الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Worldwide, hepatocellular carcinoma (HCC) is the fifth most common cancer and the third most common cause of cancer-related death. A majority of HCC patients (>80%) presents with advanced or unresectable disease. Many different drugs have been evaluated as systemic chemotherapeutic agents for the treatment of HCC. The results have been disappointing and there are probably several reasons for this. First, the tumor nodules have a slow doubling time that makes them relatively resistant. Second, this resistance is further enhanced by the expression of the multidrug resistance gene and there is a low hepatic extraction of chemotherapeutic agents. Additionally, resistance of HCC cells to apoptosis is a crucial aspect in cancer treatment because it impairs the efficacy of different therapy regimens.
Doxorubicin (DOX) is commonly used to treat liver cancer. The immense value of doxorubicin in treating a variety of solid and hematologic malignant conditions is unquestioned. However, doxorubicin has fatal cardiotoxicity and doxorubicin-induced cardiomyopathy (DIC) can develop as late as 10 years after discontinuation of the agent. Although the mechanism of DIC has not been clearly elucidated, the formation of oxidative stress is considered the most probable one among the proposed mechanisms, which include inhibition of nucleic acids and protein synthesis, reactive oxygen species (ROS) formation, release of vasoactive amines and induced apoptosis.
The aforementioned information prompted us to study the feasibility of a combined therapeutic intervention in which lower doses of DOX could be used. This therapeutic scheme would likely decrease the toxic effects of DOX, while improving the effectiveness of the therapeutic response. Herein, we used three drugs (captopril, rosuvastatin and omega 3) to evaluate and compare their potential antitumoral and cardioprotective effects when combined with DOX in experimentally- induced HCC.
Six to eight weeks old male albino mice were divided into six groups:-
Group I (Negative control): Mice treated with a weekly intraperitoneal injection of phosphate buffer saline (PBS).
Group II (DEN group): Mice served as positive controls and received an i.p injection of DEN in a dose of 75 mg/kg body weight once weekly for 3 weeks, followed by 100 mg/kg body weight once weekly for another 3 weeks. Following confirmation of incidence of HCC at the end of 8th week after DEN administration, mice were randomly assigned to one of the following subgroups for treatment:
Group II-a: DOX – treated group (DOX group)
Mice in this group were treated with DOX (12 mg/kg b.wt, i.p.: 3mg/kg every other day for four consecutive doses) starting from the 9th week.
Group II-b: (DOX +ROS) – treated group (ROS group)
Mice in this group were treated with DOX (similar to group II-a) and with a daily oral dose of rosuvastatin (ROS) (20 mg/kg b.wt.) for 3 weeks via an oral gavage syringe starting from the 9th week.
Group II-c: (DOX + Omega-3) – treated group (OMEGA group)
Mice in this group were treated with DOX (similar to group II-a) and with a daily oral dose of omega 3 (OMEGA) (1gm /kg b.wt.) for 3 weeks via an oral gavage syringe starting from the 9th week.
Group II-d: (DOX+CAP) – treated group (CAP group)
Mice in this group were treated with DOX (similar to group II-a) and with a daily oral dose of captopril (CAP) (25mg /kg b.wt.) for 3 weeks via an oral gavage syringe starting from the 9th week.
At the end of the treatment period, animals were sacrificed by cervical dislocation. Tumor tissues and hearts were isolated, immediately chilled on ice and weighed. Each excised liver or heart was divided into several parts for further assessment. Noticeably, some assessments were carried out immediatly after excision. The remaining portions of liver or heart specimens were snap-frozen and kept at - 80°C till used.
Liver histopathology revealed that, mice treated with DEN had developed either a well or moderately differentiated HCC at the end of the 8th week of DEN administration with 100% tumor incidence (8/8) . It showed large irregular nuclei with coarse chromatin & prominent nucleoli or had pleomorphic large irregular nuclei with prominent nucleoli & pseudoinclusion with no to low apoptotic cells. Similarly, no significant apoptosis was noticed after doxorubicin treatment. In contrast, addition of rosuvasatin, captopril or omega3 to doxorubicin caused pronounced apoptosis. Moreover, ROS and CAP groups produced more significant apoptosis than OMEGA group.
The vascular endothelial growth factor (VEGF) was used for the assessment of angiogenesis in mouse liver. The highest concentration of hepatic VEGF level was found in the DEN group . Compared to the DEN group, administration of doxorubicin (12 mg/kg i.p.) resulted in a significant decrease of hepatic VEGF level. Furthermore, CAP (25mg/kg, oral), ROS (20mg/kg, oral) and OMEGA (1gm/kg, oral) treated groups were shown to have more pronounced reduction of hepatic VEGF measured at the end of the 11th week following DEN administration. Furthermore, CAP and ROS treated groups showed a more significant decrease in the level of hepatic VEGF compared to all other treated groups.
The percentage of tumor cell viability was measured by using the trypan blue exclusion test. As expected, the percentage of tumor cell viability in mice livers treated only with DEN ranged from (95%-99%). However, the percentage of tumor cell viability was decreased significantly in livers of DOX - treated group when compared to the DEN group. Furthermore, the percentage of tumor cell viability was decreased significantly in CAP, ROS and OMEGA groups in comparison with the DOX group. Additionally, ROS and CAP treated groups showed the highest decrease in tumor cell viability compared to other treated groups.
Expression of the anti- and pro-apoptotic molecules (Bcl-X and Bak) was assessed immunohistochemically in tumor tissue sections. Bak expression was zero in HCC (induced by DEN injection for 6 weeks). However, intensity of Bak expression was increased significantly in HCC treated with rosuvastatin, captopril, omega 3 and DOX -treated group (+3, +3, +2 and +1 respectively). However, Bcl-X expression was not significantly affected in all treated groups. There was no significant modification in the expression of Bcl-X was evident in the tumors from DOX, CAP, ROS and OMEGA groups as well as the DEN group.
During the treatment period, the mortality rate was approximately 90% in the DOX group and approximately 70% in OMEGA group. However, the highest decrease in mortality rate occurred in CAP and ROS groups (50%). All animals of DOX-treated group produced characteristic signs as dyspnea, enlarged abdomen, enlarged kidneys and liver, fluid accumulation in the peritoneal cavity, and animals looked weaker and lethargic. DOX-treated mice showed a significant loss in their heart weights and in the heart to body weight ratio. Addition of rosuvastatin, capoten or omega 3 to doxorubicin treatment significantly suppressed the reduction in heart weight as well as the decrease in the heart/body weight ratio as compared to DOX-treated group.
Heart histopathology revealed that, the heart tissue sections of DOX group showed cardiotoxicity of doxorubicin as evidenced by degenerated necrotic purkinje fibers (absence nuclear staining). However, addition of rosuvasatin, captopril or omega 3 to doxorubicin greatly lowered its cardiotoxicity.
Tumor necrosis factor-alpha (TNF-α), malondialdehyde (MDA) as an indicator of oxidative stress, antioxidant activity of superoxide dismutase (SOD) and the level of reduced glutathione (GSH) were measured in mouse heart for determination of the cardiotoxicity of doxorubicin. The level of TNFα (pg/ml) was increased significantly in hearts of DOX - treated group in comparison with DEN - treated group. In contrast, administration of captopril, rosuvastatin and omega 3 resulted in a significant decrease in the level of cardiac TNF-α in mice compared to the DOX-treated group. The highest significant reduction in the level of cardiac TNF-α expression was noticed in mice treated with captopril. However, there was no significant difference of cardiac TNF-α level between ROS or OMEGA groups. Additionally, a marked increase in cardiac MDA level and a significant decrease in GSH level and SOD activity were observed in DOX - treated group compared to the DEN group. MDA level was increased significantly after omega-3 treatment, in contrast, administration of captopril and rosuvastatin resulted in a significant reduction when compared to DOX – treated group. All tested regimens including captopril, rosuvastatin or omega 3 showed a significant increase in cardiac GSH level and SOD activity compared to the DOX - treated group. Noticeably, OMEGA group showed a marked increase in cardiac GSH and SOD activity that was significantly different from that of CAP or ROS group.
We can conclude that addition of rosuvastatin, captopril and omega 3 to doxorubicin might increase the apoptotic and antiangiogenic effects of doxorubicin and reduce its cardiotoxicity at different levels, the most promising data were conducted with rosuvastatin or captopril treatment.