Ribozyme targeting demonstrates that the nuclear receptor coactivator AIB1 is a rate-limiting factor for estrogen-dependent growth of human MCF-7 breast cancer cells

Human breast tumorigenesis is promoted by the estrogen receptor pathway, and nuclear receptor coactivators are thought to participate in this process. Here we studied whether one of these coactivators, AIB1 (amplified in breast cancer 1), was rate-limiting for hormone-dependent growth of human MCF-7 breast cancer cells. We developed MCF-7 breast cancer cell lines in which the expression of AIB1 can be modulated by regulatable ribozymes directed against AIB1 mRNA. We found that depletion of endogenous AIB1 levels reduced steroid hormone signaling via the estrogen receptor alpha or progesterone receptor beta on transiently transfected reporter templates. Down-regulation of AIB1 levels in MCF-7 cells did not affect estrogen-stimulated cell cycle progression but reduced estrogen-mediated inhibition of apoptosis and cell growth. Finally, upon reduction of endogenous AIB1 expression, estrogen-dependent colony formation in soft agar and tumor growth of MCF-7 cells in nude mice was decreased. From these findings we conclude that, despite the presence of different estrogen receptor coactivators in breast cancer cells, AIB1 exerts a rate-limiting role for hormone-dependent human breast tumor growth.     

Interconnections between E2-dependent regulation of cell cycle progression and apoptosis in MCF-7 tumors growing on nude mice

Growth of human breast adenocarcinoma MCF-7 cells as a tumor on nude mice is dependent on estrogen. It has been shown that estrogen withdrawal (EW) induces a partial regression of the tumor via an inhibition of cell proliferation and an induction of apoptosis. We investigated in this in vivo model the underlying molecular mechanisms of the hormone-dependent regulation of cell cycle machinery and apoptosis. We found that, 2 days after EW, the tumor protein levels of p21 rose, whereas those of Rb proteins decreased in parallel with the decrease in the proportion of tumor cells in S phase and the increase of the tumor apoptotic index. Between 3 and 7 days after EW, apoptosis was inhibited and tumor proliferation returned to the control value. There was a concomitant decline in p21 and an elevation of Rb tumor protein content. Slight variations of cyclin D protein level were observed in MCF-7 tumors over the time course following EW treatment. Bcl-2 overexpression not only inhibited apoptosis induced by EW but also modulated hormone-dependent cell cycle regulation. First, the analysis of phosphorylation status of Rb protein and the measurement of the proportion of tumor cells in S phase indicated that Bcl-2 overexpression results in a decrease of DNA synthesis induced by estradiol. Furthermore, after EW, Bcl-2-induced inhibition of hormone-dependent apoptosis was associated with an inhibition of Rb protein downregulation, a sustained level of p21 protein, and a prolonged inhibition of cell cycle progression. These results suggest that, in human hormone-dependent breast cancers, cross-talk exists between the signaling pathways which lead to regulation of cell cycle progression and apoptosis.     

Selective inhibition of carbonic anhydrase-IX by sulphonamide derivatives induces pH and reactive oxygen species-mediated apoptosis in cervical cancer HeLa cells

Selective inhibition with sulphonamides of carbonic anhydrase (CA) IX reduces cell proliferation and induces apoptosis in human cancer cells. The effect on CA IX expression of seven previously synthesised sulphonamide inhibitors, with high affinity for CA IX, as well as their effect on the proliferation/apoptosis of cancer/normal cell lines was investigated. Two normal and three human cancer cell lines were used. Treatment resulted in dose- and time-dependent inhibition of the growth of various cancer cell lines. One compound showed remarkably high toxicity towards CA IX-positive HeLa cells. The mechanisms of apoptosis induction were determined with Annexin-V and AO/EB staining, cleaved caspases (caspase-3, caspase-8, caspase-9) and cleaved PARP activation, reactive oxygen species production (ROS), mitochondrial membrane potential (MMP), intracellular pH (pHi), extracellular pH (pHe), lactate level and cell cycle analysis. The autophagy induction mechanisms were also investigated. The modulation of apoptotic and autophagic genes (Bax, Bcl-2, caspase-3, caspase-8, caspase-9, caspase-12, Beclin and LC3) was measured using real time PCR. The positive staining using γ-H2AX and AO/EB dye, showed increased cleaved caspase-3, caspase-8, caspase-9, increased ROS production, MMP and enhanced mRNA expression of apoptotic genes, suggesting that anticancer effects are also exerted through its apoptosis-inducing properties. Our results show that such sulphonamides might have the potential as new leads for detailed investigations against CA IX-positive cervical cancers.     

Antisense-mediated Inhibition of the plasma membrane calcium-ATPase suppresses proliferation of MCF-7 cells

Alterations in Ca2+ signaling may contribute to tumorigenesis and the mechanism of action of some anti-cancer drugs. The plasma membrane calcium-ATPase (PMCA) is a crucial controller of intracellular Ca2+ signaling. Altered PMCA expression occurs in the mammary gland during lactation and in breast cancer cell lines. Despite this, the consequences of PMCA inhibition in breast cancer cell lines have not been investigated. In this work, we used Tet-off PMCA antisense-expressing MCF-7 cells to assess the effects of PMCA inhibition in a human breast cancer cell line. At a level of PMCA inhibition that did not completely prevent PMCA-mediated Ca2+ efflux and did not induce cell death, a dramatic inhibition of cellular proliferation was observed. Fluorescence-activated cell sorting analysis indicated that PMCA antisense involves changes in cell cycle kinetics but not cell cycle arrest. We concluded that modulation of PMCA has important effects in regulating the proliferation of human breast cancer MCF-7 cells.     

Selective cytotoxicity of synthesized procyanidin 3-O-Galloylepicatechin-4b,8-3-O-galloylcatechin to human cancer cells

Cocoa-derived flavanols and procyanidins have been previously reported to exhibit anti-oxidant and anti-tumor properties. In this study, we have investigated the cellular growth inhibitory effect of chemically-synthesized procyanidin 3-O-Galloylepicatechin-4b,8-3-O-galloylcatechin (GECGC) on a variety of human cancer cell lines. Among 16 human cancer cell lines tested, GECGC selectively inhibited proliferation of a subset of human cancer cell lines, especially those of short doubling time. In contrast, all 6 normal cell lines tested including human mammary epithelial cells and skin fibroblast were resistant to GECGC’s cytotoxicity. Cell cycle analysis and apoptosis assay showed that GECGC increased sub-G1 population and increased the population of propidium iodide and Annexin V staining cells in GECGC-sensitive cell lines, suggesting that cell growth inhibition by GECGC may be mediated through both apoptotic and non-apoptotic mechanisms. Further characterization of GECGC cytotoxicity on 30 genetically modified cell lines with overexpression or depletion of key proteins involved in cell cycle regulation and signal transduction pathways suggested that GECGC-mediated cell death involves IKKα and IKKγ. Collectively, our observations indicate that synthesized GECGC has selective anti-proliferative effect on human cancer cells and warrant further evaluation as a preventive and chemotherapeutic reagent to human malignancies.     

Induction of apoptosis in breast cancer cells by apomine is mediated by caspase and p38 mitogen activated protein kinase activation

The 1,1-bisphosphonate ester family member apomine (SR-45023A) is known to have anti-tumour activity in various cancer cell types. The aims of this study were to determine the effect of apomine on the growth of two breast cancer cell lines, MCF-7 and MDA-MB-231, to ascertain whether any growth inhibitory effects found were due to induction of apoptosis, and to investigate the mechanism of action of apomine. Apomine caused significant growth inhibition of both cell lines after 72h of treatment. Apomine-induced growth inhibition was associated with caspase and p38 MAPK activation and DNA fragmentation. Apomine had no effect on Ras localisation, nor did addition of mevalonate to treatment media prevent apomine-induced apoptosis. We conclude that apomine induces apoptosis in breast cancer cells, an effect that is independent of oestrogen receptor status and is not via inhibition of the mevalonate pathway. Our study suggests apomine is a potential anti-neoplastic drug in breast cancer treatment.     

Apoptosis and Autophagy in Breast Cancer Cells following Exemestane Treatment

Aromatase inhibitors (AIs), which block the conversion of androgens to estrogens, are used for hormone-dependent breast cancer treatment. Exemestane, a steroidal that belongs to the third-generation of AIs, is a mechanism-based inhibitor that binds covalently and irreversibly, inactivating and destabilizing aromatase. Since the biological effects of exemestane in breast cancer cells are not totally understood, its effects on cell viability, cell proliferation and mechanisms of cell death were studied in an ER-positive aromatase-overexpressing breast cancer cell line (MCF-7aro). The effects of 3-methyladenine (3-MA), an inhibitor of autophagy and of ZVAD-FMK, an apoptotic inhibitor, in exemestane treated cells were also investigated. Our results indicate that exemestane induces a strong inhibition in MCF-7aro cell proliferation in a dose- and time-dependent manner, promoting a significant cell cycle arrest in G(0)/G1 or in G(2)/M phases after 3 and 6 days of treatment, respectively. This was accompanied by a decrease in cell viability due to activation of cell death by apoptosis, via mitochondrial pathway and the occurrence of autophagy. Inhibition of autophagy by the autophagic inhibitor, 3-MA, resulted in a reduction of cell viability and activation of caspases. All together the results obtained suggest that exemestane induced mitochondrial-mediated apoptosis and autophagy, which act as a pro-survival process regulating breast cancer cell apoptosis.     

Estrogen receptor β agonists affect growth and gene expression of human breast cancer cell lines

Expression of estrogen receptor β (ERβ) has been described to reduce growth of cancer cell lines derived from hormone-dependent tumors, like breast cancer. In this study we tested to what extent two ERβ agonists, androgen derivative 3β-Adiol and flavonoid Liquiritigenin, would affect growth and gene expression of different ERβ-positive human breast cancer cell lines. Under standard cell culture conditions, we observed 3β-Adiol to inhibit growth of MCF-7 cells in a dose-dependent manner, whereas growth of BT-474 and MCF-10A cells was suppressed by the maximum concentration (100 nM) only. When treated in serum-free medium, all cell lines except of MDA-MB-231 were responsive to 1 nM 3β-Adiol, and ZR75-1 cells exhibited a dose-dependent antiproliferative response. Providing putative mechanisms underlying the observed growth-inhibitory effect, expression of Ki-67 or cyclins A2 and B1 was downregulated after 3β-Adiol treatment in all responsive lines. In contrast, treatment with lower doses of Liquiritigenin did not affect growth. In MCF-7 cells, the highest dose of this flavonoid exerted proliferative effects accompanied by increased expression of cyclin B1, PR and PS2, indicating unspecific activation of ERα. In conclusion, the ERβ agonists tested exerted distinct concentration-dependent and cell line-specific effects on growth and gene expression. The observed inhibitory effects of 3β-Adiol on breast cancer cell growth encourage further studies on the potential of this and other ERβ agonists as targeted drugs for breast cancer therapy.     

Synthesis, characterization and anti-tumor activity of moxifloxacin-copper complexes against breast cancer cell lines

Novel moxifloxacin-copper complexes were synthesized, characterized and screened for anti-proliferative and apoptosis-inducing activity against multiple human breast cancer cell lines (hormone-dependent MCF-7 and T47D as well as hormone-independent MDA-MB-231 and BT-20). The results indicated that the parent compound moxifloxacin (1) does not exert any inhibitory activity against breast cancer cell lines examined. On the other hand, the copper conjugate 2 and its nitrogen adducts 3-5 exerted growth inhibitory and apoptosis-inducing activity against breast cancer cell lines without any substantial effect on non-tumorigenic breast epithelial cells MCF-10A at equimolar concentration, suggesting a cancer cell-specific activity. BT-20 cells were more sensitive to compounds 2 and 3, while compounds 4 and 5 exerted significant anti-proliferative and apoptosis-inducing effects on T47D, MDA-MB-231 and BT-20 cell lines. Our results suggest that these novel compounds could be useful for the treatment of breast cancer in the future.     

视黄酸对胃癌细胞周期的调控

Retinoic acid can induce growth inhibition and apoptosis, and regulate cell cycle in many types of cancer cell lines. In this study, we investigated the role of all-trans retinoic acid (ATRA) and its mechanism of action in human gastric cancer cell lines. Our results demonstrated that ATRA effectively inhibited growth in three of four gastric cancer cell lines by induction of G0/G1 arrest, and did not induce apoptosis in four gastric cancer cell lines. In RA-sensitive cell lines, ATRA-induced G0/G1 arrest is associated with down regulaton of c-myc and hyperphosphorylated Rb expression, and up regulation of p21WAF1/CIP1 and p53 expression. There were no significant changes in cyclin D1 or CDK4 expression induced by ATRA. Futhermore, expression of these genes were not regulated by ATRA in ATRA-resistant gastric cancer cell line. These results indicate that growth inhibition, rather than apoptosis, is correlated with G0/G1 arrest of these cell lines, more important molecules related cell cycle, including c-myc, p21WAF1/CIP1, p53 and Rb, are involveed in regulation of cell cycle in gastric cancer cells.     

Effect of Medrogestone on 17beta-hydroxysteroid dehydrogenase activity in the hormone-dependent MCF-7 and T-47D human breast cancer cell lines

Estradiol (E2) is one of the most important hormones supporting the growth and evolution of breast cancer. Consequently, to block this hormone before it enters the cancer cell, or in the cell itself, has been one of the main targets in recent years. In the present study we explored the effect of Medrogestone (Prothil) on 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activities of the hormone-dependent MCF-7 and T-47D human breast cancer cell lines. Using physiological doses of estrone ([3H]-E1: 5 x 10(-9) mol/l) this estrogen is converted in a great proportion to E2 in both cell lines. After 24 h of the cell culture, Medrogestone significantly inhibits this transformation in a dose-dependent manner by 39% and 80% at 5 x 10(-8) M and 5 x 10(-5) M, respectively in T-47D cells; the effect is less intense in MCF-7 cells: 25% and 55% respectively. The IC50 values are 0.45 micromol/l in T-47D and 17.36 micromol/l in MCF-7 cells. It is concluded that the inhibition provoked by Medrogestone on the reductive 17beta-HSD activity involved in the local biosynthesis of the biologically active estrogen estradiol, may constitute a new therapeutic approach for the treatment of breast cancer.     

视黄酸对胃癌细胞周期的调控

视黄酸(RA)能够抑制许多类型癌细胞生长、诱导细胞凋亡和调节细胞周期。本文研究了全反式视黄酸(ATRA)对人胃癌细胞的作用机理。结果表明,ATRA通过诱导细胞滞留在G_0/G_1期而显著抑制胃癌细胞生长,但ATRA不能诱导胃癌细胞凋亡;ATRA调控细胞周期与c-myc、磷酸化Rb水平的下调和p21~(WAF1/CIP1)、p53水平的上调有关,而cyclinD_1和CDK_4水平没有明显变化。在RA抗性细胞中,ATRA不能调节这些基因表达。结果证实,ATRA对胃癌细胞生长抑制与其诱导细胞滞留在G_0/G_1期有关,而与细胞凋亡的诱导无关,许多重要的、与周期相关的分子,包括cmyc、p21~(WAF1/CIP1、p53和Rb等参与细胞周期的调控。     

Effects of olive oil polyphenols on fatty acid synthase gene expression and activity in human colorectal cancer cells

Oleuropein (OL) and hydroxytyrosol (HT), the main olive oil polyphenols, possess anti-proliferative effects in vitro. Fatty acid synthase, a key anabolic enzyme of biosynthesis of fatty acids, plays an important role in colon carcinoma development. Our aim was to investigate whether gene expression of FAS, as well as its enzymatic activity, is regulated by HT and OL in two human colon cancer cell lines, as HT-29 and SW620. In addition, we investigated the effects of these polyphenols on growth and apoptosis in these cells. FAS gene expression and activity in treated HT-29 and SW620 cells were evaluated by real-time PCR and radiochemical assay, respectively. Cell growth and apoptosis, after polyphenols treatment, were measured by MTT test and flow cytometry, respectively. The inhibition of proliferation, detected after HT treatment, was mediated by an inhibition of FAS expression and its enzymatic activity in SW620 cells, while the anti-proliferative effect in HT-29 cells seems to be independent from FAS. OL exerted an anti-proliferative effect only on SW620 cells with a mechanism which excluded FAS. Olive oil polyphenols used were able to induce apoptosis in both cell lines studied. The increase of apoptosis in these cells was accompanied by the block of cell cycle in the S phase. This study demonstrates that HT and OL may induce anti-proliferative and pro-apoptotic effects only in certain human colorectal cancer cell types. These effects are FAS mediated only in SW620 cells after treatment with HT.     

Regulation of transforming growth factor alpha and transforming growth factor beta messenger ribonucleic acid abundance in T-47D, human breast cancer cells

Both transforming growth factor beta (TGF beta) and TGF alpha mRNA are expressed in human breast cancer cell lines. We have investigated the relationship of mRNA abundance for these growth modulators to the proliferation rate of a number of human breast cancer cell lines. Furthermore, we have investigated the relationship of regulation of TGF beta and TGF alpha mRNA to growth inhibition caused by progestins and nonsteroidal antiestrogens in T-47D human breast cancer cells. The abundance of TGF beta and TGF alpha mRNA in human breast cancer cell lines was not related directly to proliferation rate of the cells in culture or estrogen receptor positivity or negativity. The relationship of TGF beta and TGF alpha mRNA to growth inhibition caused by antiestrogens and progestins was investigated in T-47D human breast cancer cells. We observed that in T-47D human breast cancer cells the abundance of TGF beta mRNA is decreased in a time- and dose-dependent fashion by progestins but remains unaltered by nonsteroidal antiestrogens. Treatment of T-47D cells for 24 h with 10 nM medroxyprogesterone acetate (MPA) reduced the level of TGF beta mRNA to one third that present in untreated cells. The same treatment increased TGF alpha mRNA 3-fold above untreated controls in a time- and dose-dependent fashion and nonsteroidal antiestrogens caused a small decrease. The regulation of both TGF alpha and TGF beta mRNA was not directly related to inhibition of growth by progestins and antiestrogens in T-47D cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human umbilical cord Wharton's jelly stem cell (hWJSC) extracts inhibit cancer cell growth in vitro

Umbilical cord mesenchymal stem cells (MSCs) have been shown to inhibit breast cancer cell growth but it is not known whether this effect is specific to only breast cancer cells. We compared the effects of human Wharton's jelly stem cell (hWJSC) extracts [conditioned medium (hWJSC‐CM) and cell lysate (hWJSC‐CL)] on breast adenocarcinoma (MDA‐MB‐231), ovarian carcinoma (TOV‐112D), and osteosarcoma (MG‐63) cells. The cells were treated with either hWJSC‐CM (50%) or hWJSC‐CL (15 µg/ml) for 48–72 h and changes in cell morphology, proliferation, cycle, gene expression, migration, and cell death studied. All three cancer cell lines showed cell shrinkage, blebbing, and vacuolations with hWJSC‐CL and hWJSC‐CM compared to controls. MTT and BrdU assays showed inhibition of cell growth by 2–6% and 30–60%, while Transwell migration assay showed inhibition by 20–26% and 31–46% for hWJSC‐CM and hWJSC‐CL, respectively, for all three cancer cell lines. Cell cycle assays showed increases in sub‐G1 and G2/M phases for all three cancer cell lines suggestive of apoptosis and metaphase arrest. AnnexinV‐FITC and TUNEL positive cells seen in TOV‐112D and MDA‐MB‐231 suggested that inhibition was via apoptosis while the presence of anti‐BECLIN1 and anti‐LC3B antibodies seen with MG‐63 indicated autophagy. Upregulation of pro‐apoptotic BAX and downregulation of anti‐apoptotic BCL2 and SURVIVIN genes were observed in all three cancer cell lines and additionally the autophagy genes (ATG5, ATG7, and BECLIN1) were upregulated in MG‐63 cells. hWJSCs possess tumor inhibitory properties that are not specific to breast cancer cells alone and these effects are mediated via agents in its extracts. J. Cell. Biochem. 113: 2027–2039, 2012. © 2012 Wiley Periodicals, Inc.     

Bax translocation mediated mitochondrial apoptosis and caspase dependent photosensitizing effect of Ficus religiosa on cancer cells

The main aim of the present work was to investigate the potential effect of acetone extract of Ficus religosa leaf (FAE) in multiple apoptosis signalling in human breast cancer cells. FAE treatment significantly induced dose and time dependent, irreversible inhibition of breast cancer cell growth with moderate toxicity to normal breast epithelial cells. This observation was validated using Sulforhodamine B assay. Cell cycle analysis by Flow cytometry showed cell cycle arrest in G1 phase and induction of sub-G0 peak. FAE induced chromatin condensation and displayed an increase in apoptotic population in Annexin V-FITC/PI (Fluorescein isothiocyanate/Propidium iodide) double staining. FAE stimulated the loss of mitochondrial membrane potential in multiple breast cancer cell lines when compared to normal diploid cells. To understand the role of Bax in FAE induced apoptosis, we employed a sensitive cell based platform of MCF-7 cells expressing Bax-EGFP. Bax translocation to mitochondria was accompanied by the disruption of mitochondrial membrane potential and marked elevation in LEHDase activity (Caspase 9). Consistent with this data, FAE induced Caspase activation as evidenced by ratio change in FRET Caspase sensor expressing MCF-7 cell line and cleavage of prominent Caspases and PARP. Interestingly, FAE accelerated cell death in a mitochondrial dependent manner in continuous live cell imaging mode indicating its possible photosensitizing effect. Intracellular generation of reactive oxygen species (ROS) by FAE played a critical role in mediating apoptotic cell death and photosensitizing activity. FAE induced dose and time dependent inhibition of cancer cell growth which was associated with Bax translocation and mitochondria mediated apoptosis with the activation of Caspase 9 dependent Caspase cascade. FAE also possessed strong photosensitizing effect on cancer cell line that was mediated through rapid mitochondrial transmembrane potential loss and partial Caspase activation involving generation of intracellular ROS.     

Antiproliferative activity and cell cycle analysis of 2-(3,5-dihydroxyphenyl)-6-hydroxybenzothiazole on MCF-7 breast and HCT-15 colon cancer cell lines

The antiproliferative activity of 2-(3,5-Dihydroxyphenyl)-6-hydroxybenzothiazole (DHB) is reported here. DHB inhibits the growth of human colon cancer HCT-15 with a 50% cell growth inhibition value of 23?μM and breast cancer MCF-7 with a 50% cell growth inhibition value of 41?μM in a dose/time dependent manner by using sulforhodamine B assay. Cell cycle analysis by flow cytometry showed that DHB-induced growth arrest could be associated with apoptosis in both cell lines. Moreover, suppression of clonogenic activity occurs after exposure to DHB at a concentration of 25?μM for HCT-15 and of 40?μM for MCF-7.