الفهرس 
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Abstract
Cancer is a major public health problem with a high mortality rate. It is characterized by uncontrolled growth, a lack of differentiation, and the spread of abnormal cells, and it can occur in any tissue or organ at any time. Hepatocellular carcinoma (HCC) is a primary cause of cancer-related death in the world. Despite the fact that there are many methods to treat HCC, the 5-year survival rate of HCC is still at a low level. In addition, colorectal cancer (CRC) is the third most frequently occurring cancer and a common cause of cancer-related death worldwide. Chemotherapy is one of the most widely used therapeutic strategies for HCC and CRC; nevertheless, it has some limitations, including established systemic toxicity, unsatisfactory response rate, unpredictable innate and acquired resistance, and limited tumor-specific selectivity. Developing new drugs or alternative strategies to refine or even substitute existing HCC or CRC chemotherapy is therefore highly desirable. Despite advances in medical research aimed at understanding the molecular and physiological aspects of cancer, cancer remains a medical challenge as it frequently includes multiple intrinsic cancer-driven pathways, and thus modulation of a single target alone is likely to be therapeutically ineffective. Several tyrosine kinases are abnormally active in the majority of cancers, eventually leading to the mediation of key tumor-driven pathways. Ponatinib (a third-generation TKI) is a multi-targeted tyrosine kinase inhibitor used to treat chronic myelogenous leukemia, acute myeloid leukemia, and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia. Ponatinib substantially disrupted proliferation and triggered cell apoptosis in neuroblastoma cell lines. It has been shown to inhibit other targets such as platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor-2 (VEGFR2), and fibroblast growth factor receptor-1 (FGFR1). Despite its anticancer benefits, the clinical application of ponatinib is hampered by its adverse side effects, the most serious of which is the risk of vascular occlusive diseases and heart failure. Therefore, combining ponatinib with other treatments targeting other RTKs or cancer-related pathways may offer a chance to prevent cancer progression, while decreasing its dose and, hence, potential toxicity. The use of phytochemicals may open up new opportunities in the search for strategies to combat tumor cell growth by targeting epigenetic changes that occur during carcinogenesis. Gossypol, a naturally occurring polyphenolic yellow pigment found in cottonseeds and cotton plants derived products. It has antifertility, antiviral, antidiabetes, antiparasitic, antioxidant, and anticancer properties. In terms of anti-cancer properties, gossypol, a small BH3-mimetic compound, could enhance ponatinib’s apoptotic activity by blocking the interaction between Bcl-2/Bcl-xL and Beclin-1 or Bax, hence boosting caspase activation and cancer cell apoptosis. Based on the aforementioned, we anticipate that by including gossypol as an adjuvant therapy, we will improve ponatinib’s anticancer efficacy by triggering apoptosis and increasing its antiproliferative/antiangiogenic properties while decreasing its dose and, consequently, its potential toxicity. Based on the aforementioned, the current study aimed to investigate the potential anticancer effect of ponatinib and/or gossypol in solid Ehrlich carcinoma (SEC) bearing mice and then on human HCC and CRC cancer cell lines (HepG-2, HCT-116, and Caco-2) to clarify the detailed pharmacologic, drug interaction, and mechanistic research involved in any potential therapeutic effects. Furthermore, this study examined the effectiveness of gossypol in terms of potentiating the anti-tumor action of ponatinib to evaluate if gossypol might be used as an adjuvant drug in cancer treatment. To accomplish these goals, this study was classified into two phases I & II: In phase (I), a model of solid Ehrlich carcinoma (SEC) was induced in female Swiss albino mice by implanting 0.2 ml viable EAC cells (106 cells/mouse) subcutaneously (S.C.) into the right thigh of the mice’s lower limb. A total of 60 female Swiss albino mice weighing 23 ± 2 g and carrying SEC were randomly assigned to six groups of ten mice each. These six groups were given DMSO+PEG+saline as a control (I), ponatinib at doses of 10 and 15 mg/kg (II, III), respectively, gossypol at a dose of 4 mg/kg (IV), and ponatinib at doses of 10 and 15 mg/kg, respectively in addition to gossypol at a dose of 4 mg/kg (V, VI). All treatments initiated on day 12 after EAC implantation and were administered in daily doses through intraperitoneal injections for 3 weeks. At the end of the experiment (i.e., 21 days post-drugs injection), all mice were sacrificed by cervical dislocation under anesthesia with thiopental (50 mg/kg, i.p.) and the solid tumors were excised and weighted. One portion of the solid tumors was fixed in 10% neutral buffered formalin (pH 7.4) for subsequent histopathological examination (H & E), and immunohistochemical detection of p53 and VEGF. The remaining portions were snap frozen in liquid nitrogen and stored at −80C for further investigation of Bcl-2, Bax, FGFR4 and caspase-9, as well as oxidative stress markers such as malondialdehyde (MDA), superoxide dismutase (SOD), reduced glutathione (GSH), and catalase, The time-course effects of different treatments on the volume of solid Ehrlich carcinoma (SEC) were also assessed. In phase (II), cancer cell lines such as HepG-2 (HCC cell line), HCT-116 and Caco-2 (colorectal cancer cell line) were used. These cells were treated with increasing concentrations of ponatinib and/or gossypol to assess synergistic drug interactions as indicated by the combination index (CI), cell viability, FGF19/FGFR4, apoptotic and autophagic cell death. Results of Phase I: Figure (S-1): Summary of in vivo results Summary & Conclusions 163 In vivo testing of the potential antitumor activity of ponatinib and/or gossypol against solid Ehrlich carcinoma in female mice revealed the following results: 1. Treatment of SEC-bearing mice with ponatinib at doses of 10 or 15 mg/kg in addition to gossypol enhanced the reduction (P < 0.05) in TWs by 74% and 85%, respectively, when compared with the untreated control. Furthermore, when ponatinib (10 or 15 mg/kg) and gossypol were combined, TWs were substantially decreased (P < 0.05) by {(45%, 69%) or (33%, 62%) and (63%, 79%)} when compared to ponatinib (10 or 15 mg/kg) and gossypol alone, respectively. 2. Treatment of SEC-bearing mice with ponatinib at doses of 10 or 15 mg/kg in combination with gossypol resulted in the greatest reduction (P < 0.05) in SEC TVs by 69%, 70% &72% and 76%, 79% & 81% on days 19, 26 and 33, respectively. Additionally, tumors were much smaller in their both weights and visible sizes, consistent with the tumor growth curve data. 3. Treatment of SEC-bearing mice with ponatinib at doses of 10 or 15 mg/kg in addition to gossypol resulted in the greatest reduction (P < 0.05) in FGFR4 and Bcl-2 gene expressions by 95% & 99.6% and 81% & 98%, respectively, when compared with the untreated control. This was associated with a 2.69 and 3.42 fold increase in Bax gene expression, as well as a 5.84 and 7.89 fold rise in caspase-9 protein expression, respectively. When ponatinib (10 or 15 mg/kg) and gossypol were combined, there was a greater decrease in FGFR4 and Bcl- 2 gene expression as well as a greater increase (P < 0.05) in Bax gene expression and caspase-9 protein expression when compared to ponatinib (10 or 15 mg/kg) and gossypol alone. There was a significant (P < 0.0001) negative correlation between Bcl-2 and Bax gene expression. 4. Mice treated with ponatinib at doses of 10 or 15 mg/kg in addition to gossypol had the best results (P < 0.05) in terms of MDA, SOD, GSH, and catalase levels. 5. Histopathological examination of solid Ehrlich tumor sections from mice treated with ponatinib in doses of 10 or 15 mg/kg and gossypol (4 mg/kg) showed extensive areas of necrosis revealing degenerative and necrotic changes and many fibroblasts, lymphocytes and neutrophils, as well as ghost bodies with many apoptotic bodies and decrease of neoplastic Ehrlich’s cells as foci with marked vacuolation. 6. Immunohistochemical staining of SEC sections from mice treated with ponatinib in doses of 10 or 15 mg/kg and gossypol (4 mg/kg) displayed enhanced p53 gene expression (59.08 ± 0.65 and 83.11 ± 1.77, respectively) as demonstrated by strongly positive cytoplasmic brown staining within the neoplastic cells, and they were significantly different when compared to untreated control SEC bearing mice, ponatinib (10 or 15mg/kg) and gossypol alone, respectively. 7. Immunohistochemical staining of SEC sections from mice treated with ponatinib (10 and 15 mg/kg) and gossypol (4 mg/kg) showed a pronounced reduction (P < 0.05) in VEGF expression (24.87 ± 1.61 and 14.96 ± 0.34), respectively, as compared to the values of untreated control SEC carrying mice (73.49 ± 1.59), with scanty expression within neoplastic cells and blood capillaries, and they were significantly different when compared to the values of ponatinib (10 or 15 mg/kg), and gossypol alone, respectively. 8. The combination of ponatinib and gossypol was demonstrated to be safe and tolerable, with mortality rates comparable between untreated and combinationtreated animals. Furthermore, they have no detrimental effects on the liver or kidney, as shown by normal levels of ALT and AST, which are biomarkers of liver function, and creatinine, a biomarker of kidney function (data not shown).