Rapamycin And Cisplatin In Breast Cancers

Rapamycin And Cisplatin In Breast Cancers Recent gene expression profiling has identified five breast cancer subtypes, of which the basal-like breast cancers are the most aggressive and possess serious clinical challenges as there are currently no targeted therapies available. Although there is increasing evidence that these tumors confer specific sensitivity to cisplatin, its success is often compromised due to its dose-limiting nephrotoxicity and development of drug resistance. To overcome this limitation, our focus is to maximize the benefits associated with cisplatin therapy through drug combination strategies. Using a well-validated kinase inhibitors library, we showed that inhibition of mTOR, TGFà ¯Ã‚ Ã‚ ¢RI, NFà ¯Ã‚ Ã‚ «B, PI3K/AKT and MAPK pathway sensitized the basal-like MDA-MB-468 cells to cisplatin treatment. Further evaluation demonstrated that combination of mTOR inhibitor, rapamycin, and cisplatin generated significant drug synergism specifically in basal-like cells (MDA-MB-468, MDA-MB-231 and HCC1937). Th ese synergistic effects were not observed in the luminal-like T47D and MCF-7 cells. We further showed that the synergistic effects of rapamycin and cisplatin is mediated through p73. Treatment of rapamycin induced p73 upregulation and synergized cisplatin activity through activation of the p73 pathway. Depletion of endogenous p73 in basal-like cells abolished these synergistic effects suggesting that p73 is required for the rapamycin and cisplatin synergism. In conclusion, combination of mTOR inhibitors and cisplatin may be a useful therapeutic strategy in basal-like breast cancers. INTRODUCTION Recent identification of novel breast cancer subtypes with distinct biological features promises a more specific, effective and less toxic therapies to the patients. Through gene profiling, breast cancer can be categorized into five different subtypes with distinct clinical outcome. The five major subtypes of breast cancer are luminal A, luminal B, human epidermal growth factor receptor-2 (HER2) overexpressing, normal-like and basal-like breast cancer [1, 2]. Of particular important is the basal-like breast cancer which accounts for 15-20% of breast cancers overall and confers a remarkably poor prognosis compared to other subtypes. Majority of basal-like breast cancers exhibit a triple-negative phenotype, characterized by the lack of expression of estrogen receptor (ER), progesterone receptor (PR) or HER2 amplification, and often have high frequency of p53 mutation [3, 4]. Due to the lack of expression of these receptors (ER, PR and HER2), patients with basal-like breast cancers usua lly do not response to hormonal therapy, Herceptin or chemotherapy [5, 6]. As a consequence, the mortality rate of basal-like breast cancer is relatively high in comparison with the non-basal subtype [1]. Numerous clinical studies are currently ongoing to identify novel therapy for treatment of basal-like breast cancers. These include the use of specific targeted therapeutic agents (e.g. Cetuximab, Dasatinib, Bevacizumab, Abraxane and Erlotinib) or conventional chemotherapeutics agents (e.g. cisplatin, doxorubicin, and paclitaxel), either as single agent or in combination, as first line therapy for basal-like breast cancers [7-9]. Cisplatin, a chemotherapeutic agent not commonly used for breast cancer, come to light in the management of basal-like breast cancer on account of evidence that breast cancer cells with basal-like phenotype confer a selective sensitivity towards cisplatin as compared to other chemotherapeutic agents. A variety of evidence suggests that basal-like breast cancers may share defects in BRCA1-associated pathways, of which DNA repair mechanism has been compromised [10]. Indeed, recent clinical studies have demonstrated the clear advantage of cisplatin in treatment of basal-like breast cancer compared to other chemotherapeutic agents [11, 12]. Nevertheless, dose-limiting toxicity including nephrotoxicity, neurotoxicity and ototoxicity have withold the wide-spread use of cisplatin in treating breast cancers in the clinic. To address this problem, we developed a high-throughput screening assay to rapidly identify new therapeutic agents that could synergize the antitumor effects of cisplatin in basal-like breast cancers. Through the use of a small chemical library that targets some of the most relevant oncogenic pathways in basal-like breast cancer, we show that inhibition of mTOR by rapamycin incurred a specific synergistic effect with cisplatin in basal-like breast cancer cells. This synergistic effect is mediated in part through the induction and activation of p73 in the presence of rapamycin and cisplatin, respectively. Together, our findings demonstrate evidence of a synergistic relation between rapamycin and cisplatin in both inhibition of cell growth and induction of apoptosis. This suggests that rapamycin and cisplatin may be a rational combination of a targeted therapy for the refractory basal-like breast cancers. Materials and Methods Cell lines and cell culture The human breast carcinoma cell lines MCF-7, T47D, MDA-MB-231, MDA-MB-468 and HCC1937 were obtained from the American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 100 IU/ml penicillin and 100 ÃŽÂ ¼g/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C with 5% CO2. MTT cell proliferation assay Dose-response curves and IC50 values were determined using the methyl thiazolyl tetrazolium (MTT) cell viability assay as described previously [4, 13]. Cells were seeded into 96-well plates for 24 hours at a density of 5 ÃÆ'- 103 cells/well. Serial drug dilutions were prepared in medium immediately before each assay, and viable cell masses following 3 days of drug exposure were determined by cell-mediated MTT reduction. Cell growth as well as drug activity was determined by measuring absorbance at 550 nm using an Anthos systems plate reader. Construction of IC50 mean graph The IC50 mean graph was constructed as defined by the Developmental Therapeutics Program of the National Cancer Institute (http://dtp.nci.nih.gov). The mean graph consists of positive (more sensitive) and negative (less sensitive) delta values, generated from a set of IC50 values by using a three-step calculation. The IC50 values for each of cell line against the tested compound were converted to log(IC50) values. For each tested compound, the log(IC50) values are averaged. Finally, the individual IC50 value is then subtracted from the average to generate the delta value. Positive delta values project to the right of the vertical line and represent cellular sensitivities to the test agent that exceed the mean. Negative values project to the left and represent cell line sensitivities to the test agent that are less than the average value. Library screening The Inhibitor Select„ ¢ chemical library which consists of 160 well-characterized, cell-permeable inhibitors was purchased from EMD Chemicals, USA. MDA-MB-468 cells at the logarithmic phase of growth were seeded into 96-well plate at a density of 5 ÃÆ'- 103 cells/well. Each compound was added to a final concentration of 10  µM in the absence or presence of 1  µM cisplatin. Plates were incubated for 72h at 37 °C. Cell proliferation was examined using MTT assay as described previously. Combination treatments that induce growth inhibition higher than those of the same doses used alone (p Drug interaction analysis Drug combination analysis was performed by using the method as described by Chou and Talalay [14]. Briefly, cells were seeded at 5 ÃÆ'- 103 cells/well in 96-well plates and treated with various concentrations of cisplatin and compound alone or in combination for 72h. Cell proliferation was measured in each well by MTT assay. Multiple drug dose-effect calculations and the combination index plots were generated using Calcusyn software (Biosoft, Cambridge, UK). Combination index, CI 1 indicate synergism, additive effect and antagonism, respectively. Apoptosis assays Quantitation of apoptosis by annexin V/PI staining was performed as described previously [3, 4]. Briefly, both floating and attached cells were collected 72h after drug treatments. Apoptotic cell death was determined using the BD ApoAlert annexin V-FITC Apoptosis Kit (BD Biosciences, USA) according to the manufacturers instructions, and cells were analyzed on a FACSCalibur flow cytometer using CellQuest Pro software (version 5.1.1; BD Biosciences, USA). Quantitative PCR (qPCR) analysis Total RNA from cells was extracted using Qiagen RNA isolation kit (Qiagen, Valencia, CA, USA) according to the manufacturers protocol. First-strand cDNA was synthesized from total RNA using random hexamer primers and the SuperScript II system for RT-PCR (Invitrogen, Carlsbad, USA). Gene expression levels were measured by qPCR using the iQ SYBR Green Supermix reagent and an Biorad iQ5 real-time PCR detector system (Bio-Rad, Richmond, CA, USA). Data analysis was performed using Opticon Monitor Analysis Software V1.08. The expression of each gene was normalized to ÃŽÂ ²2M as a reference. The relative copy numbers were calculated from an 8-point standard curve generated from a 10-fold serial dilution of full-length cDNA constructs as described previously [3, 4]. Specific forward and reverse primer sequences are as follows : TAp73fwd, 5-GCACCACGTTTGAGCACCTCT-3; TAp73rev, 5- GCAGATTGAACTGGGCCATGA-3; ÃŽÂ ²2Mfwd, 5-AGCTGTGCTCGCGCTACTCTC-3; ÃŽÂ ²2Mrev, 5-CACACGGCAGGCATACTCATC-3; PUMA fwd PUMArev NOXAfwd NOXArev. The conditions for all QRT-PCR reactions were as follows: 3 minutes at 94 °C followed by 40 seconds at 94 °C, 40 seconds at 60 °C, and 25 seconds at 72 °C for 40 cycles. All PCR products were confirmed by the presence of a single peak upon melting curve analysis and by gel electrophoresis. No-template (water) reaction mixtures and no-RT mixtures were performed on all samples as negative controls. All experiments were performed in duplicate. Protein isolation and Western blot analysis Protein lysates from cells were extracted in ice-cold lysis buffer (0.75% NP-40, 1 mM DTT, and protease inhibitors in PBS). Total protein (25 ÃŽÂ ¼g) was subjected to SDS-PAGE followed by immunoblotting with the following antibodies: p73 (diluted 1:1,000, Ab-2; CalBiochem); pS6K (diluted 1:1,000; Cell Signaling Technology); S6K (diluted 1:1,000, Ab9645; Abcam); and ÃŽÂ ²-tubulin (diluted 1:2,500, D-10; Santa Cruz Biotechnology). Lentiviral production and infection The shRNA lentiviral constructs were created by transferring the U6 promoter-shRNA cassette into a lentiviral backbone, and high-titre lentiviral stocks were generated by co-transfection with packaging vectors into 293T cells as described previously [3, 4, 13]. The shRNA target sequences for TAp73 was 5-GGATTCCAGCATGGACGTCTT-3. The TAp73 targeted sequence is found within p73 exon 3. Therefore, this shRNA does not target ΆNp73 [4]. RESULTS Selective sensitivity of basal-like breast cancer toward cisplatin To gain an overview of the selectivity of chemotherapeutic agents for basal-like breast cancer cells, we compared their antiproliferative properties in a panel of basal-like and luminal-like breast cancer cell lines which has been validated previously through gene profiling [15]. All cells were treated with increasing concentrations of cisplatin, paclitaxel or doxorubicin for 72 hours and growth measured using the MTT assay. Figure 1A and B summarizes the results from these breast cancer cell lines in which basal-like breast cancer cells demonstrated selective sensitivity to cisplatin. This selectivity was absence in cells treated with paclitaxel or doxorubicin suggesting that basal-like breast cancer cells confer selective sensitivity towards cisplatin (Figure 1A, B and Supplement Table 1). Small chemical library screening identify rapamycin as synergistic agents for cisplatin Although cisplatin is currently one of the most used agents in the treatment of cancer, the use of cisplatin is hampered by its side effects, especially neurotoxicity, nephrotoxicity and rug resistance [16]. Hence, the present study was aimed to identify chemosensitizers that could synergize the effects of cisplatin for treatment of basal-like breast cancers. To identify small molecules that enhance sensitivity of basal-like breast cancer cells to cisplatin, a cell-based high-throughput screen was performed using MDA-MB-468 cell line and a small chemical library consisting of 160 well validated specific inhibitors. The screens were done in 96-well plates to which compounds were added at 10  µM, followed by cisplatin at 1  µM. Cell viability was measured 72 hours later by MTT assay. Each plate included controls of untreated cells, cells treated with compounds or cisplatin only, and cells treated with a combination of both agents. Combinations of the treatments that induced growth inhibition higher than those of the same doses used alone (p The molecules identified in this screen includes rapamycin, [3-(Pyridin-2-yl)-4-(4-quinonyl)]-1H-pyrazole (LY364947), 4-(3-Chloroanilino)-6,7-dimethoxyquinazoline (AG1478), (E)3-[(4-Methylphenyl)sulfonyl]-2-propenenitrile (BAY11-7082), 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) and 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580). Structures of these compounds and their growth inhibitory effects were shown in Figure 2. The molecular target of these compounds was listed in Table 1. The 6 compounds identified are specific inhibitors of the mTOR, TGF-à ¯Ã‚ Ã‚ ¢, EGFR, NFà ¯Ã‚ Ã‚ «B PI3K/AKT and MAPK pathways which have been previously reported to be upregulated specifically in basal-like breast cancers [7, 17-20]. However, when tested for synergism with cisplatin at fixed concentration ratio, only rapamycin showed synergism when combined with cisplatin in MDA-MB-468 cells using the isobologram method that simulate the median-dose combination index (CI) [14]. The mean combination index at ED50, ED75, and ED90 of rapamycin (10:1 cisplatin:rapamycin) when combined with cisplatin was 0.52 à ¯Ã¢â‚¬Å¡Ã‚ ± 0.06, where combination index Specific synergistic effects of rapamycin and cisplatin in basal-like breast cancer cells Next, we sought to investigate if combination of cisplatin with rapamycin exhibit specific synergism in basal-like breast cancers by comparing their effects in a panel of breast cancer cell lines. The antiproliferative effect of treatment was evaluated using MTT assays and drug interaction was assessed by the isobologram method as described in the previous section. The results demonstrated that combination of rapamysin and cisplatin exhibited selective synergistic effects only in the basal-like MDA-MB-468, MDA-MB-231 and HCC1937 cells, but not in the luminal-like T47D or MCF-7 cells (Figure 3A and Table 2). To ensure that the lack of synergistic effects of rapamycin and cisplatin observed in luminal-like cells is not due the general lack of sensitivity of luminal-like cells to cisplatin per se, we compared the apoptotic effects of cisplatin and rapamycin alone or in combination in MDA-MB-231 and T47D cells using an equal potent dose of cisplatin (IC50 dose). Both cell lines exhibited similar amount of apoptosis following treatment with their respective IC50 dose of cisplatin. Interestingly, when both cell lines were treated with combination of cisplatin and rapamycin, synergistic effects were evidenced only in MDA-MB-231 cells but not in T47D cells. The potentiation effects of rapamycin observed in MDA-MB-231 cells were further enhanced by sequential treatment with rapamycin for 6 hours followed by cisplatin (data not shown). These results demonstrated that rapamycin is capable of sensitizing basal-like breast cancer cells to cisplatin, suggesting that the synergistic effects of rapamyc in and cisplatin may be mediated through a common pathway. Rapamycin sensitizes basal-like cells to cisplatin through p73 up regulation Previous study has shown that inhibition of mTOR by rapamycin up regulate p73 in breast cancer cells [21]. We and others have also demonstrated that p73 is overexpressed in a subset of triple-negative breast tumors and that p73 is required for cisplatin sensitivity in basal-like breast cancer cells [4]. On the basis of these findings, we ask if the synergistic effects of rapamycin and cisplatin combination could be explained by the activation of the p73 pathway. To test this notion, MDA-MB-231 cells were treated with 10  µM of cisplatin and 100 nM rapamycin alone or in combination for 48 hours. mTOR inhibition was confirmed using phospho-S6K as a marker of mTOR activity. Quantitation of the mRNA and protein expression of the TAp73 was performed using qPCR and immunoblotting, respectively. The results showed that treatment of rapamycin or cisplatin alone did not change the expression of TAp73 mRNA but induced significant up regulation of TAp73 protein expression in MDA-MB-231 cells (Figure 4A and B). Next, we evaluated the expression of the two potent pro-apoptotic BH3 only proteins, PUMA and NOXA, which has been identified as specific p73 target genes, using qPCR [22, 23]. As expected, treatment of cells with cisplatin for 48 hours induced mRNA expression of PUMA and NOXA in both MDA-MB-231 and MDA-MB-468 cells. Significant induction of PUMA and NOXA were also observed in MDA-MB-231 cells but not in MDA-MB-468 cells following treatment with rapamycin alone. When cells were treated with combination of cisplatin and rapamycin, the expression of PUMA and NOXA was further enhanced, corroborated with the massive induction of apoptosis as shown in Figure 3B. Together, these results suggest that rapamycin synergize cisplatin activity in basal-like cells through induction of p73 pathway. p73 is required for the synergistic effects of rapamycin and cisplatin in basal-like breast cancer cells To further evaluate whether p73 is the effector of the synergistic effects of rapamycin and cisplatin in basal-like breast cancer cells, we generated a series of isogenic cell lines that have been depleted for TAp73 by stably expressing a shRNA species that target specifically human TAp73. Unlike MDA-MB-231 cells which express one predominant TAp73 isoform (TAp73à ¯Ã‚ Ã‚ ¢) isoform, MDA-MB-468 cells express high levels of two p73 isoforms, TAp73à ¯Ã‚ Ã‚ ¡ and TAp73à ¯Ã‚ Ã‚ ¢ [21]. Figure 5A showed efficient knock-down of TAp73 isoforms in MDA-MB-231 and MDA-MB-468 cells. As expected, treatment of cisplatin alone induced significant amount of apoptosis in MDA-MB-468 vector control cells. This apoptotic effects were further enhanced in the presence of rapamycin, consistent with our previous observations (Figure 3B). In stark contrast, depletion of TAp73 not only reduced the amount of apoptosis following treatment of cisplatin alone but also completely abrogated the synergistic effects of rapamycin (Figure). This result is further supported by the isobologram analysis which showed a lack of synergism of rapamycin-cisplatin treatment in the TAp73 depleted cells. Together, these results suggest that TAp73 is required for the synergistic effects of rapamycin and cisplatin in basal-like breast cancers. DISCUSSION By gene profiling, breast cancers can be classified into 5 molecularly distinct subtypes: luminal A, luminal B, HER2+, basal-like and normal breast cancers. The basal-like subtype, which represents 15-20% of breast cancers, has been subjected to extensive investigation in recent years due to its association with poor patient survival [1, 2, 20, 24]. Unlike many breast cancers, patients diagnosed with basal-like breast cancers are not eligible for molecular targeted therapy that target ER (e.g. tamoxifen, aromatase inhibitors) or HER2 (e.g. Herceptin) as they do not express the estrogen receptor (ER) or progesterone receptor (PR), nor do they have amplified HER2 [1, 24]. The treatment option therefore is relied on aggressive conventional chemotherapies which have limited efficacy, many side effects and often high rate of relapse. Hence, development of an effective therapeutic strategy remains an important goal in the management of basal-like breast cancer. Several lines of evidence has suggested a link between basal-like breast cancers and BRCA1 deficiency [7, 25, 26]. In most cases, the clinical features and outcomes for women with sporadic basal-like breast cancers are broadly similar to those with BRCA1-related cancers including high tendency of developing high grade, high mitotic index tumors, shorter time of relapse, similar pattern of metastatic spread and cytogenetic changes associated with frequent loss of X-chromosome inactivation [7, 27-32]. The majority of BRCA1-associated cancers are also triple-negative (ER, PR and HER2 negative), express basal cytokeratins and other markers commonly seen in basal-like breast cancers (e.g. p53, P-cadherin and EGFR) [7]. Gene expression profiling also demonstrated that BRCA1-associated cancers segregate strongly with basal-like breast cancers [2, 29, 33, 34]. Although BRCA1 somatic gene mutations are uncommon in sporadic basal-like cancers, these tumors have been shown to have a dysfunction al BRCA1 pathway due to BRCA1 gene promoter methylation and/or BRCA1 pathway transcriptional inactivation [7, 25, 26]. The fundamental biological similarities between hereditary BRCA1-related breast cancers and basal-like cancers suggest that strategies targeting the dysfunctional BRCA1 pathway may be effective in basal-like breast cancers. There is increasing evidence that the DNA repair defects characteristic of BRCA1 related cancers, especially defective homologous recombination, confer sensitivity to certain systemic agents, such as platinum-based chemotherapy and poly(ADP-ribose) polymerase (PARP) inhibitors [34-38]. Indeed, recent clinical studies revealed that sporadic basal-like cancers responded to platinum-based chemotherapy and were associated with a high rate of complete pathologic response [9, 11, 38]. Consistent with the clinical data, our in vitro study also reveals that basal-like breast cancer cells confer specific sensitivity to cisplatin as compared to other chemotherapeutic agents (e.g. doxorubicin or paclitaxel) (Figure 1), further support research into the utility of platinum-ba sed agents in basal-like breast cancers. Given the high specificity and response rate of basal-like breast cancers toward platinum-based therapy, our focus is to maximize the benefits associated with this therapy through drug combination strategies. Using a small chemical library consisted of 160 well-validated and specific inhibitors that target the human kinome, we have identified 6 compounds that significantly potentiate the antiproliferative effects of cisplatin in basal-like breast cancer cells. These compounds include rapamycin, LY364947, AG1478, BAY11-7082, LY294002 and SB203580 which targets the mTOR, TGFà ¯Ã‚ Ã‚ ¢RI, NFà ¯Ã‚ Ã‚ «B, PI3K/AKT and MAPK pathway respectively. Of note, these pathways have been reported previously to be over activated in basal-like breast cancers [7, 17, 18, 20]. To further investigate the mode of interaction between these compounds and cisplatin, we performed a drug combination study using the isobologram approach as described previously (Ref). Out of the 6 compounds identified, rapamycin showed the strongest synergistic effects with cisplatin while others (LY364947, AG 1478, BAY11-7082, LY294002 and SB203580) showed mainly additive effects. This result is consistent with other studies which show that inhibition of mTOR by RNAi or small molecules (e.g. rapamycin, CCI-779, RAD001) enhances cisplatin chemosensitivity in ovarian [39-41], endometrial [42], head and neck [43, 44], lung [45], skin [46, 47] and liver [48] cancers. We next compared the synergistic effects of rapamycin in combination with cisplatin in a panel of luminal-like and basal-like breast cancer cell lines that has been previously validated by gene profiling [15]. Intriguingly, the synergistic effects were observed only in MDA-MB-468, MDA-MB-231 and HCC1937 basal-like cells, but not in MCF-7 or T47D luminal-like cells. Several models have been proposed to explain the synergistic effects of rapamycin and cisplatin in cancer cells. Beuvnk et al., 2005 showed that RAD001 (Everolimus), a rapamycin derivative, dramatically enhances cisplatin-induced apoptosis in wild-type p53 but not mutant p53 tumor cells by inhibiting p53-induced p21 expression [49]. Wangpaichitr et al., 2008 demonstrated that inhibition of mTOR by CCI-779 decreased levels of the anti-apoptotic proteins, BCL2/BCLxL, and increasing apoptosis in lung cancer cells that is resistance to cisplatin [50]. Although these models provide important evidence for mTOR inhibition and cisplatin synergism in cancer cells, it fails to explain the specific synergism we observed in basal-like breast cancer cells, as the basal-like cells that we tested are p53 mutated and do not express high level of BCL2/BCLxL (data not shown). This led us to postulate that a common signal transduction pathway inhibited by rapamycin may be an important component that sy nergizes cisplatin sensitivity in basal-like cells. Since p73 has been reported to mediate cisplatin sensitivity in a subset of triple-negative breast cancer cells [4] and that inhibition of mTOR by rapamycin or RNAi lead to upregulation of p73 [21], we postulated that activation of the p73 pathways might be important for the synergistic effects of rapamycin. To test the role of p73 in rapamycin and cisplatin synergism, we first evaluated the expression of p73 mRNA and protein levels following treatment with cisplatin or rapamycin alone or in combination in MDA-MB-231 cells. Consistent with previous studies, treatment of cells with cisplatin or rapamycin alone induces p73 protein expression followed by transcriptional activation of the 2 potent pro-apoptotic p73 target genes, PUMA and NOXA. When MDA-MB-231 cells were co-treated with rapamycin and cisplatin, the elevation of p73 and its pro-apoptotic target genes were synergistically enhanced. The observed changes in p73 protein in MDA-MB-231 cells, however, were not due to parallel changes in p73 RNA levels, suggesting that inhibition of mTOR might lead to inactivation of a yet unknown p73 specific protein degradation pathway. To validate that the rapamycin and cisplatin synergism is mediated by p73, we generated isogenic MDA-MB-231 and MDA-MB-468 cells that were depleted for p73 using a lentiviral-shRNA that target specifically the transactivating isoform of p73 (TAp73). Indeed, depletion of TAp73 in MDA-MB-231 and MDA-MB-468 cells completely abrogated the synergistic effects of rapamycin suggesting that the synergism between rapamycin and cisplatin required p73 function. Although the combination of cisplatin and rapamycin has not been previously investigated in clinical study, it is worth noting that a phase II neo-adjuvant clinical trial of cisplatin and RAD001 (Everolimus), in patients with triple-negative breast cancers has recently open for recruitment (ClinicalTrials.gov Identifier: NCT00930930), and will be able to address the potential of cisplatin and mTOR inhibitors combination therapy directly. It would be equally intriguing to determine the role of p73 related pathway as potential biomarkers that might predict response to treatment given the pivotal role of p73 in the synergistic effects of mTOR inhibition and cisplatin sensitivity. In conclusion, combination of mTOR inhibitors and cisplatin may be a useful therapeutic strategy in basal-like breast cancers.

John Carlos Story Essay -- Sports

The â€Å"John Carlos Story† is a book about the struggles of growing up black in America at a time when much of the nation was still segregated. John Carlos was a member of the â€Å"Olympic Project for Human Rights.† After winning the bronze medal, John Carlos and a friend and teammate, Tommy Smith, who won the gold medal, raised their fists in opposition of racial inequality and in unity of civil rights. This book shows examples of sociology in everyday life which can be explained through theories and concepts, which centers mainly on the event surrounding their win at the Olympic Games. A seemingly non-emotional, non-verbal, non-violent protest over racism was met with a great deal of negativity at the Olympic Games. Because of the location of their â€Å"protest† over racial inequality, many people did not agree with what they were doing. It was not considered socially acceptable to let their opinion on the matters of race play a part of the â€Å"opinion-free† Olympic Games. Instantly, the audience at the game did not like their way of letting the people know of their beliefs as the crowd went silent. As John Carlos stated in his book, â€Å"There’s something awful about hearing fifty thousand people go silent, like being in the eye of a hurricane. Then, as the national anthem played in full force, the calm before the storm ended and the ‘boos’ started coming down† (Carlos 121). Smith and Carlos were then able to walk off the field but it just got worse, â€Å"the shock was gone and it was officially getting ugly† (Carlos 121). The audience started to yell at them and called them â€Å"anti-American† (Carlos 121). Because of the social setting, people did not understand the purpose of what they were doing or what it had represented. There was ta... ... period of time (1968) in which there was no race cohesion and blacks were not viewed positively by some Americans. Also, the place was likely not the proper location for such a â€Å"demonstration.† Again, people had different reactions and to some, Carlos and Smith were heroes. This can be considered â€Å"labeling theory† which focuses on â€Å"how people define deviance – what is or is not ‘normal’-which is a core issue† (Ballantine/Roberts 175). In the end, those that thought he was a hero were helpful to John Carlos and with his book, society accepts him for his efforts and if there are individuals that still do not, they are the minority in society. Works Cited Ballantine, Jeanne and Roberts, Keith. Our Social World, Introduction to Sociology. Pine Forge Press, 2011. Carlos, John. The John Carlos Story. (With Dave Zirin). Haymarket Books, Chicago, IL 2011.

Mus 100 Study Guide

MUS 100 FINAL STUDY GUIDE CHAPTER 17: – Fortepiano: early piano, named for its range of dynamic levels; it was smaller and less sonorous than the modern instrument. – Classical style: restrained, objective style of art. Classical refers to Western music characteristic of the period from 1750-1825. Composers: – Mozart: Invested much of his music with a degree of emotion expression unusual for his time. Never allowed emotion to dominate his art. – Haydn: Wrote pleasant, good-natured music throughout his long life. Wrote masses, oratorios, and other religious compositions for church and for concert performance. Beethoven: Wrote masses, oratorios, and other religious compositions for church and for concert performance. CHAPTER 18: – Form: organization and design of a composition, or of one movement within a composition. – Symphony: multimovement orchestral form. – Sonata-Allegro: â€Å"first movement form†. The 3 sections: expositio n, development, and recapitulation-form a binary design. – Exposition: first section of a fugue or of a sonata-allegro. – Development: 2nd section of the sonata-allegro; it moves through many keys. – Recapitulation: 3rd section of the sonata-allegro.Reviews the material of the exposition, presenting it in a new light. – Coda: Meaning, â€Å"tail†; a closing section. – Minuet and Trio: ABA. Often the 3rd movement of a symphony, sonata, or string quartet. Consists of two minuets, the second (trio) lighter and more lyrical than the first. – Cadenza: extended passage for solo instrument; typical feature of a solo concerto. – Rondo: ABACA. Form in which various episodes alternate with the opening material. The tempo is usually fast, and the mood merry. – String Quartet: chamber ensemble consisting of two violins, a viola, and a cello. Sonata (classical period): a multimovement composition for one or two solo instruments. CHAP TER 19: – Overture: introductory orchestral piece. – Comic Opera (ope’ra comique, singspiel, opera buffa): Operas light in mood, modest in performing requirements, written in the vernacular language of the intended audience. – Requiem: mass for the dead. – Ensemble Finale: final scene of a musical show in which several soloists simultaneously express, in different words and music, their individual points of view. CHAPTER 20: – Motive: short melodic phrase that may be effectively developed. Art song: concert setting of a poem, usually by a well-known poet, to music. – Lieder: German art songs. – Song cycle: sets of songs by one composer, often using texts all by the same poet. Composers: – Schubert: earliest master of romantic art son. Composed 143 songs at 18. â€Å"Godfather† of the romantic period genre. CHAPTER 21-22: – Cyclic form: multimovement form unified by recurrence of the same or similar melodic material in two or more movements. – Absolute music: instrumental music having no tended association with a story, poem, idea or scene; non-program music. Concert overture: one movement orchestral composition, often inspired by literature and dramatic in expression, yet generally subject to analysis according to classical principles of form. – Program symphony: symphony (composition for orchestra in several movements) related to a story, idea, or scene, in which each movement usually has a descriptive title. – Idee fixe: single melody used in several movements of a long work to represent a recurring idea. – Thematic transformation: variation of thematic or melodic material for programmatic purposes.Sometimes called metamorphosis. – Dies irae: Gregorian chant for the dead. – Symphonic poem (tone poem): programmatic composition for orchestra in one movement, which may have a traditional form (such as sonata/rondo) or an original irregular form . Composers: – Brahms: misplaced classicist. Poured the warmest Romantic emotional content into his classical forms. He based his music on models from the past. – Berlioz: his works were based on unrequited love. Used the idee fixe, which was a melodic reference to his beloved. CHAPTER 23: Character piece: relatively short piano piece in a characteristic style or mood. – Nocturnes: Piece expressing the â€Å"character† of night. – Prelude: short independent or introductory piece for keyboard. – Etude: a virtuosic instrumental study or â€Å"exercise† intended for concert performance. – Rubato: romantic technique of â€Å"robbing† from the tempo at some points and â€Å"paying back† at others. Composers: – Chopin: only great composer who wrote almost exclusively for piano. Most pieces are miniatures. Virtuoso pianist, most famous for lyrical and melancholic melodies.CHAPTER 25: -Post-romanticism: general ter m for several romantic styles that succeeded the dominance of German Romanticism and preceded the return of classicism to the arts. – Atonality: avoidance of a tonic note and of tonal relationships in music. – Impressionism: style of painting and music that avoids explicit statement, instead emphasizing suggestion and atmosphere. – Primitivism: style inspired by primitive works of art and by the relaxed life of unsophisticated cultures. – Pizzicato: technique of plucking string instruments.Composers: – Mahler: post-romantics. Wrestled with conflicting romantic and classical ideals. – Strauss: leader of post-romantic composers. Strictly classical style but developed romantic techniques. – Debussy: first musician labeled an impressionist. Developed unusual harmonies and exotic timbres. – Schoenberg: inventor of the 12-tone method (serialism) > Using the 12 pitches equally. > 12 tone row: playing the 12 pitches in whatever order; no repeated tones until the row has been fully played. > Wrote in a free atonal style gt; Drifted away from traditional harmony and experimented other styles – Stravinsky: went through an early ballet period before the war. He went through a neo-classical period. > Primitivism: movement in the second decade of the 20th century. Reveals romanticism characteristics. Characterized by strong savage rhythms, dissonant combinations of sound and narrow melodies. > â€Å"Rite of Spring†: controversial piece, ballet, and scandal piece CHAPTER 27: – Experimentalism: exploration of previously unknown aspects of musical sound. Polytonality: two or more keys at the same time. – Tone cluster: chord built on seconds. – Prepared piano: piano whose timbre and pitches have been altered by the application of foreign materials on or between the strings. – Twelve-tone technique: arrangement of the twelve chromatic pitches into a tow that provides the melodic and harmonic basis for a music composition. Row: series of tones on which a serial composition is based. Composers: – Schoenberg: inventor of the 12-tone method (serialism) – Weberm: developed his own styles: lean, clean, delicate, and strong. Ives: invented polytonality (incorporating of two different keys). – Cowell: invented the plucking of a piano sound. – Cage: 1912-1992 not trained as a musician. Brought up in Los Angeles. Became a composer. > Alatoric: predetermined sounds and just guessed when it should be played. > Conceptual art: piece called 4 minutes a 33 seconds – just the sounds in CHAPTER 28: – Neoclassicism: 12th century version of classicism in music. – Neoromanticism: 12th century version of a romantic approach to music. –Minimalism: style of music based on many repetitions of simple melodic lines that gradually change and slowly evolve patterns and rhythmic patterns. Composers: – Copland: American nationa list composer > â€Å"Dean of American Music† – Gershwin: Best known of all American opera, filled with the characteristic sounds of jazz, including syncopated rhythms, expressive vocal catches and slides. – Prokofiev: focused on neoclassical music. – Barber: focused on neoromanticism. > Adagio for string orchestra (tonal piece) – Reich: focused on minimalism. – Glass: focused on minimalism.

Hawaiian Punch Essay

In July 2004, Kate Hoedebeck was promoted to director of marketing for the Hawaiian Punch brand. The CEO of the parent company, Cadbury Schweppes Americas Beverages, set a goal to create a premier marketing and sales organization that capitalizes on the terrific brand recognition. To reach this goal each brand is required to meet the desires of their customers; bottlers, distributors, retailers, and consumers. When creating the 2005 business marketing plan Kate faced a problem. The Hawaiian Punch brand had two manufacturing, sales, and distrution networks. Her objective was to determine the role each network played in the future sales and profitability of the company. III.Alternate Solutions a. Product Positioning Hawaiian Punch needs to clairify the positioning of its brand. The positioing statement needs to clairify its target markets, and focus on the values of its customers. The majority of the Hawaiian Punch juice drinkers ranged from 6 – 18 years of age. Adolescences purchase the drink most frequently from the soda aisle. Of cosnumers aged 6-12, the majority of the purchases were made in the juice aisle by parental/guardian figures. Including the fun consumption experience, its unique taste, and high viatim C content is the posotion the brand wants to own. To out perform the competitors it is necessary to establish these elements and clearly define where Hawaiian Punch sits. Its more than your normal juice, but healthier than soda. b. Innovations Hawaiian Punch would benefit from launching new sizes and flavors. Retailers believed the potential of market growth hinged on creating new packing and flavors. Hawaiian Punch recently experienced slowed growth due to the decrease in the juice market. Hawaiian Punch maintained market share, and Trade Customers believed the market would rebound. To take advantage of the market portential, Hawaiian Punch should innovate its product size offering. Currently Hawaiian Punch is packaged in 1-gallon bottle, a half-gallon bottle, a 2-liter bottle, a 20-ounce bottle, a 6.75-ounce single serve standup pouch, and 12-ounce cans. According to customer insights, 77% of Hawaiian Punch buyers purchased only size. According to reasearch, 68% of buyers that purchaed the 1-gallon bottle and 53% of buyers that purchased  the half-gallon size were exclusive in their size purchases. The 2-liter and 20-ounce bottle purchasers experienced the least amount of size loyalty. Despite having customer enjoying the different size offerings the numerous amount of sizes caused distribution constraints and shelving issues at numerous locations. By eliminating certain size offerings with low size loyalty and creating new ones while maintaining variety will elliviate contraints and provide new opportunties for customers. The brand would also benefit from new flavor innovation. Hawaiian Punch currently has 11 product flavor offerings. The most popular and original flavor maintains to be fruit Punch. Out of the 11 product offerings there has been a wide variety of success. Hawaiian Punch needs to establish stable products in both their Finished goods and Direct-Store Delivery networks. Products that do not show profitabilty should be elimiated. Introducing new flavors to the Direct-Store Delivery will benefit because of the large market potential and smaller size offerings. c. Allowances and Advertising Hawaiian Punch should increase its advertising budget and allowances for marketing. Currently Hawaiian Punch spends $2.2 Million on advertising a year. This accounts for 1% of advertising by Hawaiian Punch’s competitve juice brands collectively. Their current advertising mix consists of only radio and magazine print ads. The largest amount of advertising dollars spent is in television which currently Hawaiian Punch does not participate. This media outlet accounts for roughly 82% of total media dollars by competitors. Increasing media dollars will help reach new customers, and enforce new flavors to the brand. Of the advertising dollars spent much should be included in recreating the â€Å"Punchy’ Image. Past years have downplayed this aspect of the product brand, but still plays a large part in its recognition. By including Punchy in their advertising they should focus their attention on children between 8-18 years old. Punchys image will play a large role in delivery the benefits of the fruit drink and the position in the market. IV.Selected Solution It is recommended that Hawaiian Punch position their brand and maximize profit by increasing advertising and innovating their products. Having Dual distribution networks it is necessary to establish market segments. The  Finished Goods Network buyer is mainly mothers/guardians. This segment presents the largest amount of repurchasing and brand loyalty. Focusing advertising on the energic times and healthy benefits will persuade mothers ino purchasing the drink because it offers V.Conclusions Entering into a licensing agreement is not a good decision in the European market, the three year agreement is too short and more importantly Baxton is allowing too much of the revenue to go to Bar Maisse, they need an arrangement where they receive a larger portion of the proceeds. At the same time, choosing to enter the European market is too risky and has too many unknowns. The expertise of Bar Maisse is needed for success to be likely; otherwise there are too many unknowns with where operations should be based on what markets are more likely to yield successful sales. While focusing on Europe and entering the market at this time does appear to be the correct decision, it should not be done at the expense of increasing growth in the United States. That market is under developed and is not being capitalized, as it should be.

Individualism

Individualism 1) An individualist is considered to be someone with personality and character, someone who is not easily intimidated by social pressure or customs, someone with a personal opinion and a singular view of the world. Because modern society finds it important that people think independently, decide autonomously and take personal initiatives, the concept of individualism has acquired a positive connotation. However, individualism is also linked with the tendency to withdraw from social life and turn in towards oneself. 2) Which drives people to withdraw into a small, enclosed world consisting of their family and a few select friends, leaving the rest of society to its own devices. 3) Courageous individualism refers to the dedication shown by people who have independently chosen for something or someone and work energetically for its sake in spite of opposition from family or society; indifferent individualism refers to people who have either lost all hope in their fellow human beings or are no longer interested in initiatives of a social nature, with the result that they are prepared to withdraw from society at all costs.Individualism4) people who have received less education and who find themselves in a precarious economic situation are more quickly inclined to believe that it is 'everyone for himself' in our society: on this view, politicians pay no attention to the needs of the people, society develops in a chaotic and unpredictable manner, the welfare we once enjoyed belongs definitively to the past, life has little meaning, and there are no longer any people or associations to which one can make an appeal. 5)On the other hand, whoever considers work not only as one's own achievement but also as an opportunity provided by the community, and whoever sees it as one's duty as a citizen to ensure opportunities for fellow citizens...

Implementation and evaluation plan Assignment Example | Topics and Well Written Essays - 1000 words

Implementation and evaluation plan - Assignment Example The evidences focus on the systematic reviews of the recommendation and literature according to evidence level, using criteria that are pre-defined. The treatment lines are flagged out based on methods that included expert clinical support and evidence. Interpersonal Therapy (IPT) and Cognitive-Behavioral Therapy (CBT) have the most evidence efficiently. Additionally, both maintenance and acute MDD phases, and have undergone study in combination with the anti-depressants. Cognitive-Behavioral Therapy is well researched in connection with bibliography and Computer-Developed methods. The evidence of Cognitive-Behavioral Analysis Systems of Psychotherapy and Behavioral Activation are significant. However, the evidences need replication. Modern psychotherapies that include Commitment Therapy and Acceptance, Motivational Interviewing, and Mind-fullness-Based Cognitive therapy have no relevant evidence as psychodynamics therapy and acute treatments. Although various forms of psychotherapy have been studied, only a few types have been subjected to evaluation in RCTs. Evidence of combination of various kinds of antidepressant and psychotherapy medication is confined despite the vast use of the therapies concomitantly. Lamotrigine is appropriate to the client's problem because according to RCT, almost half of the patients responded positively. This is two times experienced with the placebo group according to level 1 (22, 23). Additionally, recent RCT in clients who responded positively on bipolar depression under lithium revealed that Lamotrigine add-on is superior compared to placebo add-on when used to treat depressive symptoms. This is evident by greater rise in the MADRS scores. According to large RCT, combining olanzapine and fluoxetine is slightly but significantly better compared to lamotrigine immunotherapy. However, Lamotrigine is highly tolerated. Combination of fluoxetine and Olanzapine was linked with statistical significance and positive results in maniac a nd depressive systems when compared to Lamotrigine. On the other hand, the size of its effect was small, and no difference was recorded in their response rate. According to the guidelines of 2005 bipolar, only a single RCT had shown antidepressant efficiency of quetiapine monotherapy in treating bipolar depression (25). Its rate of remission was 52.9 percent in the group consuming 600mg per day and 300 mg per day of quetiapine relative to 28.4% for placebo (Sagar et al., 2009). At earlier stages of testing, quetiapine monotherapy was thought to be immature as an antipsychotic depressant for bipolar. The second RCT confirmed the efficiency of quetiapine monotherapy (26). The study recommended quetiapine monotherapy as bipolar depression first-line option. Further evaluations (25) showed an improvement in health-related standard of life. Client's input can be obtained by conducting a randomized controlled trial. This involves assessing a period's systematic intervention program. This includes carrying out a psycho education program of structured group, monthly telephone evaluation and monitoring of medication adherence and mood symptoms, feedback during the treatment of mental health providers. Additionally, facilitate proper follow-up care and as-required crisis and outreach intervention. The patients can be monitored by augmenting their self-management skill through psycho education. Consequently, the decision support of the provider by

Math Edu Essay Example | Topics and Well Written Essays - 500 words

Math Edu - Essay Example First, students can be shown a square along with the length of its sides. Ask students to calculate its area. Next, pass out several cubes to the class so that there are enough for everyone to look at. These cubes should preferably have the same approximate side measurements as the square previously displayed. After explaining that surface area is the total area of the surface on the outside of an object, ask students how many sides there are, and what the shape of each side is. Ask them to make the connection themselves as to how to find the surface area of the cube. If they do not get it at first, hold up a picture of the square they just found the area of and compare it to the sides of the cube. They should be able to recognize that to find the surface area of the cube you multiply the area of the square by the number of squares/sides on the cube. Another good introduction or way to reiterate an understanding of how to find the surface area of a cube is to transform a two-dimensional drawing into a three-dimensional object. Pass out pieces of paper with six large squares on them. These squares should be arranged in a way that four are next to each vertically and one square is on each horizontal side of the third square. (It should resemble a cross).