Living MCF-7 Cells Research Articles

Screening of bioactive compounds and research of possible targets based on hollow fiber cell fishing with high performance liquid chromatography

In this study, hollow fiber cell fishing with high performance liquid chromatography (HFCF-HPLC) is further extended and verified. The surface properties of the hollow fiber seeded human breast cancer cell MCF-7, the survival rate of cells seeded, the cell death rate after screening of active compounds, the non-specific binding between active centres on the fiber and the target compounds, the positive and negative controls and the repeatability of HFCF-HPLC were investigated in detail. The target fishing factor of active compounds was defined and improved. The procedure was employed to screen and fish simultaneously phenolic acid and flavonoid active compound groups from water decoctions of traditional Chinese medicines (TCMs). Some of the structures of active compounds screened and fished were identified by comparing with the retention time of the reference substances. And the hollow fibers filled with the receptors (estrogen receptor, epidermal growth factor receptor, and cyclooxygenase-2) associated with cancer cells as well as the cell membranes and cell organelles separated from live MCF-7 cells were used respectively in preliminary research of the binding sites and the targets of potential phenolic acids and flavonoids on MCF-7 cells. The active compounds' abilities to interact with their targets were compared and are discussed. Results demonstrate that HFCF-HPLC is a simple, quick, stable and reliable method to screen and analyze bioactive compounds as well as research the possible targets of active compounds, and it provides us with a good possibility to research and prove the effects of multiple components and multiple targets for TCMs.

New imaging reagents for lipid dense regions in live cells and the nucleus in fixed MCF-7 cells.

Two new uracil (U) and 5-flurouracil (5-FU) labeled ruthenium(ii)-polypyridyl based cellular imaging reagents are reported. Confocal laser scanning microscopic images with live and paraformaldehyde (PFA) fixed MCF-7 cells are examined using these two low-cytotoxic reagents. Experimental results show that these two complexes, appropriately functionalized with U (1) and 5-FU (2), have specific affinity for the lipid dense regions like the endoplasmic reticulum, cell membrane, and cytoplasmic vacuoles in live MCF-7 cells, and dye internalization in these regions happened following an endocytosis pathway. Interestingly, these two complexes are found to be localized in the nucleus of the PFA fixed cells. For fixed cells, presumably the lipid layer disruption helped in the explicit localization of the complexes 1 and 2 in the cell nucleus through specific interaction with cellular DNA. Poor and non-specific internalization of an analogous model complex 3, without having a U or 5-FU moiety, reveals the definite influence of U or 5-FU as well as the role of lipophilicity of the respective complex 1 and 2 in the cellular internalization process. Apart from these, a large Stokes shift (∼160 nm) and an appreciably long lived 3MLCT excited state (∼320 ns) in aq. buffer medium (pH 7.4) are other key features for complexes 1 and 2. Unlike the common nuclear DNA staining reagents like DAPI, these low-cytotoxic reagents are found to be highly stable towards photo-bleaching upon irradiation with 455 nm at the MLCT band for these complexes.

Fluorescent carbon dot (C-dot) nanoclusters

Fluorescent carbon dot (C-dot) nanoclusters composed of C-dot-loaded hollow silica spheres are obtained via the dehydration of mannose, which is adsorbed onto hollow silica spheres or poly(ethylene glycol)-graft-hollow silica spheres (PEG-g-hollow silica). The structure of C-dot nanoclusters are confirmed using 1H NMR, FTIR, TEM and TGA. C-dot nanoclusters show a redshifted fluorescence emission with an increased excitation wavelength. Passivation with PEG diamines improve the quantum yields to ∼2%. Confocal laser scanning microscopy (CLSM) results reflect the fact that C-dot nanoclusters can provide good cytoplasm imaging of live Hep G2 cells and live MCF-7 cells, and the imaging obtained is brighter and more even than those from free C-dots. With their combination of good photostability and low cytotoxicity, C-dot nanoclusters are promising for the production of higher quality bioimaging.

Aurora A orchestrates entosis by regulating a dynamic MCAK-TIP150 interaction.

Entosis, a cell-in-cell process, has been implicated in the formation of aneuploidy associated with an aberrant cell division control. Microtubule plus-end-tracking protein TIP150 facilitates the loading of MCAK onto the microtubule plus ends and orchestrates microtubule plus-end dynamics during cell division. Here we show that TIP150 cooperates with MCAK to govern entosis via a regulatory circuitry that involves Aurora A-mediated phosphorylation of MCAK. Our biochemical analyses show that MCAK forms an intra-molecular association, which is essential for TIP150 binding. Interestingly, Aurora A-mediated phosphorylation of MCAK modulates its intra-molecular association, which perturbs the MCAK-TIP150 interaction in vitro and inhibits entosis in vivo. To probe if MCAK-TIP150 interaction regulates microtubule plasticity to affect the mechanical properties of cells during entosis, we used an optical trap to measure the mechanical rigidity of live MCF7 cells. We find that the MCAK cooperates with TIP150 to promote microtubule dynamics and modulate the mechanical rigidity of the cells during entosis. Our results show that a dynamic interaction of MCAK-TIP150 orchestrated by Aurora A-mediated phosphorylation governs entosis via regulating microtubule plus-end dynamics and cell rigidity. These data reveal a previously unknown mechanism of Aurora A regulation in the control of microtubule plasticity during cell-in-cell processes.

Synthesis of a near-infrared fluorescent probe and its application in imaging of MCF-7 cells

IR-789, a novel near-infrared fluorescent probe, was designed, synthesized, and applied to living cells. The probe exhibited better response fluorescence characteristics than the only FDA-approved agent, indocyanine green. Cell experiments showed that the probe had high affinity and without apparent cytotoxicity. Fluorescent image experiments in living MCF-7 cells (human breast adenocarcinoma cell line) further demonstrated the potential applications of the probe in biological systems. The probe effectively prevented the influence of autofluorescence and native cellular species in biological systems. It also exhibited high sensitivity, good photostability, and excellent cell membrane permeability.

In-situ detection of resveratrol inhibition effect on epidermal growth factor receptor of living MCF-7 cells by Atomic Force Microscopy

A new method based on Atomic Force Microscopy (AFM) was developed to real-time and in-situ detect epidermal growth factor receptor (EGFR) expression levels on living MCF-7 cells for evaluating the anticancer activity of resveratrol. Here, the inhibition effect of resveratrol on EGFR expression levels on MCF-7 cells was probed by epidermal growth factor (EGF)-functionalized tips for the first time. Changes in morphology and stiffness of single cell stimulated by resveratrol at different concentrations were detected by AFM. The consequences showed that resveratrol influenced the cellular state and reduced expression of EGFR on the cell surface, which were also interpreted by MTT assay and confocal microscopy assay. AFM, which was used to investigate potential targets for anti-tumor drug on living cells and realize a better understanding of drug action mechanism, was expected to be developed into a promising tool for screening of drugs.

SU-8 bonding protocol for the fabrication of microfluidic devices dedicated to FTIR microspectroscopy of live cells

Here we present a new bonding protocol for SU-8 negative tone photoresist that exploits the chemical modifications induced in the resin by exposure to 254 nm (UVC) light. Fourier Transform Infrared microspectroscopy (μ-FTIR) was used to carry out a thorough study on the chemical processes and modifications occurring within the epoxy resin by exposure to 365 nm and 254 nm light. In particular, we established that UVC light promotes the opening of the epoxy rings bypassing the post-exposure bake. The possibility to promote a further activation of the resin, already patterned with standard UV lithography, was exploited to produce closed microfluidic devices. Specifically, we were able to fabricate fluidic chips, characterized by broadband transparency from mid-IR to UV and long term stability in continuous flow conditions. CaF2 was used as substrate, coated by sputtering with a nanometric silicon film, in order to make surface properties of this material more suitable for standard fabrication processes with respect to the original substrate. The fabricated microfluidic chips were used to study by μ-FTIR the biochemical response of live breast cancer MCF-7 cells to osmotic stress and their subsequent lysis induced by the injection of deionized water in the device. μ-FTIR analyses detected fast changes in protein, lipid and nucleic acid content as well as cytosol acidification.

Confocal Raman data analysis enables identifying apoptosis of MCF-7 cells caused by anticancer drug paclitaxel

Confocal Raman microscopy is a noninvasive, label-free imaging technique used to study apoptosis of live MCF-7 cells. The images are based on Raman spectra of cells components, and their apoptosis is monitored through diffusion of cytochrome c in cytoplasm. K-mean clustering is used to identify mitochondria in cells, and correlation analysis provides the cytochrome c distribution inside the cells. Our results demonstrate that incubation of cells for 3 h with 10 μM of paclitaxel does not induce apoptosis in MCF-7 cells. On the contrary, incubation for 30 min at a higher concentration (100 μM) of paclitaxel induces gradual release of the cytochrome c into the cytoplasm, indicating cell apoptosis via a caspase independent pathway.

A Synthetic Dolastatin 10 Analogue Suppresses Microtubule Dynamics, Inhibits Cell Proliferation, and Induces Apoptotic Cell Death

We have synthesized eight analogues (D1-D8) of dolastatin 10 containing several unique amino acid subunits. Of these agents, D5 was found to be most effective in inhibiting both HeLa cell proliferation and microtubule assembly in vitro. At low nanomolar concentrations, D5 inhibited the proliferation of several types of cancer cells in culture. D5 bound to tubulin with a dissociation constant of 29.4 ± 6 μM. D5 depolymerized microtubules in cultured cells and produced mulitpolar spindles. At its half-maximal inhibitory concentration (15 nM), D5 strongly suppressed the dynamics of individual microtubules in live MCF-7 cells. D5 increased the accumulation of checkpoint proteins BubR1 and Mad2 at the kinetochoric region and caused G2/M block in these cells. The blocked cells underwent apoptosis with the activation of Jun N-terminal kinase. The results suggested that D5 exerts its antiproliferative action by dampening microtubule dynamics.

Caspase 3 cleavage of the inositol 1,4,5-trisphosphate receptor does not contribute to apoptotic calcium release

An important role in the regulation of apoptotic calcium release is played by the ubiquitously expressed family of inositol 1,4,5-trisphosphate receptor (IP3R) channels. One model for IP3R activation during apoptosis is cleavage by the apoptotic protease caspase 3. Here we show that early elevations in cytosolic calcium during apoptosis do not require caspase 3 activity. We detected a robust increase in calcium levels in response to staurosporine treatment in primary human fibroblasts and HeLa cells in the presence of the caspase inhibitor Z-VAD, indicating that calcium release during the initiation of apoptosis occurs independently of caspase 3. Similar results were obtained with MCF-7 cells which lack caspase 3 expression. Stable expression of caspase 3 in MCF-7 cells and TAT-based transduction of the active recombinant caspase 3 directly into living MCF-7 cells had marginal effects on the early events leading to cytosolic calcium elevations and irreversible commitment to apoptotic cell death. Significantly, blocking IP3 binding to the IP3R with an IP3 sponge resulted in suppression of staurosporine-induced calcium release and cell death. Collectively, our results suggest that generation of IP3 is sufficient for the initiation of apoptotic calcium signaling, and caspase 3-mediated truncation of IP3R channel is a consequence, not causative, of apoptotic calcium release.

An Antitubulin Agent BCFMT Inhibits Proliferation of Cancer Cells and Induces Cell Death by Inhibiting Microtubule Dynamics

Using cell based screening assay, we identified a novel anti-tubulin agent (Z)-5-((5-(4-bromo-3-chlorophenyl)furan-2-yl)methylene)-2-thioxothiazolidin-4-one (BCFMT) that inhibited proliferation of human cervical carcinoma (HeLa) (IC50, 7.2±1.8 µM), human breast adenocarcinoma (MCF-7) (IC50, 10.0±0.5 µM), highly metastatic breast adenocarcinoma (MDA-MB-231) (IC50, 6.0±1 µM), cisplatin-resistant human ovarian carcinoma (A2780-cis) (IC50, 5.8±0.3 µM) and multi-drug resistant mouse mammary tumor (EMT6/AR1) (IC50, 6.5±1µM) cells. Using several complimentary strategies, BCFMT was found to inhibit cancer cell proliferation at G2/M phase of the cell cycle apparently by targeting microtubules. In addition, BCFMT strongly suppressed the dynamics of individual microtubules in live MCF-7 cells. At its half maximal proliferation inhibitory concentration (10 µM), BCFMT reduced the rates of growing and shortening phases of microtubules in MCF-7 cells by 37 and 40%, respectively. Further, it increased the time microtubules spent in the pause (neither growing nor shortening detectably) state by 135% and reduced the dynamicity (dimer exchange per unit time) of microtubules by 70%. In vitro, BCFMT bound to tubulin with a dissociation constant of 8.3±1.8 µM, inhibited tubulin assembly and suppressed GTPase activity of microtubules. BCFMT competitively inhibited the binding of BODIPY FL-vinblastine to tubulin with an inhibitory concentration (Ki) of 5.2±1.5 µM suggesting that it binds to tubulin at the vinblastine site. In cultured cells, BCFMT-treatment depolymerized interphase microtubules, perturbed the spindle organization and accumulated checkpoint proteins (BubR1 and Mad2) at the kinetochores. BCFMT-treated MCF-7 cells showed enhanced nuclear accumulation of p53 and its downstream p21, which consequently activated apoptosis in these cells. The results suggested that BCFMT inhibits proliferation of several types of cancer cells including drug resistance cells by suppressing microtubule dynamics and indicated that the compound may have chemotherapeutic potential.

Differences in the Mechanisms of Proapoptotic BH3 Proteins Binding to Bcl-XL and Bcl-2 Quantified in Live MCF-7 Cells

Overexpression of antiapoptotic proteins including Bcl-XL and/or Bcl-2 contributes to tumor initiation, progression, and resistance to therapy by direct interactions with proapoptotic BH3 proteins. Release of BH3 proteins from antiapoptotic proteins kills some cancer cells and sensitizes others to chemotherapy. Binding of Bcl-XL and Bcl-2 to the BH3 proteins Bad, Bid, and the three major isoforms of Bim was measured for fluorescent protein fusions in live cells using fluorescence lifetime imaging microscopy and fluorescence resonance energy transfer. In cells the binding of the proteins at mitochondria is similar to the results from in vitro measurements. However, mutations in the BH3 region of Bim known to inhibit binding to Bcl-XL and Bcl-2 in vitro had much less effect in MCF-7 cells. Moreover, the BH3 mimetic ABT-737 inhibited Bad and Bid but not Bim binding to Bcl-XL and Bcl-2. Thus, the selectivity of ABT-737 also differs markedly from predictions made from in vitro measurements.

Abstract 4070: Treatment with the MEK inhibitor U0126 induces increased lactate production in prostate and breast cancer cell lines

Abstract Introduction. In this study we investigated the metabolic consequences of U0126, a highly selective MEK1/2 inhibitor, in human prostate and breast cancer cell lines using 1H and 13C magnetic resonance spectroscopy (MRS) and gene expression data. Methods. Prostate (PC3 and LNCaP) and breast (MCF7) human cancer cells were treated for 48 h with the IC50 dose of U0126. 1H MRS data were acquired from cell extracts and repeated univariate statistical testing with false discovery rate correction was performed to determine significant changes in metabolite levels. The rate of glycolysis was determined from dynamic studies in live cells using 13C MRS to monitor the metabolism of 1-13C-labeled glucose. Protein and gene expression levels were measured using Western blotting and microarray analysis. Results and Discussion. The effect of U0126 was confirmed by western blot analysis demonstrating a drop in p-ERK. The univariate statistical analysis revealed that the 48 h drug treatment induced several metabolic changes including depletion of intracellular aspartate, succinate and phosphocholine in the prostate cells. However, one of the most significant metabolic change in all three cell lines was a substantial increase in intracellular lactate levels following treatment, with lactate up 134±16% (p=0.002), 149±20% (p=0.001) and 213±43% (n=2) of control in PC3, LNCaP and MCF7 cells respectively. Dynamic studies in live MCF7 cells were in line with the extract data and showed that the rate of lactate production increased following treatment 169±43% from 260±98 fmol/cell/hour to 439±114 fmol/cell/hour (p=0.03). Preliminary studies in PC3 cells showed a similar trend. Consistent with the metabolic observations, overexpression of genes associated with glycolysis including PFKFB3, PDK1, HK2, and GLUT1 was observed in PC3 cells pointing to an increase in glycolytic enzymes induced by U0126 treatment. In an effort to explain this observation we assessed the expression of other signaling proteins and found that although U0126 inhibits its target kinase, increased Akt phosphorylation was also observed in the PC3 cells. Importantly Akt activation was previously reported to mediate an increase in glycolysis. We therefore hypothesize that the observed increases in lactate production and its intracellular accumulation following inhibition of MAPK kinase signaling result from activation of the PI3K/Akt pathway observed in our cells. Conclusion. Deregulation of the MAPK pathway is often involved in oncogenesis, and therefore inhibitors of the pathway are being investigated as targeted cancer therapeutics. Our results highlight the value of MRS in identifying metabolic consequences of treatment that may inform on molecular drug action. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4070. doi:10.1158/1538-7445.AM2011-4070

Abstract 2369: Isolation of circulating tumor cells by size exclusion using lithography fabricated precision microfilters

Abstract Background: We present a novel precision microfilter that achieves rapid and highly efficient isolation of circulating tumor cells (CTCs) from peripheral blood. Isolation of CTCs by size exclusion is a widely researched technique with the advantage of capturing cells without reliance on cell surface expression markers. For many years CTC filtration technology has relied on track-etch microfilters with randomly located pores and low-porosity. More recently, precision microfilters with high porosity have been tested for CTC isolation, but they are not widely available. We have developed a new method of fabricating precision, high porosity microfilters that are capable of high volume manufacturing. These microfilters are strong, clear, non-fluorescent, and have high CTC capture efficiencies. Methods: We describe an assay to capture and enumerate prefixed and live CTCs. MCF-7 human breast adenocarcinoma cells (ATCC) were cultured in DMEM until confluent. Cells were stained in a fixative/staining solution containing paraformaldehyde, acridine orange, and DAPI dilacetate. After incubation, a cell sample was placed on a microscope slide and cells counted to obtain the exact input. Cells were then washed into 7.5 mL whole human blood with 7.5 mL PBS and placed into a syringe. An 8 µm pore precision microfilter was placed into Swinnex filter holder, and the sample drawn by negative pressure through the filter at ∼10 mL/min. The microfilter was mounted onto a microscope slide and counted using a fluorescence microscope under FITC and DAPI settings. Cells were defined by their shape and coloration under FITC fluorescence and nuclear signature under DAPI fluorescence. The procedure was then repeated using a staining solution without fixative to capture live MCF-7 cells. Both live and fixed MCF-7 cell isolation by microfiltration were performed in triplicate. Results: Precision microfilters were able to capture MCF-7 cells spiked into 7.5 mL human blood with a recovery rate of 98±3% for fixed cells, and 90±2% for live cells. The filtration was performed in 2 minutes, and cell counts were obtained in 10 minutes. By contrast, track-etched microfilters captured only 73% of fixed cells and cell counts required 20 minutes. Conclusions: Creatv's precision microfilters are shown to recover MCF-7 cells more efficiently than track-etch filters. The pore uniformity and low fluorescent background allowed for faster and more accurate counts of spiked CTC in human peripheral blood compared to track-etch microfilters. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2369. doi:10.1158/1538-7445.AM2011-2369

Requirement of Caspase-3 for Apoptotic Calcium Release

Calcium is known to be an important second messenger in regulation of various physiological processes including execution of programmed cell death. Calcium release from endoplasmic reticulum (ER) triggers activation of multiple pro-apoptotic factors resulting in apoptosis progression and, ultimately, cell death. Apoptotic calcium release from ER stores is mediated by inositol 1,4,5-trisphosphate receptor (IP3R) channels. Different models have confirmed the importance of IP3R channels in modulation of apoptosis, however many details of IP3R-dependent calcium release still remain enigmatic. Previously, we have demonstrated that calcium release from IP3R channels in response to apoptotic stimuli occurs in two temporally distinct phases. A rapid oscillatory cytosolic calcium release during initiation of apoptotic signaling is followed by delayed and sustained increase in calcium levels associated with persistent opening of IP3R channels. Here we show that early elevations in cytosolic calcium do not require caspase-3 activity. Using MCF-7 cells deficient in caspase-3, we detected robust increase in calcium levels in response to staurosporine treatment indicating that calcium release during initiation of apoptosis occurs independently of caspase-3. To further determine contribution of caspase-3 in regulation of calcium release we generated a MCF-7 cell line stably expressing pro-caspase-3. Additionally, we used TAT-based transducing peptide to deliver active recombinant caspase-3 directly into living MCF-7 cells. Utilizing both methodologies, we found that caspase-3 has a marginal effect on the early events leading to cytosolic calcium elevations and irreversible commitment to apoptotic cell death. These results suggest that caspase-3 mediated truncation of IP3R channels is a consequence, not causative, of apoptotic signaling events.

Synthesis and characterization of DMAP-modified NPY Y1 receptor antagonists as acyl-transfer catalysts

Starting from the working hypothesis that specific chemical labelling may be an attractive approach to detect and study G protein-coupled receptors (GPCRs) we synthesized catalytically active antagonists of the neuropeptide Y1 receptor (Y1R). An argininamide-type Y1R antagonist scaffold was combined with a DMAP moiety via spacers of different length and chemical nature. These hybrid compounds have Y1R affinities in the two-digit nanomolar range and are capable of catalysing acyl-transfer reaction to surrogates of bionucleophiles, as demonstrated in the absence of cells by using esters of fluorescent dyes as substrates in buffer. By contrast, selective staining of Y1Rs on living MCF-7 cells was not achieved due to significant non-catalysed (Y1R ligand independent) reaction with biomolecules and the limited density of Y1R on the cell surface. Although this may also depend on insufficient selectivity of the staining reagents, the results of this study suggest that the general applicability of catalytic staining to GPCRs has to be reconsidered, as this approach is hampered by a very low portion of receptor of interest compared to the total amount of membrane proteins.

Curcumin suppresses the dynamic instability of microtubules, activates the mitotic checkpoint and induces apoptosis in MCF‐7 cells

In this study, curcumin, a potential anticancer agent, was found to dampen the dynamic instability of individual microtubules in living MCF-7 cells. It strongly reduced the rate and extent of shortening states, and modestly reduced the rate and extent of growing states. In addition, curcumin decreased the fraction of time microtubules spent in the growing state and strongly increased the time microtubules spent in the pause state. Brief treatment with curcumin depolymerized mitotic microtubules, perturbed microtubule-kinetochore attachment and disturbed the mitotic spindle structure. Curcumin also perturbed the localization of the kinesin protein Eg5 and induced monopolar spindle formation. Further, curcumin increased the accumulation of Mad2 and BubR1 at the kinetochores, indicating that it activated the mitotic checkpoint. In addition, curcumin treatment increased the metaphase/anaphase ratio, indicating that it can delay mitotic progression from the metaphase to anaphase. We provide evidence suggesting that the affected cells underwent apoptosis via the p53-dependent apoptotic pathway. The results support the idea that kinetic stabilization of microtubule dynamics assists in the nuclear translocation of p53. Curcumin exerted additive effects when combined with vinblastine, a microtubule depolymerizing drug, whereas the combination of curcumin with paclitaxel, a microtubule-stabilizing drug, produced an antagonistic effect on the inhibition of MCF-7 cell proliferation. The results together suggested that curcumin inhibited MCF-7 cell proliferation by inhibiting the assembly dynamics of microtubules.

Toward the detection of cellular copper(II) by a light-activated fluorescence increase.

A new type of Cu2+ fluorescent sensor, coucage, has been prepared with a photosensitive nitrophenyl group incorporated into the backbone of a coumarin-tagged tetradentate ligand. Coucage provides a selective fluorescence response for Cu2+ over other biologically relevant metal ions. Coordination of Cu2+ dims the fluorescence output until irradiation with UV light cleaves the ligand backbone, which relieves the copper-induced quenching to provide a turn-on response. Experiments in live MCF-7 cells show that coucage can be used for detecting changes in intracellular Cu2+ upon the addition of excess exogenous copper. If improvements can be made to increase its affinity for copper, this new type of turn-on sensor could be used as a tool for visualizing the cellular distribution of labile copper to gain insight into the mechanisms of copper trafficking.

Griseofulvin stabilizes microtubule dynamics, activates p53 and inhibits the proliferation of MCF-7 cells synergistically with vinblastine

BackgroundGriseofulvin, an antifungal drug, has recently been shown to inhibit proliferation of various types of cancer cells and to inhibit tumor growth in athymic mice. Due to its low toxicity, griseofulvin has drawn considerable attention for its potential use in cancer chemotherapy. This work aims to understand how griseofulvin suppresses microtubule dynamics in living cells and sought to elucidate the antimitotic and antiproliferative action of the drug.MethodsThe effects of griseofulvin on the dynamics of individual microtubules in live MCF-7 cells were measured by confocal microscopy. Immunofluorescence microscopy, western blotting and flow cytometry were used to analyze the effects of griseofulvin on spindle microtubule organization, cell cycle progression and apoptosis. Further, interactions of purified tubulin with griseofulvin were studied in vitro by spectrophotometry and spectrofluorimetry. Docking analysis was performed using autodock4 and LigandFit module of Discovery Studio 2.1.ResultsGriseofulvin strongly suppressed the dynamic instability of individual microtubules in live MCF-7 cells by reducing the rate and extent of the growing and shortening phases. At or near half-maximal proliferation inhibitory concentration, griseofulvin dampened the dynamicity of microtubules in MCF-7 cells without significantly disrupting the microtubule network. Griseofulvin-induced mitotic arrest was associated with several mitotic abnormalities like misaligned chromosomes, multipolar spindles, misegregated chromosomes resulting in cells containing fragmented nuclei. These fragmented nuclei were found to contain increased concentration of p53. Using both computational and experimental approaches, we provided evidence suggesting that griseofulvin binds to tubulin in two different sites; one site overlaps with the paclitaxel binding site while the second site is located at the αβ intra-dimer interface. In combination studies, griseofulvin and vinblastine were found to exert synergistic effects against MCF-7 cell proliferation.ConclusionsThe study provided evidence suggesting that griseofulvin shares its binding site in tubulin with paclitaxel and kinetically suppresses microtubule dynamics in a similar manner. The results revealed the antimitotic mechanism of action of griseofulvin and provided evidence suggesting that griseofulvin alone and/or in combination with vinblastine may have promising role in breast cancer chemotherapy.

Genetically Encoded Bioluminescent Indicator for ERK2 Dimer in Living Cells

In this study, a genetically encoded bioluminescent indicator for ERK2 dimer was developed with the split Renilla luciferase complementation method, in which the formation of ERK2 dimer induces a spontaneous emission of bioluminescence in living cells. In response to extracellular stimuli, such as epidermal growth factor (EGF) or 17beta-estradiol (E2), extracellular signal-regulated kinase 2 (ERK2) is phosphorylated by its upstream kinase MEK, and also phosphorylates its substrates in various regions of the cell, including the nucleus. Phosphorylated ERK2 is led to form its dimer, thereby transporting itself into the nucleus. We demonstrated with the indicator that stimulation with EGF or E2 induces the formation of ERK2 dimer in living MCF-7 cells. The dynamics of this dimer formation was examined and discussed.