Induction of G2/M phase arrest by squamocin in chronic myeloid leukemia (K562) cells

Squamocin is one of the annonaceous acetogenins and has been reported to have anticancer activity. Squamocin was found to inhibit the growth of K562 cells in a time- and dose-dependent manner. Cell cycle analysis showed G2/M phase arrest in K562 cells following 24 h exposure to squamocin. During the G2/M arrest, cyclin-dependent kinase inhibitors (CDKIs), p21 and p27 were increased in a dose-dependent manner. Analysis of the cell cycle regulatory proteins demonstrated that squamocin did not change the steady-state levels of Cdk2, Cdk4, cyclin A, cyclin B1, cyclin D3 and cyclin E, but decreased the protein levels of Cdk1 and Cdc25C. These results suggest that squamocin inhibits the proliferation of K562 cells via G2/M arrest in association with the induction of p21, p27 and the reduction of Cdk1 and Cdc25C kinase activities.     

Involvement of caspase-3 activation in squamocin-induced apoptosis in leukemia cell line HL-60

Annonaceous acetogenins have potent antitumor effect in vitro and in vivo. Squamocin is one of the annonaceous acetogenins and has been reported to have antiproliferative effect on cancer cells. Our results from this study showed that squamocin inhibited proliferation of HL-60 cells with IC50 value of 0.17 microg/ml and induced apoptosis of HL-60 cells. Investigation of the mechanism of squamocin-induced apoptosis revealed that treatment of HL-60 cells with squamocin resulted in extensive nuclear condensation. DNA fragmentation, cleavage of the death substrate poly (ADP-ribose) polymerase (PARP) and induction of caspase-3 activity. Pretreatment of HL-60 cells with caspase-3 specific inhibitor DEVD-CHO prevented squamocin-induced DNA fragmentation, PARP cleavage and cell death. The expression levels of protein bcl-2, bax have no change in response to squamocin treatment in HL-60 cells, whereas stress-activated protein kinase (SAPK/JNK) was activated after treatment with squamocin in HL-60 cells. These results suggest that apoptosis of HL-60 cells induced by squamocin requires caspase-3 activation and is related to SAPK activation.     

Squamocin mode of action to stimulate biofilm formation of Pseudomonas plecoglossicida J26, a PAHs degrading bacterium

Squamocin, an annonaceous acetogenin (ACG) extracted from Annona cherimolia (Annonaceae), has been shown to increase biofilm production of Pseudomonas plecoglossicida J26 (closely related to P. plecoglossicida), a polycyclic aromatic hydrocarbon degrading bacterium. PAHs have become priority pollutants for bioremediation due to their carcinogenicity and toxicity. The effect of various stressful stimuli (naphthalene, octanol, HCl, and NaCl) on cell growth, biofilm formation and autoinducer production of P. plecoglossicida were evaluated and compared with the effect of squamocin to establish its mode of action on biofilm formation. All stressors that inhibited growth stimulated autoinducer production while squamocin was growth stimulant at concentrations above 2.5 μg ml⁻¹. Despite structural similarities, squamocin is not an autoinducer agonist. It indirectly stimulates autoinducer production and increases P. plecoglossicida J26 cell growth. This is the first report on an ACG mode of action in the formation of biofilm in a naphthalene-degrading strain.     

Altered Expression of Proliferation-Inducing and Proliferation-Inhibiting Genes Might Contribute to Acquired Doxorubicin Resistance in Breast Cancer Cells

This study was designed to investigate the molecular changes that may develop during exposure of breast cancer cells to anticancer agents and that may lead to acquired resistance. We used two breast cancer cell lines, a parental (MCF7/WT) and a doxorubicin-resistant (MCF7/DOX) one. Cell survival, cell cycle distribution and RT-PCR expression level of genes involved in DNA damage response, MDR1, GST and TOPOIIα were measured. MCF7/DOX cells were five-fold more resistant to doxorubicin (DOX) than the MCF7/WT cells. DOX treatment causes arrest of MCF7/DOX cells in G1 and G2 phases of cell cycle whereas MCF7/WT cells were arrested in S-phase. The molecular changes in both cell lines due to DOX treatment could be classified into: (1) the basal level of p53, p21, BRCA1, GST and TOPOIIα mRNA was higher in MCF7/DOX than MCF7/WT. During DOX treatment, the expression level of these genes decreased in both cell lines but the rate of down-regulation was faster in MCF7/WT than MCF7/DOX cells. (2) The expression level of MDR1 was the same in both cell lines but 48 and 72 h of drug treatment, MDR1 disappeared in MCF7/WT but still expressed in MCF7/DOX. (3) There was no change in the expression level of BAX, FAS and BRCA2 in both cell lines. Conclusively, after validation in clinical samples, overexpression of genes like BRCA1, p53, p21, GST, MDR1 and TOPOIIα could be used as a prognostic biomarker for detection of acquired resistance in breast cancer and as therapeutic targets for the improvement of breast cancer treatment strategies.     

Calcitonin binding and degradation by two cultured human breast cancer cell lines (MCF 7 and T 47D).

Two human breast cancer cell lines (MCF 7 and T 47D) possess calcitonin-responsive adenylate cyclase systems. Suspended cells of both lines specifically bound 125I-labelled salmon calcitonin with mean dissociation constants of 1.7 nM (MCF 7) and 1.4 nM (T 47D); mean receptor numbers were 5300 and 24400 per cell respectively. Measurement of specific binding to MCF 7 cells was obscured by rapid and substantial degradation of the labelled hormone. Degradation of 125I-labelled salmon calcitonin: (i) was of high capacity; (ii) lacked the specificity displayed by 125I-labelled salmon calcitonin binding to the same cells; and (iii) was not related to binding since cell incubation supernatants retained full degrading activity. The degrading activity was inhibited by corticotropin (1-24)-tetracosapeptide, insulin and bacitracin. Inclusion of bacitracin in the incubation resulted in apparently fewer numbers of lower affinity receptors on MCF 7 cells, whereas these parameters were identical to T 47D cells incubated in the presence or absence of bacitracin. Eel [2-aminosuberic acid 1,7]-calcitonin was resistant to proteolysis in the presence of either cell line. Analysis of hormone-receptor interactions with calcitonin-responsive cells should take account of potent calcitonin-degrading activities in some cell lines.     

Acetonic extract of Buxus sempervirens induces cell cycle arrest, apoptosis and autophagy in breast cancer cells

Plants are an invaluable source of potential new anti-cancer drugs. Here, we investigated the cytotoxic activity of the acetonic extract of Buxus sempervirens on five breast cancer cell lines, MCF7, MCF10CA1a and T47D, three aggressive triple positive breast cancer cell lines, and BT-20 and MDA-MB-435, which are triple negative breast cancer cell lines. As a control, MCF10A, a spontaneously immortalized but non-tumoral cell line has been used. The acetonic extract of Buxus sempervirens showed cytotoxic activity towards all the five studied breast cancer cell lines with an IC(50) ranging from 7.74 μg/ml to 12.5 μg/ml. Most importantly, the plant extract was less toxic towards MCF10A with an IC(50) of 19.24 μg/ml. Fluorescence-activated cell sorting (FACS) analysis showed that the plant extract induced cell death and cell cycle arrest in G0/G1 phase in MCF7, T47D, MCF10CA1a and BT-20 cell lines, concomitant to cyclin D1 downregulation. Application of MCF7 and MCF10CA1a respective IC(50) did not show such effects on the control cell line MCF10A. Propidium iodide/Annexin V double staining revealed a pre-apoptotic cell population with extract-treated MCF10CA1a, T47D and BT-20 cells. Transmission electron microscopy analyses indicated the occurrence of autophagy in MCF7 and MCF10CA1a cell lines. Immunofluorescence and Western blot assays confirmed the processing of microtubule-associated protein LC3 in the treated cancer cells. Moreover, we have demonstrated the upregulation of Beclin-1 in these cell lines and downregulation of Survivin and p21. Also, Caspase-3 detection in treated BT-20 and T47D confirmed the occurrence of apoptosis in these cells. Our findings indicate that Buxus sempervirens extract exhibit promising anti-cancer activity by triggering both autophagic cell death and apoptosis, suggesting that this plant may contain potential anti-cancer agents for single or combinatory cancer therapy against breast cancer.     

Combined Fourier transform infrared and Raman spectroscopic approach for identification of multidrug resistance phenotype in cancer cell lines

Cancer cells escape cytotoxic effects of anticancer drugs by a process known as multidrug resistance (MDR). Identification of cell status by less time-consuming methods can be extremely useful in patient management and treatment. This study aims at evaluating the potentials of vibrational spectroscopic methods to perform cell typing and to differentiate between sensitive and resistant human cancer cell lines, in particular those that exhibit the MDR phenotype. Micro-Raman and Fourier transform infrared (FTIR) spectra have been acquired from the sensitive promyelocytic HL60 leukemia cell line and two of its subclones resistant to doxorubicin (HL60/DOX) and daunorubicin (HL60/DNR), and from the sensitive MCF7 breast cancer cell line and its MDR counterpart resistant to verapamil (MCF7/VP). Principal components analysis (PCA) was employed for spectral comparison and classification. Our data show that cell typing was feasible with both methods, giving two distinct clusters for HL60- and MCF7-sensitive cells. In addition, phenotyping of HL60 cells, i.e., discriminating between the sensitive and MDR phenotypes, was attempted by both methods. FTIR could not only delineate between the sensitive and resistant HL60 cells, but also gave two distinct clusters for the resistant cells, which required a two-step procedure with Raman spectra. In the case of MCF7 cell lines, both the sensitive and resistant phenotypes could be differentiated very efficiently by PCA analysis of their FTIR and Raman point spectra. These results indicate the prospective applicability of FTIR and micro-Raman approaches in the differentiation of cell types as well as characterization of the cell status, such as the MDR phenotype exhibited in resistant leukemia cell lines like HL60 and MCF7.     

Differential cross-talk of estrogen and growth factor receptors in two human mammary tumor cell lines

Cultured human mammary MCF7 and T47D tumor cell lines were used to test the interference of the partial antiestrogen 4′-hydroxytamoxifen (4-OH-TAM) and the pure antiestrogen ZM 182780 with growth factor (IGF-I, heregulin) signaling pathways. Growth of both cell lines was stimulated by IGF-I (20 ng/ml) or heregulin (3 nM). ZM 182780 effectively blocked growth factor induced as well as basal proliferation of MCF7 cells while the compound was ineffective in interfering with growth factor mitogenic activity in T47D cells. On both cell lines the IGF-I or heregulin- induced proliferation was enhanced further by 4-OH-TAM. This synergism could be inhibited dose-dependently by ZM 182780. When cells were grown in the presence of estradiol plus growth factors, the antiestrogenic potencies of both compounds and the efficacy of ZM 182780 were unaffected, while the efficacy of 4-OH-TAM was reduced. Our data show cell type specific cross-talk between the receptor for estrogen and that for IGF-I or heregulin, which is different in MCF7 and T47D cells, respectively. In MCF7 cells with demonstrable cross-talk, a clear superiority exists for a pure antiestrogen over a partial agonist in interfering with growth factor mitogenic activity.     

Effects of liposome-entrapped annamycin in human breast cancer cells: interference with cell cycle progression and induction of apoptosis

The effects of liposome-encapsulated annamycin (L-Ann) were investigated in two human breast cancer cell lines, MCF7 and MDA-MB-435. For comparative purposes, doxorubicin (Dx) was used throughout the study. A 4-hour treatment with L-Ann was significantly more active in MDA-MB-435 than in MCF7 cells (IC(50) values of 0.03 and 0.08 microg/ml, respectively), whereas Dx was equally active in the two cell lines (IC(50) 0.12 microg/ml). L-Ann induced an accumulation of cells in G2M phases which was dose-dependent in MDA-MB-435 but not in MCF7 cells. Dx also caused a dose-dependent increase of G2M cell fraction in MDA-MB-435 cells, whereas a G2M cell accumulation was observed only after treatment with the highest Dx concentration in MCF7 cells. G2M phase cell accumulations induced in MCF7 cells by L-Ann or Dx were accompanied by a decrease in cdc2 kinase activity and in cyclin B1 and cdc2 expression. Conversely, in MDA-MB-435 cells exposed to L-Ann or Dx, cdc2 kinase activity, cyclin B1 and cdc2 expression increased in parallel to the increase in the number of cells accumulated in the G2M phase. L-Ann and Dx induced apoptosis in MDA-MB-435 but not in MCF7 cells. In MDA-MB-435 cells exposed to L-Ann or Dx, no change was observed in the expression of bax, but there was a p53-independent increase in p21(waf1) expression. In MCF7 cells, treatment with L-Ann or Dx induced an increase in p53 expression with a consequent transactivation of p21(waf1) and bax. Our results indicate that L-Ann is more cytotoxic than Dx in breast cancer cells and is able to induce apoptosis through p53-independent mechanisms.     

BG-1 ovarian cell line: An alternative model for examining estrogen-dependent growth in vitro

Summary Examination of estrogen-responsive processes in cell culture is used to investigate hormonal influence on cancer cell growth and gene expression. Most experimental studies have used breast cancer cell lines, in particular MCF7 cells, to investigate estrogen responsiveness. In this study we examined an ovarian cancer cell line, BG-1, which is highly estrogen-responsive in vitro. This observation, plus the fact that the cells are of ovarian rather than mammary gland origin, makes it an attractive alternative model. 17β-Estradiol, epidermal growth factor, and insulin-like growth factor induced proliferation of BG-1 and MCF7 cells. Viability was dependent on these growth factors in BG-1 cells, but not in MCF7 cells. Therefore, we examined the differences between these two cell lines with respect to estrogen and growth factor receptors. BG-1 cells have twice as many estrogen receptors as MCF7 cells, and BG-1 cells have higher insulin-like growth factor-1 and epidermal growth factor receptor levels than MCF7 cells. This may also explain why BG-1 cells proliferate 56% more robustly in serum and show more serum dependence in culture. In both BG-1 and MCF7 cells, epidermal growth factor receptor number is low (<20 000/cell), while insulin-like growth factor-1 receptor level was highest in estrogen receptor positive cell lines. For example, insulin-like growth factor-1 receptor was higher in BG-1 and MCF7 cells than in estrogen receptor negative cells (HeLa>MDA-MB-435>HBL100). In conclusion, BG-1 cells are an excellent model for understanding hormone responsiveness in ovarian tissue and an alternative for examining estrogen receptor-mediated and insulin-like growth factor-1/epidermal growth factor/estrogen cross-talk processes because of their sensitivity to these factors.     

beta-estradiol stimulation of DNA synthesis requires different PKC isoforms in HepG2 and MCF7 cells.

The role exerted by protein kinase C (PKC) on estrogen-induced DNA synthesis has been investigated in hepatic and mammary gland cells, HepG2 and MCF7. 17-beta-estradiol stimulated DNA synthesis in HepG2 and MCF7 cells, maximal effect occurring at 10 nM. DNA synthesis stimulation was prevented by anti-estrogen ICI 182,780 and by inhibitor of PKC, Ro 31-8220. The rapid estradiol effects in MCF7 cells were determined by following the inositol trisphosphate (IP(3)) production and PKC-alpha membrane translocation. After estradiol treatment the increase of IP(3) production, prevented by anti-estrogen or by phospholipase C (PLC) inhibitor (neomycin), was present in MCF7 cells. In MDA cells, devoid of estrogen receptor, no effect was observed. The PKC-alpha presence on the membranes appeared unchanged in MCF7 cells. The PLC inhibitors, neomycin and U73,122, and PKC-alpha down regulator, phorbol 12-myristate 13-acetate (PMA), were able to prevent estradiol-induced DNA synthesis in hepatoma cells, but ineffective in mammary cells; wortmannin, an inhibitor of phosphoinositide 3-kinases (PI3-K), blocked DNA synthesis in both cell lines. These data show that beta-estradiol, via an estrogen receptor-mediated mechanism, activates more signal transduction pathways, and consequently different PKC isoforms in two responsive cell lines. In both cell lines PI3-K/PKC pathway is functional to the estrogen regulation of DNA synthesis, whereas in HepG2 cells the parallel involvement of the PLC/PKC-alpha pathway is present. The reported results indicate that the DNA synthesis stimulation by beta-estradiol requires the estrogen receptor and utilises one or more activated pathways in dependence on the cell equipment.     

Heat shock protein 70 and glycoprotein 96 are differentially expressed on the surface of malignant and nonmalignant breast cells

Heat shock proteins (HSPs), which are important for a number of different intracellular functions, are occasionally found on the surface of cells. The function of heat shock protein on the cell surface is not understood, although it has been shown to be greater in some tumor cells and some virally infected cells. Surface expression of both glycoprotein 96 (gp96) and Hsp70 occurs on tumor cells, and this expression correlates with natural killer cell killing of the cells. We examined the surface expression of gp96 and Hsp70 on human breast cell lines MCF7, MCF10A, AU565, and HS578, and in primary human mammary epithelial cells by immunofluorescence microscopy and flow cytometry. The nonmalignant cell lines HS578, MCF10A, and HMEC showed no surface expression of gp96, whereas malignant cell lines MCF7 and AU565 were positive for gp96 surface expression. All of the breast cell lines examined showed Hsp70 surface expression. These results also confirm previous studies, demonstrating that Hsp70 is on the plasma membrane of tumor cell lines. Given the involvement of heat shock proteins, gp96 and Hsp70, in innate and adaptive immunity, these observations may be important in the immune response to tumor cells.     

端粒酶反义cDNA对乳腺癌细胞恶性表型的影响

 为了进一步探讨端粒酶在肿瘤发生中的作用 ,实验应用反义核酸技术研究端粒酶反义cDNA对乳腺癌细胞MCF 7恶性表型的影响 采用的方法包括 :基因重组、脂质体共转染法获得端粒酶反义重组病毒 ,病毒感染MCF 7后 ,检测细胞的生长曲线、细胞周期及集落形成能力 结果表明 ,与对照组细胞相比 ,反义病毒感染后的MCF 7细胞恶性表型明显降低 提示端粒酶RNA的反义cDNA的导入 ,可以显著抑制乳腺癌细胞恶性表型     

Vasoactive intestinal peptide-camptothecin conjugates inhibit the proliferation of breast cancer cells

The effects of vasoactive intestinal peptide-camptothecin (VIP-CPT) conjugates were investigated on breast cancer cells and cells transfected with VIP receptors (R). (Ala(2,8,9,19,24.25.27), Nle(17), Lys(28))VIP, (A-NL-K)VIP, was synthesized and Lys(28) was coupled to a linker, N-methyl-amino-ethyl-glycine, L2, which formed a carbamate bond with CPT. The resulting (A-NL-K)VIP-L2-CPT was cytotoxic for MCF7 breast cancer cells, which have VPAC(1)-R, with IC(50) values of 380 and 90 nM using the MTT and clonogenic assays, respectively. (A-NL-K)VIP, (A-NL-K)VIP-L2 and (A-NL-K)VIP-L2-CPT inhibited specific binding of (125)I-VIP to 3T3 cells transfected with VPAC(1)-R with IC(50) values of 1.9, 56 and 126 nM, respectively. In contrast, (A-NL-K)VIP, (A-NL-K)VIP-L2 and (A-NL-K)VIP-L2-CPT inhibited specific binding of (125)I-Ro25-1553 to 3T3 cells transfected with VPAC(2)-R with IC(50) values of 3.9, 3162 and 2690 nM, respectively. (A-NL-K)VIP, (A-NL-K)VIP-L2 and (A-NL-K)VIP-L2-CPT caused increased cAMP after addition to MCF7 cells. (125)I-(A-NL-K)VIP-L2-CPT was internalized by MCF7 cells at 37 degrees C but not 4 degrees C. These results indicate that (A-NL-K)VIP-L2-CPT is a VPAC(1)-R agonist which is cytotoxic for breast cancer cells.     

Modulation of protein expression levels and DNA methylation status of breast cancer metastasis genes by anthracycline-based chemotherapy and the demethylating agent decitabine

Epigenetic drugs are promising add‐ons to cancer treatment; still, adverse effects concerning tumour promotion have been reported occasionally. In this in vitro study, we investigated the effect of combination treatment of decitabine with anthracycline‐based chemotherapy [5‐fluorouracil plus epirubicine plus cyclophosphamide (FEC)] on viability and metastatic activity of breast cancer cell lines, MDA‐MB‐231 (estrogen receptor‐negative) and MCF‐7 (estrogen receptor‐positive). The effect of decitabine and its combined treatment with FEC on viability of both cancer cell lines was assessed using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazoliumbromide and adenosine triphosphate (ATP) cell survival assays. DNA methylation specific real‐time polymerase chain reaction (PCR) (Methylight®) was employed to document the methylation status of the metastasis‐relevant urokinase‐type plasminogen activator (uPA) and plasminogen activator inhibitor‐I (PAI‐1) genes. Additionally, protein expression levels of uPA and PAI‐1 were determined using enzyme‐linked immunosorbent assays. Invasion capacity of cells was assayed using Matrigel® invasion assay. Decitabine lowered the viability of MCF‐7 cells, although MDA‐MB‐231 cells were not affected. Decitabine did not augment FEC‐mediated cytotoxicity in both cell lines. In MCF‐7 cells, methylation of the uPA and PAI‐1 gene promoter was significantly reduced by decitabine or decitabine plus FEC. Protein levels of uPA and PAI‐1 were induced by all treatments. Decitabine significantly induced the invasion capacity of MCF‐7 cells, whereas all of the drugs resulted in decreased invasion capacity of MDA‐MB‐231. Our results suggest differential effects of single‐dose decitabine and its combination with FEC on the metastatic capacity and survival of breast cancer cell lines endowed with different metastatic behaviour. Copyright © 2011 John Wiley & Sons, Ltd.     

In vitro antitumor activities of 2,6-di-[2-(heteroaryl)vinyl]pyridines and pyridiniums

The in vitro antitumor activities of 2,6-di-[2-(heteroaryl)vinyl]pyridines versus the standard National Cancer Institute 60 cell lines panel and of 2,6-di-[2-(heteroaryl)vinyl] pyridinium cations versus MCF7 (human mammary carcinoma) and LNCap (prostate carcinoma) cell lines are reported. Antiproliferative effects in both series are particularly evident for MCF7 mammary adenocarcinoma cells. Multivariate analysis of DNA microarray data for responsive tumor cell lines suggest a mechanistic pathway involving polyamine biosynthesis and prolactin signal transduction.     

Synthesis and biological evaluation of new taxoids derived from 2-deacetoxytaxinine J

A small library of 2-deacetoxytaxinine J (DAT-J) 1 derivatives was synthesised and tested in vitro for their reversal activity in human mammary carcinoma MDR cell line MCF7-R. One of the new taxoids showed to be active at 0.1 microM when tested in combination with paclitaxel.     

Prolactin-dependent up-regulation of BRCA1 expression in human breast cancer cell lines.

We report experimental evidence that BRCA1, a breast and ovarian cancer susceptibility gene, is up-regulated in response to prolactin (PRL) stimulation. Expression of the BRCA1 gene was monitored in 2 human breast cancer cell lines (MCF-7 and T-47D) and in the normal mammary epithelial cell line MCF10a. Using competitive RT-PCR, we have shown that PRL induced an increase in BRCA1 mRNA level in MCF-7 and T-47D cell lines at a dose resulting in the maximal enhancement of cell proliferation. The up-regulation was 12-fold in MCF-7 cells and 2-fold in T-47D cells. No increase in BRCA1 mRNA level was observed in the MCF10a cell line. The level of BRCA1 protein was quantified using an affinity chromatography strategy. At the protein level, PRL treatment induced a 4-fold increase of BRCA1 protein expression in MCF-7 and a 6-fold increase in T-47D cells, whereas BRCA1 protein expression was not affected by PRL in MCF10a.