Evidence that low doses of Taxol enhance the functional transactivatory properties of p53 on p21 waf promoter in MCF-7 breast cancer cells

In the present study, we evidence how in breast cancer cells low doses of Taxol for 18 h determined the upregulation of p53 and p21 waf expression concomitantly with a decrease of the anti-apoptotic Bcl-2. P53 and its gene product, the mdm2 protein, in treated cells exhibits a prevalent nuclear compartmentalization, thus potentiating p53 transactivatory properties. Indeed, the most important finding of this study consists with the evidence that Taxol at lower concentrations is able to produce the activation of p21 promoter via p53. Prolonged exposure of MCF-7 cells to Taxol (48 h) resulted in an increased co-association between p21 and PCNA compared to control and this well fits with the simultaneous block of cell cycle into the G2/M phase.     

Bystander killing of breast cancer MCF-7 cells by MDA-MB-231 cells exposed to 5-fluorouracil is mediated via Fas

The major drawback with cancer therapy is the development of resistant cells within tumors due to their heterogeneous nature and due to inadequate drug delivery during chemotherapy. Therefore, the propagation of injury ("bystander effect" (BE)) from directly damaged cells to other cells may have great implications in cancer chemotherapy. The general advantage of the bystander cell killing phenomenon is the large therapeutic index that can be achieved. Experiments suggest that this phenomenon is detected in radiation therapy as well as in gene therapy in conjunction with chemotherapy. In the present study, we developed an original in vitro model dedicated to the exploration of bystander cytotoxicity induced during breast carcinoma chemotherapy. In brief, we investigated this perpetuation of injury on untreated bystander MCF-7 breast cancer cells which were coplated with 5-fluorouracil (5-FU)-treated MDA-MB-231 breast cancer cells. To achieve this goal, a specific in vitro coculture model which involved mixing of aggressive MDA-MB-231 breast cancer cells with enhanced green fluorescent protein (EGFP) expressing stable clone of non-metastatic MCF-7 breast cancer cells (MCF-EGFP), was used. A bystander killing effect was observed in MCF-EGFP cells cocultured with MDA-MB-231 cells pretreated with 5-FU. The striking decrease in MCF-EGFP cells, as detected by assaying for total GFP intensity, is mediated by activation of Fas/FasL system. The implication of Fas in MCF-EGFP cell death was confirmed by using antagonistic anti-FasL antibody that reverses bystander cell death by blocking FasL on MDA-MB-231 cells. In addition, inhibition of CD95/Fas receptor on the cell surface of MCF-EGFP cells by treatment with Pifithrin-alpha, a p53 specific transactivation inhibitor, partially abrogated the sensitivity of bystander MCF-EGFP cells. Our data, therefore, demonstrates that the Fas/FasL system could be considered as a new determinant for chemotherapy-induced bystander cell death in breast cancers.     

Down-regulation of Bcl-2 enhances estrogen apoptotic action in long-term estradiol-depleted ER<Superscript>+</Superscript> breast cancer cells

Postmenopausal women with estrogen receptor positive (ER⁺) breast cancer frequently respond paradoxically to estrogen administration with tumor regression. Using both LTED and E8CASS cells derived from MCF-7 breast cancer cells by long-term estrogen-deprivation, we previously reported that 17 -estradiol (estradiol) is a powerful, pro-apoptotic hormone which kills the cancer cells through activation of the Fas/FasL death receptor pathway. We postulated that the mitochondrial interactive protein Bcl-2 might play a role in the regulation of estradiol-induced apoptosis in both LTED and E8CASS cells. In this study, we assessed estradiol effects on cell growth, proliferation and apoptosis. Additionally we investigated the effect of estradiol on caspase activation, NF-KB and Bcl-2 expression. The functional role of Bcl-2 in estradiol-induced apoptosis was further studied by knockdown or decrease of Bcl-2 with siRNA. Our results show that estradiol significantly inhibited cell growth primarily through a pro-apoptotic action involving caspase-7 and 9 activations (p < 0.01). Basal Bcl-2 and NF-KB levels were greatly elevated and estradiol decreased NF-KB, but not Bcl-2 expression. Knockdown of Bcl-2 expression with siRNA decreased the levels of this protein by 9 fold (p < 0.01). This reduction markedly sensitized both LTED and E8CASS cells to the pro-apoptotic action of estradiol, leading to a synergistic induction of apoptosis and a concomitant reduction in cell number (p < 0.01). Therefore, down-regulation of Bcl-2 synergistically enhanced estradiol-induced apoptosis in ER⁺ postmenopausal breast cancer cells.     

Synergic prooxidant,apoptotic and TRPV1 channel activator effects of alpha-lipoic acid and cisplatin in MCF-7 breast cancer cells

Background: Resistance to cisplatin (Cisp) in the treatment of breast cancer is a major obstacle. Alpha-lipoic acid (ALA) has both antioxidant and oxidant properties. ALA has been used on stimulation mechanisms of apoptosis and oxidative stress in the treatment of cancer with a combination of chemotherapeutic agents, although its role on molecular mechanisms in the cancer cells has not been clarified yet. The aim of this study was to evaluate if a combination therapy of ALA with Cisp can alter the effect of this chemotherapy drug in the MCF-7 breast cancer cells.

Materials: The MCF-7 cells were divided into four treatment groups as control, Cisp (0.025?mM), ALA (0.05?mM), and Cisp?+?ALA.

Results: Apoptosis, mitochondrial membrane depolarization, reactive oxygen species (ROS) production, lipid peroxidation, PARP1, caspase 3 and 9 expression levels are increased through activating TRPV1 in the cells by the Cisp and ALA treatments, although cell viability, reduced glutathione and glutathione peroxidase (GPx) values were decreased by the treatments. The Cisp and ALA-induced increase of intracellular free Ca²⁺ concentration was decreased with the TRPV1 blocker, capsazepine.

Conclusions: Apoptosis and oxidant effects of Cisp were increased by activation of TRPV1 channels, but its action on the values was further increased by the ALA treatment. Combination therapy of ALA and Cisp could be used as an effective strategy in the treatment of breast cancer.     

Roscovitine-activated HIP2 kinase induces phosphorylation of wt p53 at Ser-46 in human MCF-7 breast cancer cells

Human MCF-7 breast cancer cells are relatively resistant to conventional chemotherapy due to the lack of caspase-3 activity. We reported recently that roscovitine (ROSC), a potent cyclin-dependent kinase 2 inhibitor, arrests human MCF-7 breast cancer cells in the G(2) phase of the cell cycle and concomitantly induces apoptosis. Exposure of MCF-7 cells to ROSC also strongly activates the wt p53 tumor suppressor protein in a time- and dose-dependent manner. The p53 level increased despite upregulation of Hdm-2 protein and was attributable to the site-specific phosphorylation at Ser-46. The p53 protein phosphorylated at serine 46 causes the up-regulation of the p53AIP1 protein, a component of mitochondria. In the present study we identified the pathway mediating ROSC-induced p53 activation. Exposure of MCF-7 cells to ROSC activated homeodomain-intereacting protein kinase-2 (HIPK2). The overexpression of wild-type but not kinase inactive HIPK2 increased the basal and ROSC-induced level of p53 phosphorylation at Ser-46 and strongly enhanced the rate of apoptosis in cells exposed to ROSC. We show that HIPK2 is activated by ROSC and mediates ROSC-induced P-Ser-46-p53, thereby stabilizing wt p53 and increasing the efficacy of drug-induced apoptosis in MCF-7 cells. These results identify HIPK2 as a component of the ROSC-induced signaling pathway leading to the stabilization and activation of wt p53 protein.     

PRIMA-1 cytotoxicity correlates with nucleolar localization and degradation of mutant p53 in breast cancer cells

PRIMA-1 has been identified as a compound that restores the transactivation function to mutant p53 and induces apoptosis in cells expressing mutant p53. Studies on subcellular distribution of the mutant p53 protein upon treatment with PRIMA-1^Met, a methylated form of PRIMA-1, have suggested that redistribution of mutant p53 to nucleoli may play a role in PRIMA-1 induced apoptosis. Here, we specifically investigated the influence of PRIMA-1 on cellular localization of mutated p53-R280K endogenously expressed in tumour cells. By using immunofluorescence staining, we found a strong nucleolar redistribution of mutant p53 following PRIMA-1 treatment. This subcellular localization was associated to p53 degradation via ubiquitylation. When cells were treated with adriamycin, neither nucleolar redistribution nor mutant p53 down modulation and degradation were observed. Interestingly, cells where p53-R280K was silenced were more sensitive to PRIMA-1 than the parental ones. These results indicate that in some cellular context, the cell sensitivity to PRIMA-1 could depend on the abolition of a gain-of-function activity of the mutated p53, through a protein degradation pathway specifically induced by this compound.     

Sensitization by glycerol for CDDP-therapy against human cultured cancer cells and tumors bearing mutated p53 gene

To clarify effective chemotherapeutic regimens against cancer, we examined the effects of glycerol on apoptosis induced by CDDP treatment using cultured human cancer cells (in vitro) and transplanted tumor in mice (in vivo). Human tongue cell carcinoma (SAS) cells transfected with mutated p53 gene (SAS/m p53) showed CDDP-resistance compared with the cells with neo control gene (SAS/ neo). When those cultured cells were pre-treated with glycerol, CDDP-induced apoptosis was enhanced by glycerol in SAS/m p53 cells but not in SAS/ neo cells.In tumor-transplanted mice, the glycerol treatment to tumors enhanced growth delay induced by CDDP in mp53 tumors transplanted with SAS/m p53 cells, but not in wtp53 tumors transplanted with SAS/ neo cells. When transplanted tumors were treated with CDDP alone, the cells positive for active caspase-3, 85 kDa PARP and apoptosis were observed by immunohistochemical staining in wtp53 tumors but not in mp53 tumors. When the tumors were treated with CDDP combined with glycerol, positive cells were observed not only in wtp53 tumors but also in mp53 tumors. These results showed that the CDDP-induced growth inhibition of the tumors is p53 -dependent and that the enhanced growth delay by glycerol may be due to the increased apoptosis. Glycerol might be available for cancer chemotherapy in patients with mp53 tumors.     

Complete sequence of p53 gene in 20 patients with lung cancer: comparison with chemosensitivity and immunohistochemistry

In this study the entirep53 complementary DNA has been sequenced in 20 non-small cell lung carcinomas (NSCLC) and the results correlated with chemosensitivity, immunohistochemistry and clinical data. Ten patients had mutations inp53, 8 missense mutations and 2 nonsense mutations. The method discovered two mutations never described previously and two other mutations that have never been described before in connection with NSCLC tumours. Chemosensitivity data, according to a short-term assay (FMCA), indicated that tumours with p53 mutation were more resistant to cisplatin and cyclophosphamide. Immunohistochemical studied demonstrated a 70% concordance between over-expression of p53 protein and mutation inp53. No conclusions or trends could be drawn from the immunohistochemical studies ofBcl-2 andBax.     

MicroRNA-590-3P suppresses cell survival and triggers breast cancer cell apoptosis via targeting sirtuin-1 and deacetylation of p53

Downregulation of microRNA-590-3p (miR-590-3p) is a frequently occurring, nonphysiological event which is observed in several human cancers, especially breast cancer. However, the significance of miR-590-3p still remain unclear in the progression of this disease. This study explored the role of miR-590-3p in apoptosis of breast cancer cells. Gene expression of miR-590-3p, Sirtuin-1 (SIRT1), Bcl-2 associated X protein (BAX), and p21 was evaluated with real-time polymerase chain reaction (PCR) and SIRT1 protein expression was assessed by Western blot analysis in breast cancer cell lines. Bioinformatics analysis and luciferase reporter assay were used to evaluate targeting of SIRT1 messenger RNA (mRNA) by miR-590-3p. Cells were transfected with miR-590-3p mimic and inhibitor and their effects on the expression and activity of SIRT1 were evaluated. The effects of miR-590-3p upregulation on the acetylation of p53 as well as cell viability and apoptosis were assessed by Western blot analysis, WST-1 assay, and flow cytometry, respectively. miR-590-3p expression was considerably downregulated in breast cancer cells which was accompanied by upregulation of SIRT1 expression. SIRT1 was recognized as a direct target for miR-590-3p in breast cancer cells and its protein expression and activity was dramatically inhibited by the miR-590-3p. In addition, there was an increase in p53 and its acetylated form that ultimately led to upregulation of BAX and p21 expression, suppression of cell survival, and considerable induction of apoptosis in breast cancer cells. These findings suggest that miR-590-3p exerts tumor-suppressing effects through targeting SIRT1 in breast cancer cells, which makes it a potential therapeutic target for developing more efficient treatments for breast cancer.     

Expression and clinicopathological significance of lipin-1 in human breast cancer and its association with p53 tumor suppressor gene

Breast cancer (BC) is an important cause of female cancer-related death. It has recently been demonstrated that metabolic disorders including lipid metabolism are a hallmark of cancer cells. Lipin-1 is an enzyme that displays phosphatidate phosphatase activity and regulates the rate-limiting step in the pathway of triglycerides and phospholipids synthesis. The objective of this study was to evaluate lipin-1 expression, its prognostic significance, and its correlation with p53 tumor suppressor in patients with BC. In this study, 55 pairs of fresh samples of BC and adjacent noncancerous tissue were used to analyze lipin-1, using quantitative real-time polymerase chain reaction and immunohistochemistry (IHC) staining. The expression of other clinicopathological variables and p53 was also examined using IHC technique. The cell migration was studied in MCF-7 and MDA-MB231 cells following the inhibition of lipin-1 by propranolol. Our results show that the relative expression of lipin-1 messenger RNA was significantly higher in BC tissues compared with the adjacent normal tissue and its inhibition reduced cell migration in cancer cells. This upregulation was negatively correlated with histological grade of tumor and p53 status (p = .001 and p = .034) respectively and positively correlated with the tumor size (p = .006). Our results also seem to indicate that the high lipin-1 expression is related to a good prognosis in patients with BC. The expression of lipin-1 may be considered as a novel independent prognostic factor. The inhibition of lipin-1 may also have therapeutic significance for patients with BC. The correlation between lipin-1 and p53 confirms the role of p53 in the regulation of lipid metabolism in cancer cells.     

Cordycepin induces cell cycle arrest and apoptosis by inducing DNA damage and up-regulation of p53 in Leukemia cells

Cordycepin, an adenosine analog derived from Cordyceps militaris has been shown to exert anti-tumor activity in many ways. However, the mechanisms by which cordycepin contributes to the anti-tumor still obscure. Here our present work showed that cordycepin inhibits cell growth in NB-4 and U937 cells by inducing apoptosis. Further study showed that cordycepin increases the expression of p53 which promotes the release of cytochrome c from mitochondria to the cytosol. The released cytochrome c can then activate caspase-9 and trigger intrinsic apoptosis. Cordycepin also blocks MAPK pathway by inhibiting the phosphorylation of ERK1/2, and thus sensitizes the apoptosis. In addition, our results showed that cordycepin inhibits the expression of cyclin A2, cyclin E, and CDK2, which leads to the accumulation of cells in S-phase. Moreover, our study showed that cordycepin induces DNA damage and causes degradation of Cdc25A, suggesting that cordycepin-induced S-phase arrest involves activation of Chk2-Cdc25A pathway. In conclusion, cordycepin-induced DNA damage initiates cell cycle arrest and apoptosis which leads to the growth inhibition of NB-4 and U937 cells.     

c‐erbB‐2 and p53 expression in breast cancer fine needle aspirates

The aim of this study was to co‐evaluate c‐ erbB ‐2 and p53 protein expression in breast cancer fine needle aspirates (FNA) and to compare this with histological variables and the immunohistochemical phenotype of the tumours. Furthermore, we assessed the relationship of c‐ erbB ‐2 and p53 immunocytochemical expression to tumour prognostic factors. We examined 124 breast cancer FNAs and 79 matched surgical specimens using the avidin–biotin complex (ABC) and the alkaline phosphatase immunocytochemical techniques. C‐ erbB ‐2 immunopositivity was detected in 37.9% of the FNAs, while 31.7% were positive for p53. A statistically significant correlation was observed between p53 negativity and absence of c‐ erbB ‐2 immunostaining in the FNAs ( P =0.0007). Smears from infiltrating ductal carcinomas tended to be more frequently positive for p53 (36.7%) than those from lobular carcinomas (11.7%) ( P =0.054). In matched tumour tissues, c‐ erbB ‐2 was positive in 16.7% and p53 in 19% of cases. The immunocytochemical results for both c‐ erbB ‐2 and p53 were significantly correlated with the immunohistochemical results. There was no correlation between c‐ erbB ‐2 and p53 immunostaining, in both FNAs and tissues, and patients' menopausal status, tumour size, grade and lymph node status.     

ARTIK-52 induces replication-dependent DNA damage and p53 activation exclusively in cells of prostate and breast cancer origin

The realization, that the androgen receptor (AR) is essential for prostate cancer (PC) even after relapse following androgen deprivation therapy motivated the search for novel types of AR inhibitors. We proposed that targeting AR expression versus its function would work in cells having either wild type or mutant AR as well as be independent of androgen synthesis pathways. Previously, using a phenotypic screen in androgen-independent PC cells we identified a small molecule inhibitor of AR, ARTIK-52. Treatment with ARTIK-52 caused the loss of AR protein and death of AR-positive, but not AR-negative, PC cells. Here we present data that ARTIK-52 induces degradation of AR mRNA through a mechanism that we were unable to establish. However, we found that ARTIK-52 is toxic to breast cancer (BC) cells expressing AR, although they were not sensitive to AR knockdown, suggesting an AR-independent mechanism of toxicity. Using different approaches we detected that ARTIK-52 induces replication-dependent double strand DNA breaks exclusively in cancer cells of prostate and breast origin, while not causing DNA damage, or any toxicity, in normal cells, as well as in non-PC and non-BC tumor cells, independent of their proliferation status. This amazing specificity, combined with such a basic mechanism of toxicity, makes ARTIK-52 a potentially useful tool to discover novel attractive targets for the treatment of BC and PC. Thus, phenotypic screening allowed us to identify a compound, whose properties cannot be predicted based on existing knowledge and moreover, uncover a barely known link between AR and DNA damage response in PC and BC epithelial cells.     

Induction of antiproliferative effect by diosgenin through activation of p53,release of apoptosis-inducing factor （AIF） and modulation of caspase-3 activity in different human cancer cells

Previously, we demonstrated that a plant steroid, diosgenin, altered cell cycle distribution and induced apoptosis in the human osteosarcoma 1547 cell line. The objective of this study was to investigate if the antiproliferative effect of diosgenin was similar for different human cancer cell lines such as laryngocarcinoma HEp-2 and melanoma M4Beu cells. Moreover, this work essentially focused on the mitochondrial pathway. We found that diosgenin had an important and similar antiproliferative effect on different types of cancer cells. In addition, our new results show that diosgenin-induced apoptosis is caspase-3 dependent with a fall of mitochondrial membrane potential, nuclear localization of AIF and poly (ADP-ribose) polymerase cleavage. Diosgenin treatment also induces p53 activation and cell cycle arrest in the different cell lines studied.     

Abrogation of p53 by its antisense in MCF-7 breast carcinoma cells increases cyclin D1 via activation of Akt and promotion of cell proliferation

The p53 protein has been a subject of intense research interest since its discovery as about 50% of human cancers carry p53 mutations. Mutations in the p53 gene are the most frequent genetic lesions in breast cancers suggesting a critical role of p53 in breast cancer development, growth and chemosensitivity. This report describes the derivation and characterization of MCF-7As53, an isogenic cell line derived from MCF-7 breast carcinoma cells in which p53 was abrogated by antisense p53 cDNA. Similar to MCF-7 and simultaneously selected hygromycin resistant MCF-7H cells, MCF-7As53 cells have consistent basal epithelial phenotype, morphology, and estrogen receptor expression levels at normal growth conditions. Present work documents investigation of molecular variations, growth kinetics, and cell cycle related studies in relation to absence of wild-type p53 protein and its transactivation potential as well. Even though wild-type tumor suppressor p53 is an activator of cell growth arrest and apoptosis-mediator genes such as p21, Bax, and GADD45 in MCF-7As53 cells, no alterations in expression levels of these genes were detected. The doubling time of these cells decreased due to depletion of G0/G1 cell phase because of constitutive activation of Akt and increase in cyclin D1 protein levels. This proliferative property was abrogated by wortmannin, an inhibitor of PI3-K/Akt signaling pathway. Therefore this p53 null cell line indicates that p53 is an indispensable component of cellular signaling system which is regulated by caveolin-1 expression, involving Akt activation and increase in cyclin D1, thereby promoting proliferation of breast cancer cells.     

The mechanisms of lipoxygenase inhibitor-induced apoptosis in human breast cancer cells

Previous experimental studies have shown that high dietary fat intake is associated with mammary carcinogenesis. In the current study, the effect of 5-LOX or 12-LOX inhibitors on human breast cancer cell proliferation and apoptosis, as well as the possible mechanisms were investigated. The LOX inhibitors, NDGA, Rev-5901, and baicalein all inhibited proliferation and induced apoptosis in MCF-7 (ER+) and MDA-MB-231 (ER-) breast cancer cell in vitro. In contrast, the LOX products, 5-HETE and 12-HETE had mitogenic effects, stimulating the proliferation of both cell lines. These inhibitors also induced cytochrome c release, caspase-9 activation, as well as downstream caspase-3, caspase-7 activation, and PARP cleavage. LOX inhibitor treatment also reduced the levels of anti-apoptotic proteins Bcl-2 and Mcl-1 and increased the levels of the pro-apoptotic protein bax. In conclusion, blockade of both 5-LOX and 12-LOX pathways induces apoptosis in breast cancer cells through the cytochrome c release and caspase-9 activation, with changes in the levels of Bcl-2 family proteins.