Topotecan and methotrexate alter expression of the apoptosis-related genes BCL2, FAS and BCL2L12 in leukemic HL-60 cells

The BCL2 family of genes (B-cell CLL/lymphoma 2; Bcl-2) plays a pivotal role in the highly regulated process of apoptosis. We have recently cloned a newly identified member of this family, BCL2L12, which was found to be differentially expressed in many tumors. It is known that topotecan and methotrexate act through induction of apoptosis in cancer cells. In the present study we investigated the expression profile of the novel apoptotic gene BCL2L12 in relation to other apoptotic genes in the human leukemic cell line HL-60, after treatment with topotecan or methotrexate. The kinetics of apoptosis induction and cell toxicity were investigated by DNA laddering and the MTT method, respectively. Gene expression levels were analyzed by RT-PCR using gene-specific primers. Downregulation of BCL2L12, BCL2 and FAS was observed after treatment of HL-60 cells with topotecan, while treatment with methotrexate led to downregulation of BCL2 and FAS, with no change in BCL2L12 expression. Our results support the significance of mRNA modulations in the expression of apoptosis-related genes during treatment of human leukemic cells with anticancer drugs.     

Cisplatin downregulates BCL2L12, a novel apoptosis-related gene, in glioblastoma cells

Glioblastoma progression is mainly characterized by intense apoptosis resistance and marked necrosis. Over-expression of BCL2L12, a novel member of Bcl-2 family has been shown in primary glioblastoma. BCL2L12 blocks effective caspase-3/7 maturation and inhibits p53 tumor suppressor, deriving resistance toward apoptosis and inducing extensive cell necrosis. Cisplatin is a major chemotherapeutic agent which has a broad range of anti-neoplastic activities including apoptosis induction. To investigate the effect of cisplatin on the expression of BCL2L12 in glioblastoma cells, two glioblastoma cell lines were treated with different concentrations of cisplatin for 48 h. The cell viability and IC50 was determined using MTT assay. Then, the two glioblastoma cell lines were treated with 48 h IC50 concentration of cisplatin for 24, 48, and 72 h. Apoptosis induction was analyzed by fluorescence microscopy and flow cytometry. Gene expression study was performed on BCL2L12 and TBP as target and internal control genes, respectively. The quantitative real-time polymerase chain reaction results showed that BCL2L12 gene expression was significantly (p?=?0.001) downregulated in the presence of cisplatin. In conclusion, cisplatin treatment induced a time-dependent apoptosis in glioblastoma cells, at least partially via downregulation of BCL2L12 gene expression.     

The effect of combination treatment with docosahexaenoic acid and 5-fluorouracil on the mRNA expression of apoptosis-related genes, including the novel gene BCL2L12, in gastric cancer cells

Docosahexaenoic acid (DHA) plays an important role in suppressing the growth of cancer. In this paper, the synergetic anticancer effect of combination DHA with 5-fluorouracil (5-FU) was investigated in gastric carcinoma cells. We found that DHA inhibited the growth of cultured SGC7901 cells at different concentrations in a dose- and time-dependent manner. Furthermore, the growth-inhibition activities of increasing concentration of 5-FU were markedly enhanced when different doses of 5-FU were administered in the combination with dose as low as 40 μg/ml of DHA. The early phase of apoptosis was increased in DHA- and 5-FU-treated cells. In the case of apoptotic genes expression in the combination-treated cells, BAX mRNA expression increased, whereas FAS, BCL-2, BCL2L12, and CASPASE-9 mRNA expression decreased. These results suggest that DHA strongly enhances the anticancer effect of 5-FU. Moreover, the application of both compounds on gastric cancer cells provides a new potential approach for cancer therapy.     

The function of chitosan/agarose biopolymer on Fe2O3 nanoparticles and evaluation of their effects on MCF-7 breast cancer cell line and expression of BCL2 and BAX genes

In recent decades, magnetic nanoparticles modified with biocompatible polymers have been recognized as a suitable tool for treating breast cancer. The aim of this research was to evaluate the function of chitosan/agarose-functionalized Fe₂O₃ nanoparticles on the MCF-7 breast cancer cell line and the expression of BCL2 and BAX genes. Free Fe₂O₃ nanoparticles were prepared by hydrothermal method. FTIR, XRD, SEM, DLS, VSM, and zeta potential analyses determined the size and morphological characteristics of the synthesized nanoparticles. The effect of Fe₂O₃ free nanoparticles and formulated Fe₂O₃ nanoparticles on induction of apoptosis was studied by double-dye Annexin V-FITC and PI. Also, the gene expression results using the PCR method displayed that Fe₂O₃ formulated nanoparticles induced BAX apoptosis by increasing the anti-apoptotic gene expression and decreasing the expression of pro-apoptotic gene BCL2, so the cell progresses to planned cell death. In addition, the results showed that the BAX/BCL2 ratio decreased significantly after treatment of MCF-7 cells with free Fe₂O₃ nanoparticles, and the BAX/BCL2 ratio for Fe₂O₃ formulated nanoparticles increased significantly. Also, to evaluate cell migration, the scratch test was performed, which showed a decrease in motility of MCF-7 cancer cells treated with Fe₂O₃ nanoparticles formulated with chitosan/agarose at concentrations of 10, 50, 100, and 200 μg/ml.     

The effects of the esterified Quercetin with omega3 and omega6 fatty acids on viability,nanomechanical properties,and BAX/BCL-2 gene expression in MCF-7 cells

Molecular Biology Reports - Quercetin is one of the major flavonoids and it appears to have cytotoxic effects on various cancer cells through regulating the apoptosis pathway genes such as BAX and...     

Signaling crosstalk of FHIT,CHK2 and p38 in etoposide induced growth inhibition in MCF-7 cells

FHIT (Fragile Histidin Triad) is a tumor suppressor gene involved in regulating cell death during DNA damage conditions. The exact mechanism of DNA damage-induced FHIT signaling is not well understood. It is known that p38 kinase and CHK2 kinase are being activated during stress-induced conditions and DNA damage, resulting in cell death. Since both CHK2 and FHIT are being influenced by DNA damage, we have evaluated the interplay of p38, CHK2 and FHIT in response to etoposide-induced cell death. DNA damage was induced by etoposide in MCF-7 cells and viability was examined using MTT. FHIT expression was blocked using siRNA. Protein expression was measured using western blotting. Our results indicated that etoposide induced cytotoxicity in MCF-7. Block of FHIT expression, completely reversed etoposide cytotoxicity. Besides, etoposide induced p38 and CHK2 phosphorylation and reduced FHIT expression in a time-dependent manner. The time-course study indicated that CHK2 had been phosphorylated prior to p38 activation. Knockdown of FHIT expression reduced CHK2 phosphorylation but had no significant effect on p38 activation. Inhibition of p38 kinase and CHK2 prevented etoposide induced alteration in FHIT expression. Furthermore, p38 inhibitors augmented etoposide-induced CHK2 phosphorylation. These results indicate that etoposide lowers FHIT expression and induces cell death via p38 and CHK2 phosphorylation. These results demonstrate a time dependent complex crosstalk of FHIT, p38 and CHK2 pathways in response to etoposide. Moreover, our findings suggest signaling interaction for these pathways which can be targeted for manipulating cell proliferation.     

Inducible expression of maize polyamine oxidase in the nucleus of MCF-7 human breast cancer cells confers sensitivity to etoposide

Summary. In this study, polyamine oxidase from maize (MPAO), which is involved in the terminal catabolism of spermidine and spermine to produce an aminoaldehyde, 1,3-diaminopropane and H₂O₂, has been conditionally expressed at high levels in the nucleus of MCF-7 human breast cancer cells, with the aim to interfere with polyamine homeostasis and cell proliferation. Recombinant MPAO expression induced accumulation of a high amount of 1,3-diaminopropane, an increase of putrescine levels and no alteration in the cellular content of spermine and spermidine. Furthermore, recombinant MPAO expression did not interfere with cell growth of MCF-7 cells under normal conditions but it did confer higher growth sensitivity to etoposide, a DNA topoisomerase II inhibitor widely used as antineoplastic drug. These data suggest polyamine oxidases as a potential tool to improve the efficiency of antiproliferative agents despite the difficulty to interfere with cellular homeostasis of spermine and spermidine. Authors’ address: Dr. Paraskevi Tavladoraki, Department of Biology, University ‘Roma Tre’, Viale G. Marconi 446, 00146 Rome, Italy     

Silencing of Bcl-2 expression by small interfering RNA induces autophagic cell death in MCF-7 breast cancer cells

Apoptosis (programmed cell death type I) and autophagy (type II) are crucial mechanisms regulating cell death and homeostasis. The Bcl-2 proto-oncogene is overexpressed in 50-70% of breast cancers, potentially leading to resistance to chemotherapy, radiation and hormone therapy induced apoptosis. In this study, we investigated the role of Bcl-2 in autophagy in breast cancer cells. Silencing of Bcl-2 by siRNA in MCF-7 breast cancer cells downregulated Bcl-2 protein levels (>85%) and led to inhibition of cell growth (71%) colony formation (79%), and cell death (up to 55%) by autophagy but not apoptosis. Induction of autophagy was demonstrated by acridine orange staining, electron microscopy and an accumulation of GFP-LC3-II in preautopghagosomal and autophagosomal membranes in MCF-7 cells transfected with GFP-LC-3(GFP-ATG8). Silencing of Bcl-2 by siRNA also led to induction of LC-3-II, a hallmark of autophagy, ATG5 and Beclin-1 autophagy promoting proteins. Knockdown of ATG5 significantly inhibited Bcl-2 siRNA-induced LC3-II expression and the number of GFP-LC3-II-labeled autophagosome (punctuated pattern) positive cells and autophagic cell death (p     

Silencing of Bcl-2 expression by small interfering RNA induces autophagic cell death in MCF-7 breast cancer cells

Apoptosis (programmed cell death type I) and autophagy (type II) are crucial mechanisms regulating cell death and homeostasis. The Bcl-2 proto-oncogene is overexpressed in 50-70% of breast cancers, potentially leading to resistance to chemotherapy, radiation and hormone therapy-induced apoptosis. Here, we investigated the role of Bcl-2 in autophagy in breast cancer cells. Silencing of Bcl-2 by siRNA in MCF-7 breast cancer cells downregulated Bcl-2 protein levels (>85%) and led to inhibition of cell growth (71%) colony formation (79%), and cell death (up to 55%) by autophagy but not apoptosis. Induction of autophagy was demonstrated by acridine orange staining, electron microscopy and an accumulation of GFP-LC3-II in autophagosomal membranes in MCF-7 cells transfected with GFP-LC-3(GFP-ATG8). Silencing of Bcl-2 by siRNA also led to induction of LC-3-II, a hallmark of autophagy, ATG5 and Beclin-1 autophagy promoting proteins. Knockdown of ATG5 significantly inhibited Bcl-2 siRNA-induced LC3-II expression, the number of GFP-LC3-II-labeled autophagosome positive cells and autophagic cell death (p < 0.05). Furthermore, doxorubicin at a high dose (IC(95), 1 microM) induced apoptosis but at a low dose (IC(50), 0.07 microM) induced only autophagy and Beclin-1 expression. When combined with Bcl-2 siRNA, doxorubicin (IC(50)) enhanced autophagy as indicated by the increased number cells with GFP-LC3-II-stained autophagosomes (punctuated pattern positive). These results provided the first evidence that targeted silencing of Bcl-2 induces autophagic cell death in MCF-7 breast cancer cells and that Bcl-2 siRNA may be used as a therapeutic strategy alone or in combination with chemotherapy in breast cancer cells that overexpress Bcl-2.     

Estrogen activates raf-1 kinase and induces expression of Egr-1 in MCF-7 breast cancer cells

We have demonstrated that in normal and b/b rat red blood cells (RBCs) hsp70-like protein (heat shock protein 70-like) is localized in the cytosol and it is exported via exosomes during in vivo reticulocytes maturation. As we have presumed, in the mutant (b/b) rat, hsp70-like protein transfers from cytosol to the RBC membrane. In the normal rat RBCs this happens when those cells are submitted to heat stress conditions. Our study indicates that the presence of hsp70-like protein in the b/b rat RBC plasma membrane is consistent with a primary defect and is not a consequence of life long stress, i.e. hypoxia.     

GNA13 regulates BCL2 expression and the sensitivity of GCB-DLBCL cells to BCL2 inhibitors in a palmitoylation-dependent manner

GNA13, encoding one of the G protein alpha subunits of heterotrimeric G proteins that transduce signals of G protein-coupled receptors (GPCR), is frequently mutated in germinal center B-cell-like diffuse large B-cell lymphoma (GCB-DLBCL) with poor prognostic outcomes. Due to the “undruggable” nature of GNA13, targeted therapy for these patients is not available. In this study, we found that palmitoylation of GNA13 not only regulates its plasma membrane localization, but also regulates GNA13’s stability. It is essential for the tumor suppressor function of GNA13 in GCB-DLBCL cells. Interestingly, GNA13 negatively regulates BCL2 expression in GCB-DLBCL cells in a palmitoylation-dependent manner. Consistently, BCL2 inhibitors were found to be effective in killing GNA13-deficient GCB-DLBCL cells in a cell-based chemical screen. Furthermore, we demonstrate that inactivating GNA13 by targeting its palmitoylation enhanced the sensitivity of GCB-DLBCL to the BCL2 inhibitor. These studies indicate that the loss-of-function mutation of GNA13 is a biomarker for BCL2 inhibitor therapy of GCB-DLBCL and that GNA13 palmitoylation is a potential target for combination therapy with BCL2 inhibitors to treat GCB-DLBCL with wild-type GNA13.Subject terms: B-cell lymphoma, B-cell lymphoma, Translational research     

Genistein induces apoptosis and autophagy in human breast MCF-7 cells by modulating the expression of proapoptotic factors and oxidative stress enzymes

Breast cancer is one of the common tumors occurring in woman and despite treatment, the prognostic is poor. Genistein, a soy isoflavone, has been reported to have chemopreventive\chemotherapeutic potential in multiple tumor types. Here, we investigated the genistein antiproliferative effect in MCF-7 breast cancer, underlying the molecular mechanisms involved in this effect. MCF-7 cancer and CCD1059sK fibroblast cells were treated with estradiol (10 nM) or genistein (0.01–100 μM) for 24, 48, and 72 h and the cell proliferation was investigated by MTT; membrane cell permeability was evaluated by LDH and PI incorporation; apoptosis was investigated by externalization of phosphatidylserine by FACS; and presence of autophagy was detected by LC3A/B immunostaining. The expression of apoptotic proteins and antioxidant enzymes was evaluated by qPCR. The results demonstrate that genistein (100 μM) for 72 h of treatment selectively reduced MCF-7 cell proliferation independent of estrogen receptor activation, while no cytotoxicity was observed in fibroblast cells. Further experiments showed that genistein induced phosphatidylserine externalization and LC3A/B immunopositivity in MCF-7 cells, indicating apoptosis and autophagy cell death. Genistein increased in three times proapoptotic BAX/Bcl-2 ratio and promoted a parallel downregulation of 20 times of antiapoptotic survivin. In addition, genistein promoted a decrease of 5.5, 9.3, and 3.6 times of MnSOD, CuZnSOD, and TrxR mRNA expression, respectively, while the GPx expression was increased by 6.5 times. These results suggest that the antitumor effect of genistein involved the modulation of antioxidant enzyme and apoptotic signaling expression, which resulted in apoptosis and progression of autophagy.     

In vitro effects of 2-methoxyestradiol on MCF-12A and MCF-7 cell growth, morphology and mitotic spindle formation

The influence of 2-methoxyestradiol (2ME) was investigated on cell growth, morphology and spindle formation in a tumorigenic (MCF-7) and non-tumorigenic (MCF-12A) epithelial breast cell line. Inhibition of cell growth was more pronounced in the MCF-7 cells compared to the MCF-12A cells following 2ME treatment. Dose-dependent studies (10(-5)-10(-9) M) revealed that 10(-6) M 2ME inhibited cell growth by 44% in MCF-12A cells and by 84% in MCF-7 cells (p-value < 0.05). 2ME-treated MCF-7 cells showed abnormal metaphase cells, membrane blebbing, apoptotic cells and disrupted spindle formation. These observations were either absent or less prominent in MCF-12A cells. 2ME had no effect on the length of the cell cycle between S-phase and the time a mitotic peak was reached in either cell line but MCF-7 cells were blocked in mitosis with no statistically significant alterations in the phosphorylation status of Cdc25C. Nevertheless, Cdc2 activity was significantly increased in MCF-7 cells compared to MCF-12A cells (p-value < 0.05). The results indicate that 2ME disrupts mitotic spindle formation and enhances Cdc2 kinase activity, leading to persistence of the spindle checkpoint and thus prolonged metaphase arrest that may result in the induction of apoptosis. The tumorigenic MCF-7 cells were especially sensitive to 2ME treatment compared to the normal MCF-12A cells. Therefore, differential mechanism(s) of growth inhibition are evident between the normal and tumorigenic cells.