الفهرس 
Hepatocellular carcinomaOnly 14 pages are availabe for public view
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Abstract
The new incidence of cancer is projected to rise from 170 million to 260 million between 2018 and 2040. Both breast and liver cancers represent a major portion in cancer patients worldwide. The first (Breast cancer) is the most frequently diagnosed cancer and the leading cause of death in women, where it accounts 23% of cancer diagnoses and 14% of cancer deaths each year (DeSantis et al., 2019). Also, liver cancer represents one of the major health problems in many nations. Due to their heavy burden, identification of alternative approaches of drugs or therapeutic strategies is imperative. The use of chemotherapy represents a constitutional paradigm in the treatment of cancer patients. According to The New Global Cancer data (Bray et al., 2018), 9.8 million cancer patients require first-course chemotherapy annually and the number will increase to 15 million by 2040 (Wilson et al., 2019). In the past few decades, nanoformulation of water insoluble drugs was discovered and used to develop drug delivery system capable of improving its therapeutic index, minimize the associated side effects of the organic solvents it contains and thus enhancing the responsiveness of cancer cells. Paclitaxel (PTX), for example, is commonly used as antibreast cancer chemotherapy. Due to it’s low water solubility (< 0.1 μg/ml) and limited bioavailability, it is commercially formulated in an organic solubilizer containing Cremophor EL and ethanol. Unfortunately, the severe side effects of this formulation were attributed to cytotoxic effect of these solvents. Moreover, PTX was found to activate PTX resistance gene, P-glycoprotein (P-gp) that actively pumps PTX out of the treated cells and induces drug resistance. This triggered the notion of PTX nanoformulation, where albumin-bound PTX nanoparticle was the first nanotechnology-based drug approved by the Food & Drug Administration (FDA) in the treatment of breast and ovarian cancers (Palumbo et al., 2016). However, there are many concerns about its efficacy such as instability when co administered with other drugs (Mizuta et al., 2017). In another context, glucocorticoids are efficacious in reducing the chemotherapy adverse effects and enhance their intrinsic anticancer activity (LeVee et al., 2009). Pretreatment with dexamethasone (DEX), for example, increased the antitumor activity of some anticancer drugs such as carboplatin and gemcitabine and decreased host toxicity in nude mouse xenograft models of human cancer. The underlining mechanisms were attributed to DEX inhibitory effect on cytokines produced by the tumor, increasing tumor necrosis factor (TNF) and decreasing the expression of IL-1ß and vascular endothelial growth factor (VEGF) (Wang et al., 2007). Additionally, DEX administration attenuated the severity of PTX- associated acute pain syndrome in patients with non-squamous lung carcinoma (Saito et al., 2019) and enhanced the anti-tumor effects of PTX in orthotopic mouse breast cancer (Popilski et al., 2018). In another context, cytochromes P450 (CYPs), a large superfamily of membrane-bound monooxygenases, are involved in phase I oxidative biotransformation of a wide range of xenobiotics including anticancer drugs (Zerilli et al., 1998). Approximately 60 human cytochrome CYPs genes were identified (Nelson et al., 1996). There is unlimited number of reports that investigated the expression of different CYPs in both hepatic and extrahepatic tissues with respect to their roles in drug metabolism (Rendic and Di Carlo, 1997). The expression levels of CYPs genes play a major role in cell responsiveness to chemotherapeutics (LeBlanc and Waxman, 1989). Dexamethasone was able to induce the expression of many CYPs involved in drug metabolism including CYP3A4 and CYP2C8 (Lindley et al., 2002). Because PTX for instance is endogenously metabolized by CYP3A4 and CYP2C8 (Cresteil, et al., 1994), it is anticipated that DEX may enhance the metabolism of PTX through their stimulatory effect on expression of such metabolising enzymes. The overall picture depicts a complex relation that involves drug formulation (standard PTX, nanoformulated PTX and solvent containing PTX) from one side and cotreatment of cancer cells with dexamethasone. Drug formulation, however, is not the only factor that determines drug ’s therapeutic efficacy. Other factors are host (cellular) related, such as genes involved in drug resistance and metabolism. These genes are translated into proteins that greatly affect cellular response to anticancer drugs. This triggers the interest of exploring the relations among: PTX formulations, DEX cotreatment and drug metabolizing and resistance genes. Hence, in this thesis three approaches were undertaken. In the first part, PTX was nanoformulated with poly-lactic-coglycolic acid (PLGA) and the nanoparticles (PTX-NPs) were used to investigate to how far cotreatment with glucocorticoids such as dexamethasone can compete the nanoformulation of PTX. The second approach was designated to explore the effect of PTX-NPs on the viability of breast adenocarcinoma cells (MCF-7) and the associated changes in the expression of Taxol resistance gene I (Txr1) and PTX metabolizing genes (CYP3A4 and CYP2C8). The third part, however, aimed to compare the responsiveness of human hepatocellular carcinoma cells (HepG2) to PTX or solvent containing PTX (Taxol) in combination with dexamethasone and the associated expression pattern of hepatic Taxol resistance gene (Txr1) and PTX metabolizing genes. To establish these goals we utilized paclitaxel in three forms including pure (water-insoluble) PTX, organic solvent containing form (Taxol) in addition to PTX nanoformulated with poly-lactic-co-glycolic acid (PLGA) as a carrier. The later (PTX-NPs) was subjected to the constitutional physical characterization including FT-IR analysis, determining the size and surface charge on the nanoparticles and the drug release. The study utilized two cell lines (MCF-7 and HepG2). The first was used as the drug (PTX) is predominantly used as anti-breast cancer chemotherapy, whereas the second (HepG2) was used as liver in the main site of PTX metabolism where it hosts CYP3A4 and CYP2C8 involved in PTX microsomal biotransformation. Dexamethasone was used with fixed concentration in combination with any of PTX forms mentioned above. A list of experimental protocols were employed to determine the cytotoxic effect, determination the percent of apoptotic cells by flow cytometry and the expression level of Taxol resistance gene 1 (Txr 1), CYP3A4 and CYP2C8. The obtained results demonstrated a proper nanoformulation of PTX as validated by the nanoparticle size (less than 200 nm) measured by Transmission electron microscopy (TEM) and increased the anionic surface charge (zeta potential) to -10 mEv compared to -2.4 mEv of blank PLGA nanoparticles. The proper conjugation of PTX with PLGA in the nanoparticles was validated by FT-IR spectroscopy. The encapsulation efficiency was 99% and progressive drug release over the first 70 hrs, followed by sustained releasing phase. In breast cancer cells (MCF-7), the initial IC50 of PTX was 19.3 g/ml and cotreatment of cells with PTX+DEX has minimized the IC50 to 5.22 g/ml, whereas PTX-NPs alone inhibited cell proliferation with IC50 6.67 g/ml. Also, in presence of DEX, PTX-NPs mildly decreased the IC50 to 4.96 g/ml. In parallel, cotreatment has increased the responsiveness of cells to the drug without potentiating its apoptotic effect. Moreover, DEX (combined with PTX or PTX-NPs) downregulated the both TRG1 (by 26% and 28.4% respectively and CYP3A4 genes. CYP2C8, in contrast, was upregulated only in cells treated with DEX and PTX. Cells were more responsive to PTX-NPS compared to standard PTX and apoptosis was induced in 47.8 % and 51.7%, respectively. In hepatoma cells, initially, HepG2 cells were more resistant to PTX than Taxol. Also, cells became more responsive to the standard PTX and Taxol in the presence of DEX, where the IC50 values decreased from 42.5 g/ml to 13.07 g/ml and from 6.5 g/ml to 3.6 g/ml, respectively. Apoptosis was the main mechanism of cytotoxicity in HepG2 cells treated with PTX or Taxol. Involvement of DEX, however, decreased the percent of apoptotic cells. Moreover, the expression of Txr1 decreased by 18% and 35% in cells cotreated with (PTX+DEX) or (Taxol+DEX). In parallel, the expression of paclitaxel metabolizing genes (CYP3A4 and CYP2C8) was increased compared to DEX untreated cells. Conclusively, chemotherapy represents the most crucial protocol that commonly applied in cancer treatment and to prohibit its recurrence. Although nanoformulation of PTX with PLGA provides a good chance to overcome the side effects associated with the currently used formulation, the study demonstrated that cotreatment of breast cancer cells with free PTX with DEX potentiated the drug anticancer effect. More importantly, cotreatment with DEX demonstrated anticancer effect similar (even better) than PTX-NPs effect, where The IC50 values indicated that this cotreatment was lower than that induced by PTX-NPs alone. This may indicate the dual beneficial role of DEX in promoting the anticancer effect of PTX. Also, it minimized the required dose to one third from one side and its clinical benefits in minimizing some postoperative and post chemotherapy complications. Additionally this in vitro study reports the associations between the enhanced responsiveness of hepatoma cells to PTX or Taxol in presence of DEX, associated with a decrease in drug resistance and upregulation of the paclitaxel metabolism similar to the changes seen in breast cancer cells.