The Stone Age Essay Example | Topics and Well Written Essays - 750 words

The Stone Age - Essay Example During this time, people made their tools and weapons mainly with the help of stones when metal working had not taken place yet. Geographically primeval culture has spread to all continents except Antarctica. The Stone Age is divided into three eras, which differ from each other with own peculiarities and conditions. The Stone Age brought humans to a totally new stage of their development. When climate drastically changed and glaciers melted, nomadic people reached the territory between Israel and Iraq, which hills were full of verdurous trees and wild animals. The Middle East became very attractive to humans who found favorable conditions for living here. Ancient people discovered that grain could give a lot of harvest, so eventually they found a way to gather crop with the help of appropriate stone tools. According to the archaeobotanist, George Willcox (2003), it had been noticed that during three-week period of time one person could gather enough grains for being able to feed fou r people for the entire year (Stories from the Stone Age). In additional, ancient people realized the efficiency of grain that could be stored for a long period of time without decay in case it was kept in a dry place. Consequently, discovering a very profitable place full of potential food, people at last found a reason to stop their nomadic life and settle down. Moreover, their decision to stay on chosen territory was also influenced with the fact that all tools made of stones and intended for gathering harvest were quite heavy, therefore people’s inability and lack of desire to carry their burdens eventually managed to change their lives irrevocably. Settling down in the Middle East made ancient people to face several challenges, one of which was finding ways to produce food out of plants. Also humans managed to develop and widen variety of tools for hunting realizing how precarious this activity was. Hence, people started to use stones connected by cords for catching wild animals and double-bladed sickles for cutting grass. In addition, people discovered usage of grains and began to produce so-called ancestor of bread. Gathering harvest and sharing tools helped people to unite their strength in a way when priority moved from individual prosperity to community’s wellbeing. People of that era had a special ritual for burial. They put dead person into the hole, placed a big stone on a chest and laid some personal possessions and various offerings. As archeologists found out, with the lapse of time this funeral rite faced some changes, like later n people started to took the scalp of the dead person out of a hole and bring it back into the world. Stones which had been found on the mentioned territories contained some scratches, which nowadays archeologists interpret as a first attempt to represent numbers. This fact has a great importance in a formation of ancient culture. Despite the fact that ancient people at last found an ideal place to stay on, they again were forced to return to their former nomadic lifestyle later on. Hence, the climate change brought draught which in its turn caused a dreadful shortage of food. So this was another challenge which ancient people were forced to overcome. All population split for small groups and began searching for food. One part of people finally settled down in Jordan near the

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.

Telling America s Story Essay -- William F. Lewis Essays

Telling America 's Story The three essays of rhetorical criticism, Telling America 's Story: Narrative Form and the Regan Presidency by William F. Lewis, The "Promiscuous Audience" Controversy and the Emergence of the Early Woman 's Rights Movement by Susan Zaeske, and Medicine, Rhetoric, and Euthanasia: A Case Study in the Workings of a Postmodern Discourse by Michael J. Hyde each employ a variety of strategies to examine the rhetoric of three distinct situations. This paper will attempt to dissect each of the essays in a comparative manner. Specifically, it will evaluate the introductions, how effectively they are constructed, and how the essays follow according to the expectations set forth in the introductions. In Telling America 's Story, Lewis starts by providing a quick explanation of the country's status as Regan entered office. The reader is instantly introduced to "the Western world's most gifted communicator." Lewis does not leave the reader with that viewpoint for long as he goes on to explain the views of Regan's critics. Arguments of Regan's "ideology without ideas" and the New Republic editor's comment that "Ronald Regan has never let the facts get in the way of a good story" begin to steer the reader toward a more objective state of mind. A fair balance of the two viewpoints is maintained throughout the piece, but the overall feel of it seems to be geared toward the latter. It is not until half way down the second page of his essay that Lewis clearly states his purpose, to "account for the distinctive reputation, style, and effect of Ronald Regan's discourse". He even lists the two objectives that his essay will accomplish, which are explained later under headings that bear the same name as the stated ... ...ike Lewis's topic. The question/answer method is effective in this essay because it gives the reader something to prepare for. The reader knows what to look for in the rest of essay and will be more attentive when reading. She does not leave the reader hanging; the rest of the essay is distinctly laid out and easily answers all questions. The review of these essays showed that while rhetorical criticism does need to have formal structure, there are many ways for a critic to accomplish their objectives within the confines that basic structure. Although it is not always best choice for every situation, I feel that a shorter, more direct approach to an introduction, as in Hyde's piece, is the most effective. I also thought that the question/answer format worked well for Zaske. It may have been the topic, but I found that the Hyde essay was the most engaging overall. Telling America 's Story Essay -- William F. Lewis Essays Telling America 's Story The three essays of rhetorical criticism, Telling America 's Story: Narrative Form and the Regan Presidency by William F. Lewis, The "Promiscuous Audience" Controversy and the Emergence of the Early Woman 's Rights Movement by Susan Zaeske, and Medicine, Rhetoric, and Euthanasia: A Case Study in the Workings of a Postmodern Discourse by Michael J. Hyde each employ a variety of strategies to examine the rhetoric of three distinct situations. This paper will attempt to dissect each of the essays in a comparative manner. Specifically, it will evaluate the introductions, how effectively they are constructed, and how the essays follow according to the expectations set forth in the introductions. In Telling America 's Story, Lewis starts by providing a quick explanation of the country's status as Regan entered office. The reader is instantly introduced to "the Western world's most gifted communicator." Lewis does not leave the reader with that viewpoint for long as he goes on to explain the views of Regan's critics. Arguments of Regan's "ideology without ideas" and the New Republic editor's comment that "Ronald Regan has never let the facts get in the way of a good story" begin to steer the reader toward a more objective state of mind. A fair balance of the two viewpoints is maintained throughout the piece, but the overall feel of it seems to be geared toward the latter. It is not until half way down the second page of his essay that Lewis clearly states his purpose, to "account for the distinctive reputation, style, and effect of Ronald Regan's discourse". He even lists the two objectives that his essay will accomplish, which are explained later under headings that bear the same name as the stated ... ...ike Lewis's topic. The question/answer method is effective in this essay because it gives the reader something to prepare for. The reader knows what to look for in the rest of essay and will be more attentive when reading. She does not leave the reader hanging; the rest of the essay is distinctly laid out and easily answers all questions. The review of these essays showed that while rhetorical criticism does need to have formal structure, there are many ways for a critic to accomplish their objectives within the confines that basic structure. Although it is not always best choice for every situation, I feel that a shorter, more direct approach to an introduction, as in Hyde's piece, is the most effective. I also thought that the question/answer format worked well for Zaske. It may have been the topic, but I found that the Hyde essay was the most engaging overall.

Filipino people Essay

Women have always enjoyed greater equality in Philippine society than was common in other parts of Southeast Asia. Since pre-Spanish times, Filipinos have traced kinship bilaterally. A woman’s rights to legal equality and to inherit family property have not been questioned. Education and literacy levels in 1990 were higher for women than for men. President Aquino often is given as an example of what women can accomplish in Philippine society. The appearance of women in important positions, however, is not new or even unusual in the Philippines. Filipino women, usually called Filipinas, have been senators, cabinet officers, Supreme Court justices, administrators, and heads of major business enterprises. Furthermore, in the early 1990s women were found in more than a proportionate share of many professions although they predominated in domestic service (91 percent), professional and technical positions (59. 4 percent), and sales (57. 9 percent). Women also were often preferred in assembly-type factory work. The availability of the types of employment in which women predominated probably explains why about two-thirds of the rural to urban migrants were female. Although domestic service is a low-prestige occupation, the other types of employment compare favorably with opportunities open to the average man. This favorable occupational distribution does not mean that women were without economic problems. Although women were eligible for high positions, these were more often obtained by men. In 1990 women represented 64 percent of graduate students but held only 159 of 982 career top executive positions in the civil service. In the private sector, only about 15 percent of top-level positions were held by women. According to many observers, because men relegated household tasks to women, employed women carried a double burden. This burden was moderated somewhat by the availability of relatives and servants who functioned as helpers and child caretakers, but the use of servants and relatives has sometimes been denounced as the equivalent of exploiting some women to free others. Since the Spanish colonial period, the woman has been the family treasurer, which, at least to some degree, gave her the power of the purse. Nevertheless, the Spanish also established a tradition of subordinating women, which is manifested in women’s generally submissive attitudes and in a double standard of sexual conduct. The woman’s role as family treasurer, along with a woman’s maintenance of a generally submissive demeanor, has changed little, but the double standard of sexual morality is being challenged. Male dominance also has been challenged, to some extent, in the 1987 constitution. The constitution contains an equal rights clause–although it lacks specific provisions that might make that clause effective. As of the early 1990s, divorce was prohibited in the Philippines. Under some circumstances, legal separation was permitted, but no legal remarriage was possible. The family code of 1988 was somewhat more liberal. Reflective of Roman Catholic Church law, the code allowed annulment for psychological incapacity to be a marital partner, as well as for repeated physical violence against a mate or pressure to change religious or political affiliation. Divorce obtained abroad by an alien mate was recognized. Although the restrictive divorce laws might be viewed as an infringement on women’s liberty to get out of a bad marriage, indications were that many Filipinas viewed them as a protection against abandonment and loss of support by wayward husbands. http://www. mongabay. com/history/philippines/philippines-the_role_and_status_of_the_filipina. html La Mujer Indigena – The Native WomanA description of the Filipino Woman during Pre-Spanish Timeby Lorna S. Torralba Titgemeyer| | Introduction:When Sr. Mary John Mananzan came to Vienna to give a seminar on the comparative role and status of the Filipino woman in the family and society, past and present, initially I was not so sure of participating, for reasons difficult to explain. Partly because I was confident of my status as woman and wife, or maybe I was afraid that my individualism and self-confidence might be influenced or could cause changes in me. But curiosity got the better of me. The day turned out to be very amusing, very interesting and very informative. The following is in part a summary of Sr. Mary John? s one-day lecture, reflecting on the status of the pre-Spanish Filipino woman, as this helped me understand why we sometimes have this strange feeling of being different from how we had been brought up†¦ that is, being meek, obedient and humble†¦ in short, a good mujer christiana. From Catalona or Babaylan, La Mujer Indigena to La Mujer ChristianaThe Philippines during the pre-colonial period was not a whole entity, the way it is now. It was made up of loosely related principalities with their own separate social, political and economic systems under their own tribal rulers. Community life and social activities were organized mainly on the basis of kinship, beliefs and economic interest. A group of elders were advisers to the tribal ruler and jointly they acted as judge and lawgiver. In some communities, the Babaylan was highly respected as priestess or religious practitioner, as well as healer, counselor and mediator in the tribe. Although differing in name, every tribe had its own religious practitioners, who were preferred to men. In fact, when a male performed the religious office of a Catalona or Babaylan, he was dressed like a woman. With this reference, I would like to present the unknown image of pre-Spanish Filipino woman, la mujer indigena totally in contradiction to the prevailing belief that the elevation of the status of women, was one of the benefits brought by Spanish colonization. The matriarchal society which many of us believed we always had in the Philippines is also a false presumption. The falsely taken patriarchal upbringing with its machismo and a touch of misogyny came uplater with the Spanish colonization. In the eraly Philippines there had always been an egalitarian relationship not only between husband and wife, but also in the upbringing of offsprings. The early Filipinos gave equal importance to both male and female offsprings. Inheritance was divided equally among them, distinguishing only primogeniture and legitimacy. Education was an opportunity for both sexes. Arranged marriage was a custom among pre-Spanish Filipinos. The groom and his family gave dowry to the bride? sparents, an amount agreed upon according to their means. When married the woman did not lose her name. In some Tagalog regions, if the woman was especially distinguished, the husband usually took her name. So it was usual to hear people refered to the husband of Ninay or the husband of Isyang. The pre-colonial Filipino wife was treated as a companion, not as slave. She enjoyed freedom in making decisions in the family. Her say was not only confined to domestic affairs like having a baby or not. Giving birth many times was disliked by women, especially those who inhabited towns near the sea, saying that in having many children, they are like pigs. For this reason they practiced abortion after having the desired number of children. What name to give a child was also her prerogative. She enjoyed a key role in the economic stability of the family. Formal contracts were done only in her presence. In fact there were only very few husbands who would dare enter into contracts without the consent or presence of their wives. It was seldom that a woman did not know how to manage the family landholdings. She had the task of agricultural production once the ground had been prepared by the man. She engaged herself in weaving and pottery-making and usually managed the trading of products and wares. The role of women in the political field, especially leadership role is a disputable subject for those who say, this was based merely on legends. Remember the legend of Queen Maniwantiwan, the wife of Datu Marikudo whose consent had to be secured before he could sell his lands to the Bornean immigrants led by Datu Puti. Another queen who is reported to have ruled Cotabato in the seventh century was Queen Sima. The practice of primogeniture with regard to inheritance regardless of sex allowed women to succeed their fathers as rulers of tribes. The most famous of the women leaders of pre-Spanish society was Princess Urduja of Pangasinan. She was supposed to be a beautiful Amazon, courageous and intelligent, possessing knowledge of languages and culture of Old Asia. In Teresita Infante? s documented study, The Woman in Early Philippines and Among Tribal Minorities, there is a description of the role of women among the Kalingas: â€Å"Kalinga women are not barred from belonging to the highest rank of society, which entitles them to the privileges equal to those of men in similar rank. Some are recognized as pact holders and as she is the one who owns the pact, only her children or relatives have the right to inherit it. † Pact holders were those who held agreement with a prominent citizen of another tribe or community in which each party agreed to give protection and aid to all members of each other? s community while they were in his/her territory. Punishment was imposed if any harm had been done to them by his/her tribe member. This important position of being a pact holder was recognized among women in the pre-Spanish society. In the event of divorce caused by childlessness, infidelity, failure to fulfill obligations towards family, etc. the dowry had to be returned by the bride? s family if she was at fault. However, if the husband was at fault, he lost any right of its return. The children were divided equally between the two regardless of sex. The conjugally-acquired property was also divided equally. This way, she possessed equal rights with regard to divorce according to law and custom. To summarize, the pre-Spanish filipino woman, the mujer indigena had an honoured position in the family and society, which was dispensed with by the Spaniards. A new Filipina was formed, a person moulded to the image and likeness of the perfect woman of the Iberian society of her time. She had to follow many rules and regulations on how to lead the life of a good mujer christiana, which meant lesser freedom and rights. | http://www. univie. ac. at/Voelkerkunde/apsis/aufi/wstat/mujer. htm.

A study into Biodiesel as an alternative fuel

Except for hydroelectricity and nuclear energy, the majority of the present-day world’s energy needs are fulfilled by fossil fuels such as petrochemical fuels, coal and various natural gases. These fossil fuels sources are finite and judging from the current usage rates are in danger of depleting shortly.Also the two renewable sources used currently i.e. hydroelectricity and nuclear energy cannot be used as fuels in transport vehicles majority of which are run using fossil fuels. Because of the large and increasing levels of energy requirement demands in both the industrial and domestic sector, as well as the increased pollution problems because of using fossil fuels have made it necessary to develop renewable energy sources that would last limitlessly and would cause less harm to the environment than the traditional fossil fuels.This requirement has fueled the interest in alternative sources to petroleum-based fuels. One such alternative is to use oils of plant origin which i s known as biodiesel. Biodiesel is a biodegradable and non-toxic fuel and has low emission profiles as compared to petroleum diesel (Meher, Vidyasagar & Naik, 2004:249-250).Using converted vegetable oils and animal fats as an alternative to Petrol-Diesel fuel has been around for decades. In fact the use vegetable oil for diesel engines is almost as old as the diesel engine itself. The original Diesel engine was designed to run on cheap vegetable oils.The engine was commonly demonstrated running on peanut oil. Most of the present day diesel engines can in fact run on biodiesel without using any special equipment. The importance of biodiesel as an attractive fuel is increasing due to the depletion of fossil fuel resources (Knothe, Dunn & Bagby, n.d:1).This dissertation is a study into the use of biodiesel as an alternative to petroleum-based fuels by focusing on the various aspects of biodiesel and its use.The study will focus on understanding the various reasons for going in for alte rnative fuels in detail, study the engine performance using biodiesel fuels, assess the compatibility of biodiesel and fossil fuels, study the production process of biodiesel in detail, analyze the emission levels using biodiesel as fuel, and study the advantages and limitations of using biodiesel.What is Biodiesel? Biofuels can be defined as liquid fuels produced from biomass for either transport or burning purposes. They can be produced from agricultural and forest products, and biodegradable portion of industrial and municipal waste.It is generally held that biofuels offer many benefits, including sustainability, reduction of greenhouse gas emissions, and security of supply. Various scenarios have led to the conclusion that biofuels will be in widespread use in the future energy systems. Biofuels can be converted into liquid and gaseous fuels through thermo-chemical and biological methods.Hence, a variety of fuels can be produced from biomass resources including liquid fuels such as methanol, biodiesel and Fischer-Tropsch diesel, and gaseous fuels such as hydrogen and methane. Biofuels are primarily use din vehicles but can also be used in engines or fuels cells for electricity generation. The figure below shows the types of biofuels.DefinitionsThe term biodiesel is a combination of two terms : the Greek word for life ‘bio’ and ‘diesel’ from the last name of the inventor Rudolf Diesel. The term refers to the diesel equivalent, processed fuel derived from biological sources. It is a cleaner-burning diesel replacement fuel made from natural and renewable sources such as new and used vegetable oils and animal fats. Biodiesel is the name for a variety of ester-based oxygenated fuels from renewable biological sources. It can be made from processed organic oils and fats.Chemically, biodiesel is defined as the monoalkyl esters of long-chain fatty acids derived from renewable biolipids. In general terms, biodiesel may be defined as a domestic, renewable fuel for diesel engines derived from natural oils like soybean oil that meets the specifications of ASTM (American Society for Testing & Materials) D 6751.While in technical terms, biodiesel is a diesel engine fuel comprised of monoalkyl esters of long-chain fatty acids derived from vegetable oils or ani mal fats, designated B100 and meeting the requirements of ASTM D 6751 (Demirbas, 2008:114; Shrivastava & Jain published in Kumar & Nehar 2007:32).Properties of BiodieselBiodiesel is a clear amber-yellow colored liquid with a viscosity similar to that of petrodiesel. Biodiesel is non-inflammable and in contrast to petrodiesel is non-explosive, with a flash point of 423K for biodiesel as compared to 337K for petrodiesel. Unlike, petrodiesel, biodiesel is biodegradable and non-toxic and also significantly reduces toxic and other emissions when burned in a fuel.However, it is more expensive than petrodiesel, which appears to be the primary factor in preventing its more widespread use (Demirbas, 2008:115). The figure below shows the main technical properties of biodiesel.