and colony formation assay showed that compound 3n inhibited the growth of MDA-MB-231 cells and prevented colony formation. Hoechst staining, flow cytometry and western blot analysis revealed that compound 3n induced caspase-dependent apoptosis in MDA-MB-231 cells. Conclusively, compound 3n was demonstrated to be a potential anti-cancer lead compound for further investigation.     

Copper complex derived from <Emphasis Type="Italic">S</Emphasis>-benzyldithiocarbazate and 3-acetylcoumarin induced apoptosis in breast cancer cell

Copper complexes have been widely studied for the anti-tumour application as cancer cells are reported to take up greater amounts of copper than normal cells. Preliminary study revealed that the newly synthesised copper complex [Cu(SBCM)₂] displayed marked anti-proliferative towards triple-negative MDA-MB-231 breast cancer cells. Therefore, Cu(SBCM)₂ has great potential to be developed as an agent for the management of breast cancer. The present study was carried out to investigate the mode of cell death induced by Cu(SBCM)₂ towards MDA-MB-231 breast cancer cells. The inhibitory and morphological changes of MDA-MB-231 cells treated with Cu(SBCM)₂ was determined by using MTT assay and inverted light microscope, respectively. The safety profile of Cu(SBCM)₂ was also evaluated towards human dermal fibroblast (HDF) normal cells. Confirmation of apoptosis and cell cycle arrest were determined by flow cytometry analysis. The expression of p53, Bax, Bcl-2 and MMP2 protein were detected with western blot analysis. Cu(SBCM)₂ significantly inhibited the growth of MDA-MB-231 cells in a dose-dependent manner with GI₅₀ 18.7?±?3.06 µM. Indeed, Cu(SBCM)₂ was less toxic towards HDF normal cells with GI₅₀ 31.8?±?4.0 µM. Morphological study revealed that Cu(SBCM)₂-treated MDA-MB-231 cells experienced cellular shrinkage, membrane blebbing, chromatin condensation and formation of apoptotic bodies, suggesting that Cu(SBCM)₂ induced apoptosis in the cells, which was confirmed by Annexin-V/PI flow cytometry analysis. It was also found that Cu(SBCM)₂ induced G₂/M phase cell cycle arrest towards MDA-MB-231 cells. The induction of apoptosis and cell cycle arrest in the present study is possibly due to the down-regulation of the mutant p53 and MMP2 protein. In conclusion, Cu(SBCM)₂ can be developed as a targeted therapy for the treatment of triple-negative breast cancer.     

Molecular docking,PASS analysis,bioactivity score prediction,synthesis, characterization and biological activity evaluation of a functionalized 2-butanone thiosemicarbazone ligand and its complexes

2-Butanone thiosemicarbazone ligand was prepared by condensation reaction between thiosemicarbazide and butanone. The ligand was characterized by ¹H NMR, ¹³C NMR, FT-IR, mass spectrometry and UV spectroscopic studies. Docking studies were performed to study inhibitory action against topoisomerase II (Topo II) and ribonucleoside diphosphate reductase (RR) enzymes. Inhibition constants (K _i ) of the ligand were 437.87 and 327.4 μM for the two enzymes, respectively. The ligand was tested for its potential anticancer activity against two cancer cell lines MDA-MB-231 and A549 using MTT assay and was found to exhibit good activity at higher doses with an IC₅₀ = 80 μM against human breast cancer cell line MDA-MB-231. On the other hand, no significant activity was obtained against the lung carcinoma cell line A549. Antibacterial activity of the ligand was tested against Staphylococcus aureus and E. coli using the disc diffusion method. Ligand did not exhibit any significant antibacterial activity. Four complexes of Co(III), Fe(II), Cu(II), and Zn(II) were prepared with the ligand and characterized by various spectroscopic studies. Low molar conductance values were obtained for all complexes displaying non-electrolyte nature except in Co(III) complex. As expected, complexation with metal ions significantly increased the cytotoxicity of the ligand against the tested cell lines viz. IC₅₀ values of <20 μM for Co, Fe, and Zn complexes and approx. 80 μM against MDA cells versus IC₅₀ value of <20 μM for Co and Cu complexes and that of 30 and 50 μM for Fe and Zn complexes, respectively, against A549 cells. The Cu complex was found to be active against E. coli and S. aureus with MIC values in the range of 6–10 mg/mL. Other than Cu, only Co complex was found to possess antibacterial activity with MIC values of 5–10 mg/mL when tested against S. aureus. Bioactivity score and Prediction of Activity Spectra for Substances (PASS) analysis also depicted the drug-like nature of ligand and complexes.     

Synthesis,characterization and biological evaluation of C5′-N-cyclopropylcarboxamido-C6-amino-C2-alkynylated purine nucleoside analogues

In an effort to develop potent antibacterial and anticancer agents, a series of C5′-N-cyclopropylcarboxamido-C6-amino-C2-alkynylated purine nucleoside analogues 11a-g were synthesized through a Sonogashira cross-coupling reaction. The nine-step synthesis is easy to perform, and employs commercially available reagents. 2-Iodo-5′-N-cyclopropylcarboxamidoadenosine (9) was used as the starting intermediate for the synthesis of title derivatives 11a-g. Synthetic intermediates (2–9) and final products (11a-g) were appropriately characterized by IR, ¹H NMR, ¹³C NMR and mass spectroscopy. The synthesized purine nucleoside analogues (11a-g) were evaluated for their in vitro antibacterial activity against two gram-positive and two gram-negative bacteria. They were then tested for cytotoxicity against MDA-MB-231 and Caco-2 cancer cell lines to determine their anti-cancer activity. Among the tested compounds, compounds 11c and 11g showed most potent antibacterial activity against S.aureus and P.aeruginosa bacterial strains. Compounds 11b and 11e displayed considerable IC₅₀^s of 7.9 and 6.8 µg/mL, respectively, vs MDA-MB-231 cell lines of 7.5 and 8.3 µg/mL, respectively, against the Caco-2 cell lines.     

H+-dependent inorganic phosphate transporter in breast cancer cells: Possible functions in the tumor microenvironment

Tumor microenvironment has a high concentration of inorganic phosphate (Pi), which is actually a marker for tumor progression. Regarding Pi another class of transporter has been recently studied, an H⁺-dependent Pi transporter, that is stimulated at acidic pH in Caco2BBE human intestinal cells. In this study, we characterized the H⁺-dependent Pi transport in breast cancer cell (MDA-MB-231) and around the cancer tissue. MDA-MB-231 cell line presented higher levels of H⁺-dependent Pi transport as compared to other breast cell lines, such as MCF-10A, MCF-7 and T47-D. The Pi transport was linear as a function of time and exhibited a Michaelis-Menten kinetic of K_m = 1.387 ± 0.1674 mM Pi and V_max = 198.6 ± 10.23 Pi × h^?1 × mg protein^?1 hence reflecting a low affinity Pi transport. H⁺-dependent Pi uptake was higher at acidic pH. FCCP, Bafilomycin A1 and SCH28080, which deregulate the intracellular levels of protons, inhibited the H⁺-dependent Pi transport. No effect on pHi was observed in the absence of inorganic phosphate. PAA, an H⁺-dependent Pi transport inhibitor, reduced the Pi transport activity, cell proliferation, adhesion, and migration. Arsenate, a structural analog of Pi, inhibited the Pi transport. At high Pi conditions, the H⁺-dependent Pi transport was five-fold higher than the Na⁺-dependent Pi transport, thus reflecting a low affinity Pi transport. The occurrence of an H⁺-dependent Pi transporter in tumor cells may endow them with an alternative path for Pi uptake in situations in which Na⁺-dependent Pi transport is saturated within the tumor microenvironment, thus regulating the energetically expensive tumor processes.     

Preclinical evaluation of 89Zr-labeled anti-CD44 monoclonal antibody RG7356 in mice and cynomolgus monkeys

RG7356 is a humanized antibody targeting the constant region of CD44. RG7356 was radiolabeled with ⁸⁹Zr for preclinical evaluations in tumor xenograft-bearing mice and normal cynomolgus monkeys to enable study of its biodistribution and the role of CD44 expression on RG7356 uptake.

Studies with ⁸⁹Zr-RG7356 were performed in mice bearing tumor xenografts that differ in the level of CD44 expression (CD44⁺ or CD44^-) and RG7356 responsiveness (resp or non-resp): MDA-MB-231 (CD44⁺, resp), PL45 (CD44⁺, non-resp) and HepG2 (CD44^–, non-resp). Immuno-PET whole body biodistribution studies were performed in normal cynomolgus monkeys to determine normal organ uptake after administration of a single dose.

At 1, 2, 3, and 6 days after injection, ⁸⁹Zr-RG7356 uptake in MDA-MB-231 (CD44⁺, resp) xenografts was nearly constant and about 9 times higher than in HepG2 (CD44^–, non-resp) xenografts (range 27.44 ± 12.93 to 33.13 ± 7.42% ID/g vs. 3.25 ± 0.38 to 3.90 ± 0.58% ID/g). Uptake of ⁸⁹Zr-RG7356 was similar in MDA-MB-231 (CD44⁺, resp) and PL45 (CD44⁺, non-resp) xenografts. Studies in monkeys revealed antibody uptake in spleen, salivary glands and bone marrow, which might be related to the level of CD44 expression. ⁸⁹Zr-RG7356 uptake in these normal organs decreased with increasing dose levels of unlabeled RG7356.

⁸⁹Zr-RG7356 selectively targets CD44⁺ responsive and non-responsive tumors in mice and CD44⁺ tissues in monkeys. These studies indicate the importance of accurate antibody dosing in humans to obtain optimal tumor targeting. Moreover, efficient binding of RG7356 to CD44⁺ tumors may not be sufficient in itself to drive an anti-tumor response.     

Radiosynthesis,biological evaluation and preliminary microPET study of 18F-labeled 5-resorcinolic triazolone derivative based on ganetespib targeting HSP90

Heat-shock protein 90 (HSP90) is a molecular chaperone that activates oncogenic transformation in several solid tumors, including lung and breast cancers. Ganetespib, a most promising candidate among several HSP90 inhibitors under clinical trials, has entered Phase III clinical trials for cancer therapy. Despite numerous evidences validating HSP90 as a target of anticancer, there are few studies on PET agents targeting oncogenic HSP90. In this study, we synthesized and biologically evaluated a novel ¹⁸F-labeled 5-resorcinolic triazolone derivative (1, [¹⁸F]PTP-Ganetespib) based on ganetespib. [¹⁸F]PTP-Ganetespib was labeled by click chemistry of Ganetespib-PEG-Alkyne (10) and [¹⁸F]PEG-N₃ (11) with 37.3?±?5.11% of radiochemical yield and 99.7?±?0.09% of radiochemical purity. [¹⁸F]PTP-Ganetespib showed proper LogP (0.96?±?0.06) and good stability in human serum over 97% for 2?h. [¹⁸F]PTP-Ganetespib showed high uptakes in breast cancer cells containing triple negative breast cancer (TNBC) MDA-MB-231 and Her2-negative MCF-7 cells, which are target breast cancer cell lines of HSP90 inhibitor, ganetespib, as an anticancer. Blocking of HSP90 by the pretreatment of ganetespib exhibited significantly decreased accumulation of [¹⁸F]PTP-Ganetespib in MDA-MB-231 and MCF-7 cells, indicating the specific binding of [¹⁸F]PTP-Ganetespib to MDA-MB-231 and MCF-7 cells with high HSP90 expression. In the biodistribution and microPET imaging studies, the initial uptake into tumor was weaker than in other thoracic and abdominal organs, but [¹⁸F]PTP-Ganetespib was retained relatively longer in the tumor than other organs. The uptake of [¹⁸F]PTP-Ganetespib in tumors was not sufficient for further development as a tumor-specific PET imaging agent by itself, but this preliminary PET imaging study of [¹⁸F]PTP-Ganetespib can be basis for developing new PET imaging agents based on HSP90 inhibitor, ganetespib.     

Affibody-displaying bionanocapsules for specific drug delivery to HER2-expressing cancer cells

A novel HER2-targeted carrier was developed using bionanocapsules (BNCs). Bionanocapsules (BNCs) are 100-nm hollow nanoparticles composed of the l-protein of hepatitis B virus surface antigen. An affibody of HER2 was genetically displayed on the BNC surface (Z_HER2-BNC). For the investigation of binding affinity, Z_HER2-BNC was incubated with the cancer cell lines SK-BR-3 (HER2 positive), and MDA-MB-231 (HER2 negative). For analysis of HER2 targeting specificity, Z_HER2-BNC or Z_WT-BNC (without affibody) was incubated with both SK-BR-3 and MDA-MB-231 cells by time lapse and concentration. For the delivery of encapsulated molecules (calcein), fluorescence of Z_HER2-BNC mixed with liposomes was also compared with that of Z_WT-BNC and nude liposomes by incubation with SK-BR-3 cells. As a result, Z_HER2-BNC-liposome complex demonstrated the delivery to HER2-expressing cells (SK-BR-3) with a high degree of specificity. This indicates that genetically engineered BNCs are promising carrier for cancer treatment.     

Synthesis and biological evaluation of 2-(3,4-dimethoxyphenyl)-6-(2-[18F]fluoroethoxy)benzothiazole ([18F]FEDBT) for PET imaging of breast cancer

Given the ever-present demand for improved PET radiotracer in oncology imaging, we have synthesized 2-(3,4-dimethoxyphenyl)-6-(2-[¹⁸F]fluoroethoxy)benzothiazole ([¹⁸F]FEDBT), a fluorine-18-containing fluoroethylated benzothiazole to explore its utility as a PET imaging tracer. [¹⁸F]FEDBT was prepared via kryptofix-mediated nucleophilic substitution of the tosyl group precursor. Fractionated ethanol-based solid-phase (SPE cartridge-based) purification afforded [¹⁸F]FEDBT in 60% radiochemical yield (EOB), with radiochemical purity in excess of 98% and the specific activity was 35 GBq/μmol. The radiotracer displayed clearly higher cellular uptake ratio in various breast cancer cell lines MCF7, MDA-MB-468 and MDA-MB-231. However, both biodistribution and microPET studies have showed an higher abdominal accumulation of [¹⁸F]FEDMBT and the tumor/muscle ratio of 1.8 was observed in the MDA-MB-231 xenograft tumors mice model. Further the lipophilic improvement is needed for the reducement of hepatobilliary accumulation and to promote the tumor uptake for PET imaging of breast cancer.     

The effect of a hyposmotic shock and purinergic agonists on K(Rb) efflux from cultured human breast cancer cells

The effect of a hyposmotic shock and extracellular ATP on the efflux of K⁺(Rb⁺) from human breast cancer cell lines (MDA-MB-231 and MCF-7) has been examined. A hyposmotic shock increased the fractional efflux of K⁺(Rb⁺) from MDA-MB-231 cells via a pathway which was unaffected by Cl⁻ replacement. Apamin, charybdotoxin or removing extracellular Ca²⁺ had no effect on volume-activated K⁺(Rb⁺) efflux MDA-MB-231 cells. An osmotic shock also stimulated K⁺(Rb⁺) efflux from MCF-7 cells but to a much lesser extent than found with MDA-MB-231 cells. ATP-stimulated K⁺(Rb⁺) efflux from MDA-MB-231 cells in a dose-dependent fashion but had little effect on K⁺(Rb⁺) release from MCF-7 cells. ATP-stimulated K⁺(Rb⁺) efflux was only inhibited slightly by replacing Cl⁻ with NO₃⁻. Removal of external Ca²⁺ during treatment with ATP reduced the fractional efflux of K⁺(Rb⁺) in a manner suggesting a role for cellular Ca²⁺ stores. Charybdotoxin, but neither apamin nor iberiotoxin, inhibited ATP-stimulated K⁺(Rb⁺) release from MDA-MB-231 cells. Suramin inhibited the ATP-activated efflux of K⁺(Rb⁺). UTP also stimulated K⁺(Rb⁺) efflux from MDA-MB-231 cells whereas ADP, AMP and adenosine were without effect. A combination of an osmotic shock and ATP increased the fractional efflux of K⁺(Rb⁺) to a level greater than the sum of the individual treatments. It appears that the hyposmotically-activated and ATP-stimulated K⁺ efflux pathways are separate entities. However, there may be a degree of ‘crosstalk’ between the two pathways.     

Induction of apoptosis by Centaurea nerimaniae extract in HeLa and MDA-MB-231 cells by a caspase-3 pathway

We investigated the cytotoxic and apoptotic effects of a methanol extract of Centaurea nerimaniae, a plant endemic in Turkey, on HeLa and MDA-MB-231 cells. Eight concentrations of C. nerimaniae extract were applied to cells, and cytotoxic effects were measured using the xCELLigence system. The TUNEL assay was used to assess apoptotic cell death and immunohistochemistry was used to determine active caspase-3 using the effective cytotoxic doses of the extract. Doses of 1.42 mg/ml C. nerimaniae inhibited the growth of HeLa cells and 3.67 mg/ml C. nerimaniae inhibited the growth of MDA-MB-231 cells in a dose- and time-dependent manner. The apoptotic indexes for HeLa and MDA-MB-231 cells were increased significantly compared to control groups. Immunohistochemistry showed that the number of caspase-3 immunostained cells increased in the extract treatment groups for both HeLa and MDA-MB-231 cells. In the MDA-MB-231 cell line, caspase-3 immunostaining was observed in nuclei and/or cytoplasm in the extract treated group. Caspase-3 activation was greater in HeLa cells than in MDA-MB-231 cells. We found that the extract of C. nerimaniae had a strong antiproliferative effect and induced apoptosis via caspase-3; MDA-MB-231 cancer cells were more resistant than HeLa cells.     

Interfering transbody-mediated Her2 gene silencing induces apoptosis by G0/G1 cell cycle arrest in Her2-overexpressing SK-BR-3 breast cancer cells

We previously isolated an interfering transbody, 4MH2, which penetrated the cytosol of living cells and preferentially hydrolyzed the target Her2 (ErbB2) mRNA, resulting in Her2 gene silencing followed by apoptotic cell death in Her2-overexpressing breast cancer cells. Here, we report the apoptotic cell death mechanism mediated by 4MH2-induced Her2 gene silencing in Her2-overexpressing SK-BR-3 breast cancer cells, in comparison with a small interfering RNA (siRNA) targeting Her2 mRNA (Her2₁₈-siRNA). 4MH2 induced G₀/G₁ cell cycle arrest to cause apoptotic cell death in SK-BR-3 cells by triggering specific signaling pathways associated with Her2 knockdown, including upregulation of G₀/G₁ cell cycle arrest-associated p21^Cip1 and p27^Kip1, downregulation of cyclin D1, inhibition of Akt phosphorylation, and downregulation of antiapoptotic Bcl-xL, which are comparable to those mediated by Her2₁₈-siRNA. Our results suggest that 4MH2-mediated Her2 gene silencing can trigger the downstream signaling pathways caused by Her2 downregulation, comparable to those mediated by the corresponding siRNA.     

Indirect organogenesis from leaf explants of Hemidesmus indicus (L.) R. Br.: an important medicinal plant

Hemidesmus indicus (Asclepiadaceae) leaf explants were utilized for establishing culture in MS medium fortified with individual cytokinins, auxins, and their combinations. Optimum response (80%) was observed in N⁶-benzyladenine (BA, 20 μM) + indole-3-acetic acid (IAA, 1 μM) with 19.67 ± 0.81 shoots per explant. Roots were induced in ¼MS + indole-3-butyric acid (IBA, 20 μM).     

A novel approach combining the Calgary Biofilm Device and Phenotype MicroArray for the characterization of the chemical sensitivity of bacterial biofilms

A rapid method for screening the metabolic susceptibility of biofilms to toxic compounds was developed by combining the Calgary Biofilm Device (MBEC device) and Phenotype MicroArray (PM) technology. The method was developed using Pseudomonas alcaliphila 34, a Cr(VI)-hyper-resistant bacterium, as the test organism. P. alcaliphila produced a robust biofilm after incubation for 16 h, reaching the maximum value after incubation for 24 h (9.4 × 10⁶ ± 3.3 × 10⁶ CFU peg^?1). In order to detect the metabolic activity of cells in the biofilm, dye E (5×) and menadione sodium bisulphate (100 μM) were selected for redox detection chemistry, because they produced a high colorimetric yield in response to bacterial metabolism (340.4 ± 6.9 Omnilog Arbitrary Units). This combined approach, which avoids the limitations of traditional plate counts, was validated by testing the susceptibility of P. alcaliphila biofilm to 22 toxic compounds. For each compound the concentration level that significantly lowered the metabolic activity of the biofilm was identified. Chemical sensitivity analysis of the planktonic culture was also performed, allowing comparison of the metabolic susceptibility patterns of biofilm and planktonic cultures.     

NHE1 mediates MDA-MB-231 cells invasion through the regulation of MT1-MMP

Na⁺/H⁺ exchanger 1 (NHE1), an important regulator of intracellular pH (pH_i) and extracellular pH (pH_e), has been shown to play a key role in breast cancer metastasis. However, the exact mechanism by which NHE1 mediates breast cancer metastasis is not yet well known. We showed here that inhibition of NHE1 activity, with specific inhibitor Cariporide, could suppress MDA-MB-231 cells invasion as well as the activity and expression of MT1-MMP. Overexpression of MT1-MMP resulted in a distinguished increase in MDA-MB-231 cells invasiveness, but treatment with Cariporide reversed the MT1-MMP-mediated enhanced invasiveness. To explore the role of MAPK signaling pathways in NHE1-mediated breast cancer metastasis, we compared the difference of constitutively phosphorylated ERK1/2, p38 MAPK and JNK in non-invasive MCF-7 cells and invasive MDA-MB-231cells. Interestingly, we found that the phosphorylation levels of ERK1/2 and p38 MAPK in MDA-MB-231 cells were higher than in MCF-7 cells, but both MCF-7 cells and MDA-MB-231 cells expressed similar constitutively phosphorylated JNK. Treating MDA-MB-231 cells with Cariporide led to decreased phosphorylation level of both p38 MAPK and ERK1/2 in a time-dependent manner, but JNK activity was not influenced. Supplementation with MAPK inhibitor (MEK inhibitor PD98059, p38 MAPK inhibitor SB203580 and JNK inhibitor SP600125) or Cariporide all exhibited significant depression of MDA-MB-231 cells invasion and MT1-MMP expression. Furthermore, we co-treated MDA-MB-231 cells with MAPK inhibitor and Cariporide. The result showed that Cariporide synergistically suppressed invasion and MT1-MMP expression with MEK inhibitor and p38 MAPK inhibitor, but not be synergistic with the JNK inhibitor. These findings suggest that NHE1 mediates MDA-MB-231 cells invasion partly through regulating MT1-MMP in ERK1/2 and p38 MAPK signaling pathways dependent manner.     

Inhibition of breast cancer growth via miR-7 suppressing ALDH1A3 activity concomitant with decreasing breast cancer stem cell subpopulation

Breast cancer patients with high expression of aldehyde dehydrogenases (ALDHs) cell population have higher tolerability to chemotherapy since the cells posses a characteristic of breast cancer stem cells (BCSCs) that are resistant to conventional chemotherapy. In this study, we found that the ALDH-positive cells were higher in CD44⁺CD24⁻ and CD44⁺CD24⁻ESA⁺BCSCs than that in both BT549 and MDA-MB-231 cell lines but microRNA-7 (miR-7) level was lower in CD44⁺CD24⁻ and CD44⁺CD24⁻ESA⁺BCSCs than that in MDA-MB-231 cells. Moreover, miR-7 overexpression in MDA-MB-231 cells decreased ALDH1A3 activity by miR-7 directly binding to the 3′-untranslated region of ALDH1A3; while the ALDH1A3 expression was downregulated in MDA-MB-231 cells, the expressions of CD44 and Epithelium Specific Antigen (ESA) were reduced along with decreasing the BCSC subpopulation. Significantly, enforced expression of miR-7 in CD44⁺CD24⁻ESA⁺BCSC markedly inhibited the BCSC-driven xenograft growth in mice by decreasing an expression of ALDH1A3. Collectively, the findings demonstrate the miR-7 inhibits breast cancer growth via suppressing ALDH1A3 activity concomitant with decreasing BCSC subpopulation. This approach may be considered for an investigation on clinical treatment of breast cancers.     

Ectophosphatase activity in the triple-negative breast cancer cell line MDA-MB-231

Breast cancer is one of the most common cancers in the female population worldwide, and its development is thought to be associated with genetic mutations that lead to uncontrolled and accelerated growth of breast cells. This abnormal behavior requires extra energy, and indeed, tumor cells display a rewired energy metabolism compared to normal breast cells. Inorganic phosphate (Pi) is a glycolytic substrate of glyceraldehyde-3-phosphate dehydrogenase and has an important role in cancer cell proliferation. For cells to obtain Pi, ectoenzymes in the plasma membrane with their catalytic site facing the extracellular environment can hydrolyze phosphorylated molecules, and this is an initial and possibly limiting step for the uptake of Pi by carriers that behave as adjuvants in the process of energy harvesting and thus partially contributes to tumor energy requirements. In this study, the activity of an ectophosphatase in MDA-MB-231 cells was biochemically characterized, and the results showed that the activity of this enzyme was higher in the acidic pH range and that the enzyme had a K_m = 4.5 ± 0.5 mM para-nitrophenylphosphate and a V_max = 2280 ± 158 nM × h⁻¹ × mg protein⁻¹. In addition, classical acid phosphatase inhibitors, including sodium orthovanadate, decreased enzymatic activity. Sodium orthovanadate was able to inhibit ectophosphatase activity while also inhibiting cell proliferation, adhesion, and migration, which are important processes in tumor progression, especially in metastatic breast cancer MDA-MB-231 cells that have higher ectophosphatase activity than MCF-7 and MCF-10 breast cells.