Leptin enhances growth inhibition by cAMP elevating agents through ...

[Cancer Biology & Therapy 8:12, 1183-1190; 15 June 2009]; ©2009 Landes Bioscience

Research Paper

Leptin enhances growth inhibition by cAMP elevating agents through apoptosis of MDA-MB-231 breast cancer cells Silvio Naviglio, Davide Di Gesto, Maria Romano, Annunziata Sorrentino, Fausto Illiano, Luca Sorvillo, Alberto Abbruzzese, Monica Marra, Michele Caraglia,* Emilio Chiosi, Annamaria Spina and Gennaro Illiano Department of Biochemistry and Biophysics; Second University of Naples; Medical School; Naples, Italy

Abbreviations: cAMP, 3'–5'-cyclic adenosine monophosphate; PKA, protein kinase A; Jak/STAT, janus kinase/signal transducer and activator of transcription; ERK, extracellular signal-regulated kinases; PI-3K, phosphoinositide 3 kinase; GSK3, glycogen synthase kinase 3; Bcl2, B-cell lymphoma2; PI, propidium iodide Key words: leptin, cAMP, PKA, Bcl2, breast cancer, targeted therapy

Elevation of cAMP inhibits the proliferation and expression of transformed phenotype in several cell types, including breast cancer cells. Leptin has been shown to act as a mitogen/survival factor in many types of cancer cells. In the present work, we have studied the impact of cAMP elevation on leptin-induced proliferation of breast cancer cells. Here we report that treatment of estrogen receptor negative human breast cancer cell line MDA-MB-231 with leptin or cAMP elevating agents has positive and negative effects on cell proliferation, respectively. Surprisingly, we find that leptin strongly potentiates the antiproliferative action of cAMP elevating agents, by concurring to cell cycle arrest at G1 phase and inducing apoptosis. Pretreatment with the PKA inhibitor KT-5720 completely prevented the anti-proliferative effects induced by the combination between leptin and cAMP elevating agents. The above anti-proliferative effects were paralleled by the decrease of cyclin D1 and A and by the increase of inhibitor p27kip1 cell cycle regulating protein levels. In these conditions we found also a strong decrease of anti-apopotic Bcl2 protein levels. Altogether, our data extend the evidence of adenylate cyclase/cAMP/PKA as a growth suppressor system and of leptin as a growth promoting factor in breast cancer cells. Remarkably, our results suggest that when cAMP levels are increased, leptin drives cells towards apoptosis, and that targeting both cAMP levels and leptin signaling might represent a simple novel way for therapeutic intervention in breast cancer.

*Correspondence to: Michele Caraglia; Department of Biochemistry and Biophysics; Second University of Naples; Medical School; Via L. De Crecchio 7; Naples 80138 Italy; Tel.: +390815665871; Fax +390815665866; Email: [email protected] Submitted: 03/02/09; Revised: 03/19/09; Accepted: 03/27/09 Previously published online as a Cancer Biology & Therapy E-publication: http://www.landesbioscience.com/journals/cbt/article/8562

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Introduction Breast cancer is one of the most common malignancies and a major cause of cancer death in women throughout the world.1 Localized breast cancer can be cured by surgery. However, the high mortality rate associated with breast cancer is due to a propensity of the tumor to metastasize when the primary tumor is small or undetectable. Although hormone therapy is effective for the treatment of most patients with estrogen receptor (ER)-positive breast cancer, resistance to hormones is frequent. In addition, a number of tumors do not express these receptors and do not respond to anti-hormone therapy.2 Drugs targeting other pathways involved in breast carcinogenesis, such as trastuzumab, an antibody against ErbB2, or oral tyrosine kinase inhibitors are actually used in therapy. However, chemotherapy is a major treatment modality for both hormone-refractory and ER-negative breast cancer. On the other hand, women with advanced metastatic breast cancer, that is resistant to hormone therapy, usually respond poorly to conventional chemotherapy and to other current targeted therapies.3 Therefore, new effective therapies are warranted for the treatment of metastatic breast cancer. Evidence suggests that different hormones and peptide growth factors might cooperate in promoting mammary carcinogenesis. Since the circulating levels of leptin are elevated in obese individuals, and excessive body weight has been shown to increase breast cancer risk in postmenopausal women, several studies have addressed the role of leptin in breast cancer.4-6 Leptin acts through binding to specific membrane receptors that belong to the class I cytokine receptor family and of which six isoforms (obR a-f ) have been identified up to now. Binding of leptin to its receptor activates different signaling pathways, including Jak/STAT, Ras/ ERK1/2 and PI-3K/Akt/GSK3 pathways.7 Expression of leptin and its receptors occurs in breast cancer cell lines and in human primary breast carcinoma.8 Leptin is able to induce the growth of breast cancer cells primarily via activation of the Jak/STAT3 and ERK1/2 pathways.9

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Leptin and cAMP in breast cancer cells

The extracellular-signal-regulated kinase (ERK)-dependent signaling pathway is relevant to breast cancer, and several studies demonstrate it is frequently activated.10 A variety of extracellular stimuli other than growth factors of the EGF gene family, including ligands of G protein coupled receptors (GPCRs) and of the cytokine receptor family, as well as estradiol, progesterone and androgens affect Ras/Raf/ERK signaling cascade.11-14 Indeed the occurrence of network between multiple signal transduction pathways and its importance in the control of proliferation, including that of breast cancer cells is largely known.10 These signaling connections play an important role in cancer biology and a combined blockade of such signaling pathways is considered a relevant strategy for therapeutic intervention.2,15 Proliferative signal transmission through the Ras/Raf/ERK pathway is blocked by the elevation of cellular cAMP levels mainly via PKA-mediated Raf inhibition.16,17 The elevation of cAMP also induces the cell cycle inhibitor p27kip1.18 A decrease in p27kip1 levels is thought to be an important factor in breast tumor progression.19 An intracellular concentration of cAMP results from the fine balance between its synthesis and degradation induced by adenylate cyclases and PDEs, respectively, and can be elevated more than twenty-fold after activation of ACs by extracellular signals.20,21 Therefore, affecting cAMP levels by targeting its synthesis and degradation represents a strategy for therapeutic intervention.22 In the present study, the impact of cAMP elevation on leptininduced proliferation was investigated in MDA-MB-231, which is an estrogen receptor negative and highly metastatic human breast cancer cell line.23-25 cAMP levels were pharmacologically increased by the addition of either 8-Br-cAMP, a cell-permeable analogue of cAMP, or forskolin, a potent stimulator of adenylate cyclase cAMP biosynthetic activity, or IBMX, a broad-spectrum phosphodiesterase inhibitor.26-28 Surprisingly, our results indicate that leptin treatment strongly enhances the anti-proliferative effect of cAMP elevating agents, by both enforcing cell cycle arrest at G1 phase and inducing apoptosis. These anti-proliferative effects are mediated by PKA and correlated with the increase of cell cycle inhibitor p27kip1 and the decrease of anti-apopotic bcl2 protein levels.

Results Leptin induces proliferative effect in MDA-MB-231 cells. Figure 1A shows that leptin stimulates the growth of MDA-MB231 cells, which confirms previous studies with this cytokine in various breast cancer models.29,30 Cells were incubated with increasing (2–100 nM) concentrations of leptin for 48 h and cell proliferation was determined by a conventional tetrazolium-based (MTT) assay. As shown in Figure 1, leptin elicited proliferative responses (about 30%) that were significantly higher than untreated cells starting from concentrations of 20 nM. To study the mechanism underlying the proliferative effect of leptin in MDA-MB-231 cells, we monitored STAT-3 and ERK1/2 phosphorylation by immunoblotting at different time points, from 30 min to 24 h, in cells treated with 40 nM leptin. As shown in Figure 1B, we found that exposure of cells to leptin resulted in a strong phosphorylation of both ERK1/2 and STAT3 that was detectable 1184

Figure 1. Effects of leptin on the proliferation of MDA-MB-231 cells and on ERK1/2 and STAT3 activation. (A) Treatments with increasing concentrations of leptin were carried out for 48 h and cell viability was measured by MTT assay. Data represent the average of three independent experiments. The means and S.D. are shown. *p < 0.01 vs. control untreated cells. (B) Cells were treated or not with 40 nM leptin for the indicated times. The activation (phosphorylation) and levels of ERK1/2 and STAT3 proteins were assessed by western blotting from 40 μg of cell extracts using antibodies against the indicated proteins. The image is representative of two different experiments with similar results.

at 30 min, maintained until 4 h and declined to basal levels at 24 h. The above findings suggest that leptin stimulates the growth of MDA-MB-231 cells and this effect could be mediated via STAT3 and/or ERK1/2 signalling pathways. cAMP elevation induces anti-proliferative effects in MDA-MB231 cells. Figure 2 shows that different cAMP elevating agents inhibit the growth of MDA-MB-231 cells. Cells were incubated for 48 h with increasing (from 10-9 to 10-4 M) concentrations of forskolin (A), or (from 0.05 to10 mM) 8-Br-cAMP (B), or (from 0.05, to 5 mM ) IBMX (C); then cell proliferation was calculated by a conventional tetrazolium-based (MTT) assay. As shown in Figure 2, all cAMP elevating compounds induced anti-proliferative effects that appeared of different extent, according to the different modes of action of the agents. In fact, the maximal growth inhibitions induced by the agents were different: 10-5 M Forskolin inhibited cell proliferation by more than 40%, whereas 2 mM 8-Br-cAMP and 2 mM IBMX induced no more than 30% and 20% cell growth arrest, respectively. Leptin enhances the anti-proliferative effects of cAMP elevating agents in MDA-MB-231 cells. To investigate whether cAMP plays a role on the growth promoting effects induced by leptin, we evaluated the effects of cAMP elevating agents on the leptin-induced proliferation in MDA-MB-231 cells. We performed a combined treatment for 48 h with a non-maximal concentration of leptin


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Figure 2. Effects of cAMP elevating agents on the proliferation of MDAMB-231 cells. Treatments with increasing concentrations of forskolin (A), 8-Br-cAMP (B), IBMX (C) were carried out for 48 h and the cell viability was measured by MTT assay. Data represent the average of three independent experiments. The means and S.D. are shown. *p < 0.01 vs. control untreated cells.

Figure 3. Effects of concurrent treatments with forskolin and leptin on the proliferation of MDA-MB-231 cells. Co-treatments with increasing concentrations of forskolin and 40 nM leptin (A) and with increasing concentrations of leptin and 10-6 M forskolin (B) were carried out for 48 h and the cell viabilitiy was measured by MTT assay. Data represent the average of three independent experiments. The means and S.D. are shown. *p < 0.01 vs. control untreated cells. www.landesbioscience.com

(40 nM) and increasing concentrations of each cAMP elevating agents, and vice versa. Unexpectedly, we found that in all combinations the presence of leptin enhanced the anti-proliferative effects of cAMP elevating compounds. In Figure 3 data regarding leptin and forskolin co-treatments are shown. Panel A shows that in the presence of leptin, the minimal effective dose of forskolin is lowered from 10-7 to 10-8 M and that the growth inhibitory effects induced by forskolin and leptin are significantly higher than those caused by forskolin alone (at 10-6 M forskolin the inhibition increases from 22% to 35%). Panel B shows that in the presence of forskolin (at 10-6 M not maximal concentration) leptin lacks to induce a proliferative effect even at higher concentrations (up to 100 nM). In fact, in these experimental conditions leptin dose-dependently enforced the antiproliferative effects caused by forskolin. Similar results were obtained with the other cAMP elevating agents, 8-BrcAMP and IBMX (data not shown). Overall, the data described above suggest that leptin enhances the anti-proliferative effects of cAMP. The anti-proliferative effects induced by the combination of leptin and cAMP elevating agents are completely abrogated by selective PKA inhibitor KT5720. cAMP, in most cells including breast cancer cells, inhibits proliferation. cAMPmediated inhibition of proliferation very often correlates with the interruption of proliferative Ras/Raf/ERK signalling pathway mainly via PKA-mediated Raf inhibition.31 To investigate whether forskolin- and forskolin/leptin-induced anti-proliferative effects were mediated by cAMP/PKA axis, we evaluated the effects of a specific PKA inhibitor, KT5720, together with forskolin, leptin and forskolin plus leptin on cell proliferation. Figure 4 shows that pretreatment of MDA-MB-231 cells with PKA inhibitor completely prevented the growth inhibition induced by forskolin and by forskolin plus leptin, while KT5720 had a minimal and insignificant impact on leptin-induced proliferative effect. In order to extend the role of cAMP/PKA and to examine the involvement of Ras/Raf/Erk cascade on the anti-proliferative effects induced by cAMP elevation, we determined the effect of forskolin treatment on phosphorylation of both ERK1/2, and CREB proteins (Fig. 5). We treated cells with forskolin for different times, from 30 min to 6 h, and then we evaluated ERK1/2 and CREB phosphorylation by immunoblotting with specific anti-phospho-ERK1/2 and anti-phospho-CREB specific antibodies. As shown in Figure 5, we found that exposure of cells to forskolin resulted in a significant time-dependent decrease of ERK1/2 activity that was almost completely abrogated at 6 h. CREB protein, a major substrate of PKA, is strongly phosphorylated in response to forskolin.16,32 As expected, the treatment of cancer cells with forskolin determined a time-dependent increase of CREB phosphorylation (Fig. 5). All the above findings offer an example of how cAMP may act as an inhibitor of MAPK and indicate that cAMP/PKA axis plays a critical role in anti-proliferative effects of forskolin and forskolin plus leptin in MDA-MB-231 cells. Leptin enhances the anti-proliferative effects of cAMP elevating agents by potentiation of cell accumulation in G1 phase and apoptosis. To explore the effects of leptin plus cAMP elevation on the cell cycle and/or apoptosis, MDA-MB-231 cells were exposed or not to leptin, forskolin and leptin plus forskolin for 24, 48 and


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72 h. Thereafter, distribution of cells in cell cycle was evaluated by FACS analysis of propidium iodide-stained cells.33 We also looked at the proportion of cells with hypoploid DNA content (sub-G1 population), characteristic of cells having undergone to DNA fragmentation which is a biochemical hallmark of apoptosis.25,34 Figure 6 shows that forskolin-treated cells strongly accumulated in G1 phase with a concomitant decrease of both S and G2/M phases of cell cycle (A and C); G1 accumulation was already evident at 24 hours (more than 80% cells in G1) and enhanced at 72 hours with ≈95% cells in G1. Moreover, a weak increase of sub-G1 population in response to forskolin was observed at 72 h. Interestingly, Figure 6 shows that forskolin/leptin combined treatment induces a time-dependent increase of sub G1 population (from 18% at 24 h, up to 38% at 72 h), whereas the treatment with leptin alone did not cause any relevant change in the sub-G1 fraction up to 72 h. The latter result is in accordance with the poor apoptosis-inducer activity reported for leptin.9 The apoptosis induced by forskolin/leptin treatment was paralleled by a clear activation of the terminal caspase 3, executioner of apoptosis. Figure 6D shows a strong decrease of the uncleaved isoform of caspase-3 in 24 h leptin/forskolin-treated cells suggesting the increase of its activity that is correlated to its fragmentation. Forskolin alone induced similar effects on caspase 3 activation but at later time points, 48 h and 72 h (Fig. 6D). Remarkably, no decrease of procaspase-3 protein level was observed in cells treated with leptin alone for up to 72 h. Finally, we have evaluated the effects of the different treatments on the fragmentation of PARP that is a substrate for caspase-3. The pattern of the PARP processing paralleled that of caspase-3 cleavage (Fig. 6D). Overall the above data suggest that anti-proliferative effects caused by cAMP elevating agent forskolin is mainly due to G1 cell cycle arrest, and that leptin potentiates such cancer growth inhibitory effects by inducing apoptosis of G1 cAMP blocked cells. The anti-proliferative effects induced by leptin and cAMP elevation is paralleled by modulation of relevant cell cycle and apoptosis regulating proteins. In order to evaluate the effects of the combination of leptin and cAMP elevation on cell cycle and/ or apoptosis of MDA-MB-231 cells, we studied the expression of p27Kip1, cyclin D1, cyclinA and Bcl2 proteins by immunoblotting. Additionally, the protein levels of cyclin-dependent kinase inhibitor p21Cip1, p53 transcription factor and of pro-apoptotic Bad, other relevant growth regulating proteins, were also assessed. Figure 7 shows that p27 protein is increased in forskolin-treated and, even more, in leptin/forskolin-treated cells, whereas p21 inhibitor is not, according to previous results showing that in MDA-MB-231 cells 8-Br-cAMP treatment resulted in elevation of p27kip protein in a sustained fashion.23 On the other hand, a decrease of both p27 and p21 proteins, is detectable in extracts from cells treated with leptin alone, according to a proliferative action by leptin on breast cancer cells.30,35 Consistently, Figure 7 also shows that both forskolin and leptin/forskolin induce a strong decrease of the positive growth regulators, cyclin D1 and cyclin A proteins. Moreover, Bcl2 anti-apoptotic protein is strongly downregulated in leptin/forskolin-treated cells and a less pronounced decrease is recorded in cells treated with forskolin alone. On the other hand, no significant changes are found in cells treated with leptin alone, 1186

Figure 4. Effects of PKA inhibitor KT5720 on leptin-, forskolin-, leptin/ forskolin-induced changes of the proliferation of MDA-MB-231 cells. Treatments with 40 nM leptin, 10-6 M forskolin, 40 nM leptin plus 10-6 M forskolin were carried out for 48 h in presence or absence of 10 μm PKA inhibitor KT5720 and cell viability was measured by MTT assay. Data represent the average of three independent experiments. The means and S.D. are shown. *p < 0.01 vs. control untreated cells.

Figure 5. Effects of forskolin treatment on ERK1/2 and CREB phosphorylation in MDA-MB-231 cells. Cells were treated or not with 10-6 M forskolin for the indicated times. The phosphorylation status and levels of ERK1/2 and STAT3 were assessed by western blotting from 40 μg of cell extracts using antibodies against the indicated proteins. The image shown is representative of two different experiments with similar results.

in agreement with the above described effects of forskolin and leptin on apoptosis of breast cancer cells. Finally, no detectable p53 protein changes were observed in response to the different treatments, whereas pro-apoptotic Bad protein levels increased in leptin/forskolin-treated cells. Altogether, these data suggest that cAMP elevation by forskolin treament causes anti-proliferative effects mainly via a cyclin D1/p27-mediated G1 cell cycle arrest, and that leptin treatment potentiates the anti-proliferative effect by cAMP elevation via a Bcl2 downregulation-mediated apoptosis.

Discussion Currently, there is no effective therapy for estrogen independent breast cancer. The activation of apoptosis of cancer cells is considered a key mechanism of anti-cancer therapy. In this study we report that in MDA-MB-231 estrogen receptor negative human breast cancer cell line, a well-established model


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significant increase of cells with hypoploid DNA content (sub-G1 population) paralleled by caspase-3 activation and PARP cleavage, whereas cAMP elevation alone (i.e., cAMP elevating agents treatments without leptin addition) results mainly in a p27/cyclin D1-mediated G1 phase cell accumulation. Remarkably, leptin-induced apoptosis is preceded by a strong increase of Bad/Bcl2 protein ratio, mainly due to a dramatic downregulation of Bcl2 content. Initial reports indicated that dibutryl-cAMP together with arginine suppresses the proliferation of MCF-7 cells.36 Subsequently, it was confirmed that the elevation of cAMP levels produces substantial effects in MCF-7 cells. Addition of 8Br-cAMP or expression of mutant (Q227L)-activated G alphas in MCF-7 cells block the ability of these cells to grow in an anchorage-independent manner, and stable transfection of activated-G alphas in MCF-7 cells reduced both EGF stimulation of MAPK in MCF-7 cells and the ability of the same cells to form tumours in nude mice.37 Subsequent studies have demonstrated that G protein alpha expression inhibits the growth of established human tumors of breast cancer cells in athymic mice by inhibiting the MAPK pathway.23 In addition, these data also imply that targeting of the cAMP/MAPK axis (i.e., by continuous elevation of cAMP) could be used to block tumor formation.10 Indeed, affecting cAMP levels by targeting its synthesis and degradation does represent a strategy for therapeutic intervention.16,22 Subtype-specific phosphodiesterase inhibitors are currently used in the treatment of some diseases.38 On the other hand, adenylyl cyclases, which synthesize cyclic AMP, are amenable to the action of specific modulators.39 Forskolin, a natural plant extract, Figure 6. Effects of concurrent treatments with forskolin and leptin on cell cycle prowas first identified as a general stimulator of adenylyl gression and apoptosis of MDA-MB-231 cells. Treatments with 40 nM leptin, 10-6 M forskolin, 40 nM leptin plus 10-6 M forskolin were carried out for 24, 48 and 72 cyclase more than 20 y ago. Recently, 6-[3-(dimethh. Panel A refers to 24 h treatments; representative FACS histograms of propidium ylamino)propionyl]forskolin, a water-soluble forskolin iodide stained cells (20,000 events/sample) are shown. The percentage of hyp- derivative with high selectivity for type 5 (cardiac) oploid sub-G1 cells and of cells in each cell cycle phase is indicated. In (B and C) adenylyl cyclase was developed and has been used in quantitative data indicating the percentage of hypoploid sub-G1 (B) and of G1 (C) the treatment of acute heart failure.27 Moreover, other cells treated at 24, 48, 72 h in three independent experiments are shown. The means and S.D. are shown. *p < 0.01 vs. control untreated cells. In (D) the effects on activa- naturally occurring molecules, such as resveratrol and tion of caspase-3 and PARP are shown. 20 μg of cell extracts from 24, 48 and 72 inorganic phosphate, have been shown to regulate adenyh treated cells were subjected to SDS-PAGE and blotted with antibodies against the late cyclase activity for controlling the proliferation of indicated proteins (α-tubulin was used as a standard for the equal loading of protein cancer cells, including breast cancer cells.40,41 In addiin the lanes). The image is representative of three immunoblotting analysis from three tion, 8-Cl-cAMP, a potent site-selective analog of cAMP, different cellular preparations with similar results. has completed several Phase I clinical studies and recently entered Phase II clinical trials as an anticancer agent.26,42 system of highly invasive breast cancer, leptin strongly enhances Unfortunately, despite their potent antiproliferative effect in many the anti-proliferative effects induced by cAMP elevation through cancer cells, substances that increase cAMP such as forskolin, apoptosis occurrence. Leptin is a well known mitogen/survival 8-br-cAMP, 8-cl-cAMP, monobutiryl or dibutiryl cAMP, and factor in breast cancer cells and several studies have addressed blockers of phosphodiesterases are not recommended to be used as the role of leptin in breast cancer pathogenesis and progression. anti-cancer drugs because of their high cytotoxicity. Interestingly, here we demonstrate that leptin causes a large proHere we describe that cAMP elevation, irrespective of the apoptotic action when used in combined treatments with cAMP cAMP elevating agent used (forskolin, or 8-br-cAMP, or IBMX) elevating agents. We demonstrate that leptin plus cAMP elevation could induce growth inhibition of MDA-MB-231 breast cancer (i.e., leptin and cAMP elevating agents co-treatments) cause a cells mainly via a cell cycle block at G1 phase. Importantly, in our www.landesbioscience.com


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study leptin was found to have a positive pharmacological interaction with cAMP elevating agents through apoptosis occurrence which allows a reduction in the effective doses of cAMP elevating drugs (by ten-fold for the forskolin). An anti-tumor effect by leptin has been observed in a murine model of hepatocellular carcinoma;43 moreover, a negative growth effect by leptin has been described to occur in pancreatic cancer cells, and also in human vascular smooth muscle cells.44,45 Moreover, a detrimental effect of leptin on human bone marrow stromal cells by apoptosis induction has been shown.46 We demonstrate that leptin/cAMP elevation causes a strong reduction of anti-apoptotic Bcl2 protein and that Bcl2 downregulation correlates to caspase-3-mediated apoptosis in response to leptin. We also provide evidence that leptin triggers Jak2/STAT3 and ERK1/2 signaling pathways and that the cAMP/PKA axis mediates the anti-proliferative effect of leptin/cAMP elevation. However, the way by which leptin drives MDA-MB-231 cells to a cAMP/PKA- and Bcl2-mediated apoptosis is not completely understood and is actually under our investigation; preliminary results suggest that the Raf/MEK/Erk pathway might play a relevant role (Naviglio S, et al. unpublished). Recently, leptin-induced Bcl2 gene upregulation has been described by microarray studies in MCF-7 breast cancer cells.47 Moreover, the Bcl2 promoter contains binding sites for CREB/ ATF1 transcription factors.48 Activation of PKA has been shown to be an important biochemical pathway for Bcl2 as well as Bad phosphorylation, reduced Bcl2-Bax dimerization and apoptosis induction triggered by microtubule-damaging drugs in MDA-MB-231 cells.49,50 Moreover, previous findings indicate that the ERK-dependent signaling pathway regulates Bcl2 expression, and vice versa that Bcl2 activates ERK signaling pathway to regulate downstream genes.51,52 The proto-oncogene Bcl2, has various functions besides its role in protecting cells from apoptosis.53,54 Drugs that target Bcl2 to induce apoptosis of cancer cells are currently in clinical development.55,56 Importantly, we demonstrate that, whatever the mechanism, leptin markedly downregulates Bcl2 protein levels when used in combination with cAMP elevating agents. In this way, the effective anti-cancer concentrations of the latter can be reduced thus decreasing their undesired side-effects. In conclusion, our data confirm evidence of adenylate cyclase/ cAMP/PKA as a growth suppressor system in breast cancer cells and, remarkably, imply that targeting of cAMP levels and leptin signalling might represent a novel simple approach to therapeutic intervention in breast cancer.

Materials and Methods Materials. All cell culture materials were from Gibco-Life Technologies (Gaithersburg, MD). Human recombinant leptin, 8-Br-cAMP, forskolin, 3-isobutyl-1-methylxanthine (IBMX), KT5720 (Protein Kinase A inhibitor) were purchased from Sigma (Sigma-Aldrich, St. Louis, MO). Anti-procaspase-3, and antipoly (ADP ribose) polymerase (PARP) antibodies were obtained from Upstate (Lake Placid, NY). Anti-tubulin antibodies were obtained from Oncogene-Calbiochem (La Jolla, CA). All other 1188

Figure 7. Effects of concurrent treatments with forskolin and leptin on levels of the indicated cell cycle and apoptosis regulating proteins. Treatments with 40 nM leptin, 10-6 M forskolin, 40 nM leptin plus 10-6 M forskolin were carried out for 48 hours. 30 μg of cell extracts were subjected to SDS-PAGE and blotted with antibodies against the indicated proteins. The image is representative of three immunoblotting analysis from three different cellular preparations with similar results.

antibodies were obtained from Santa Cruz Biotechnology (San Diego, CA). Cell culture and treatments. MDA-MB-231 human breast cancer cell line was obtained from the American Type Culture Collection (Rockville, MD). MDA-MB-231 cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 10% fetal bovine serum (FBS) and cultured at 37°C in a 5% CO2 humidified atmosphere. Typically, cells were split (8 x 105/10 cm plate) and grown in 10% serum containing medium. After 24 h, the medium was removed, cells were washed with PBS and cultured in low-serum (0.1%) fresh medium. After 36 h of serum starvation, cells were washed with PBS and exposed or not to treatments with cAMP elevating agents and/or leptin in 0.1% serum containing medium for the times indicated in Results. Floating cells were recovered from culture medium by centrifugation, and adherent cells were harvested by trypsinization. Both floating and adherent cells were used for the experiments. Leptin was prepared in HCl/NaOH solution according to preparation instructions by Sigma, 8-Br-cAMP was prepared in water, forskolin and IBMX were prepared as stock solutions in DMSO and ethanol, respectively, and dilutions were made such that final concentration of solvent(s) was kept below 0.1%. Control cells were treated with an equivalent volume of solvents. Cell proliferation assay. Cells were seeded in 96-multi-well plates at the density of 8 x 103 cells/well and managed as mentioned in the above “Cell culture and treatments” paragraph.


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Viable cells were determined by the 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, cells were treated with cAMP elevating agents and/or leptin for up to 72 h (see the fig. legends). Before harvesting, 100 μl of MTT solution (5 mg/ml) was added to each well and incubated at 37°C for 3 h, then the formazan product was solubilized by the addition of 100 μl 0.04 N HCl isopropanol. The optical density of each sample was determined by measuring the absorbance at 570 nm versus 650 nm using an enzyme-linked immunosorbent assay reader (Molecular Device). Cell proliferation assays were performed at least four times (in replicates of six-wells for each data point in each experiment). Data are presented as means ± standard deviation for a representative experiment. Preparation of cell lysates. Cell extracts were prepared as follows. Briefly, 3–5 volumes of RIPA buffer (PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing 10 μg/ml aprotinin, leupeptin and 1 mM phenylmethylsulfonyl fluoride (PMSF) were added to recovered cells. After incubation on ice for 1 h, samples were centrifuged at 18,000 g in an Eppendorf microcentrifuge for 15 min at 4°C and the supernatant (SDS total extract) was recovered. Some aliquots were taken for protein quantification according to Bradford method; others were diluted in 4x Laemmli buffer, boiled and stored as samples for immunoblotting analysis. Immunodetection of proteins. Typically, we employed 20–40 μg of total extracts for immunoblotting. Proteins from cell preparations were separated by SDS-PAGE and transferred onto nitrocellulose sheets (Schleicher & Schuell, Dassel, Germany) by a Mini Trans-Blot apparatus BioRad (Hercules, CA). II goat anti-rabbit or anti-mouse antibodies, conjugated with horseradish peroxidase (BioRad), were used as a detection system (ECL) according to the manufacturer’s instructions Amersham Biosciences (UK). Evaluation of apoptosis by flow cytometry. After drug treatment, cells were recovered as described in the above “Cell culture and treatments” paragraph, fixed by resuspension in 70% ice-cold methanol/PBS and incubated overnight at 4°C. After fixing, samples were pelleted at 400 g for 5 min, and pellets were washed once with ice-cold PBS and centrifuged for a further 5 min. Pellets were resuspended in 0.5 ml DNA staining solution (50 μg/ml of propidium iodide, PI, and 100 μg RNase A in PBS), and incubated at 37°C for 1 h in the dark. Samples were transferred to 5-ml Falcon tubes and stored on ice until assayed. Flow cytometric analysis was performed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) interfaced with a Hewlett-Packard computer (mod. 310) for data analysis. For the evaluation of intracellular DNA content, at least 20,000 events for each point were analyzed, and regions were set up to acquire quantitative data of cells with fragmented DNA (sub-G1 or apoptotic events) compared with the events that fell into the normal G1, S, G2 regions. Statistical analysis. Experiments were performed at least three times with replicate samples. Data are plotted as mean ± SD (standard deviation). The means were compared using analysis of variance (ANOVA) plus Bonferroni’s t-test. p values of less than 0.05 were considered significant. www.landesbioscience.com

Acknowledgements

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