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Abstract
Chemotherapy has been the cornerstone of cancer treatment for several decades. However, chemotherapeutic agents have considerable nonspecific toxicities. Myelosuppression is the most common dose-limiting side effect of cytotoxic agents. Moreover, development of resistance to treatment is common. Recent years have seen the advent of a new generation of agents that directly target either the malignant cell itself or cells supporting tumor growth.
Strategies targeting tumor angiogenesis have been a focus of intense research over the past decade, following observations that the growth and metastatic spread of tumors are dependent on their development of a vascular supply. This research effort has led to the isolation of an array of factors that mediate both positive and negative regulation of angiogenesis, with the most pivotal positive regulator being VEGF.
In the current study, the effects of thalidomide, rofecoxib and captopril on tumor growth, angiogenesis, VEGF and hematotoxicity either alone or in combination with cisplatin were investigated.
The objectives were:
1. To evaluate the antitumor effects of thalidomide, rofecoxib and captopril alone or in combination with cisplatin using EAC cells implanted as a solid tumor in the right flank of female Swiss albino mice. The following parameters were assessed:
• The tumor volume
• Tumor growth time (TGT) and tumor growth delay time (TGDT).
• Percentage survival of animals.
• Mean survival time (MST).
• Percentage increased life span (%ILS).
2. To assess the angiostatic activities of thalidomide, rofecoxib and captopril alone or in combination with cisplatin on EAC-bearing female Swiss albino mice. The percentage of angiogenesis was assessed according to the method of Lee et al. (1990).
3. To measure the time course levels of VEGF both in plasma and tumor tissue. VEGF was measured before and after single or combined therapy as a possible mechanism through which thalidomide, rofecoxib and captopril achieved their antiangiogenic effects.
4. To evaluate the hematotoxicity profile of thalidomide, rofecoxib and captopril in EAC-bearing female Swiss albino mice. The modulatory effects of these drugs on cisplatin-induced hematotoxicity were also assessed.
Antitumor activity:
Eighty female Swiss albino mice were used in this experiment. Each mouse was inoculated s.c. in the right flank with 2.5x106 EAC cells in 0.1 ml saline. Twenty-four hours after tumor inoculation, the animals were randomly divided into 8 groups, 10 animals each.
The treatment regimens were as follows:
1. Group I: animals that received saline and served as a control group.
2. Group II: animals that received cisplatin (i.p.) at a dose of 2 mg/kg.
3. Group III: animals that received thalidomide (i.p.) at a dose of 100 mg/kg.
4. Group IV: animals that received rofecoxib (p.o.) at a dose of 20 mg/kg.
5. Group V: animals that received captopril (p.o.) at a dose of 50 mg/kg.
6. Group VI: animals that received a combination therapy of cisplatin (2 mg/kg, i.p.) and thalidomide (100 mg/kg, i.p.).
7. Group VI: animals that received a combination therapy of cisplatin (2 mg/kg, i.p.) and rofecoxib (20 mg/kg, p.o.).
8. Group VII: animals that received a combination therapy of cisplatin (2 mg/kg, i.p.) and captopril (50 mg/kg, p. o.).
All treatments (except cisplatin) were given for 21 consecutive days starting 24 hours after tumor cells inoculation. Cisplatin was given for 3 consecutive days (Wagner et al., 1998) starting 24 hours after tumor cells inoculation. The tumor volume for each animal was measured every other day along the experiment. Tumor growth time (TGT) and tumor growth delay time (TGDT) were calculated. Animals were monitored and the mortality was recorded daily along the study period (100 days) to calculate the percentage survival of animals, mean survival time (MST) and percentage increased life span (%ILS).
Angiostatic activity:
Eighty female Swiss albino mice were used. Each mouse was inoculated i.d. at 4 sites bilaterally on the lower ventral side (after shaving this area) with 100 μl EAC suspension (2.5x106 cells/ 0.1 ml) on each site. Animals were randomly divided into 8 groups. The treatment was initiated 24 hours after tumor inoculation, which was designated day (0). Animals were given the drug regimens, mentioned under the antitumor experiment section, for 3 consecutive days. The degree of angiogenesis was assessed by measuring the tumor vascular volume on day 3.
Angiogenic marker (VEGF) and hematotoxicity:
One hundred and sixty eight female Swiss albino mice were inoculated s.c. at 2 sites bilaterally on the lower ventral side (after shaving this area) with 100 μl EAC suspension (2.5x106 cells/ 0.1 ml) on each site. Animals were randomly divided into 8 main groups, 21 mice each, and given the same treatment regimens as in the antitumor experiment section. Each group was further subdivided into 3 subgroups, A, B and C, 7 mice each. These subgroups were sacrificed on days 7, 14 and 21, respectively. Plasma levels of VEGF were determined using ELISA. Immunohistochemistry was used to determine VEGF levels in tumor tissues. Complete blood count and bone marrow examination were done.
Date were collected, tabulated, described and analyzed by:
1. One way analysis of variance (ANOVA) followed by Post Hoc test (Bonferroni ) for multiple comparison for tumor volume, TGT, TGDT, MST, % of angiogenesis, plasma VEGF levels, blood count and bone marrow cellularity.
2. Chi-square test was used to analyze the effect of different treatments on survival and tissue VEGF pattern.
The following results were obtained from the 1st experiment:
• Individual treatments with thalidomide, rofecoxib or captopril produced a significant reduction in tumor volume as compared to the control group.
• Their depressing effect on tumor volume tended to be enhanced progressively from the individual treatment to the combinations with cisplatin.
• The treatment with cisplatin, thalidomide and their combination could expand the life span of animals for 86, 96 and 100 days, respectively.
The following results were obtained from the 2nd experiment:
• Thalidomide and rofecoxib significantly inhibited the angiogenesis compared to the control group.
• The combination of cisplatin with thalidomide or rofecoxib further inhibited the angiogenesis significantly compared to the control group as well as cisplatin-treated group.
• In the contrast, a significant inhibitory effect of captopril or cisplatin on angiogenesis was not evident. However, the combination of captopril and cisplatin could produce a significant inhibition of angiogenesis.
The following results were obtained from the 3rd experiment:
• Individual treatments with thalidomide or rofecoxib and their combination with cisplatin produced a significant reduction in plasma levels of VEGF on day 7.
• Treatment with captopril, either alone or in combination with cisplatin, failed to produce a significant decrease in plasma levels of VEGF neither from the control nor cisplatin.
• Comparing the plasma levels of VEGF on days 14 and 21 post inoculation, one-way ANOVA showed a non-significant effect among all groups.
• EACs in the control group showed a strong expression of VEGF (VEGF-rich tumors).
• Individual treatments with thalidomide, rofecoxib or captopril could reduce the percentage of VEGF-rich tumors to reach 42.9%, 57.1% and 66.6% of the control, respectively.
• The combination of cisplatin with thalidomide, rofecoxib or captopril has produced a further reduction in the percentage of VEGF-rich tumors to reach 28.6%, 42.9% and 57.1%, respectively.
• A significant reduction in tumor weight was observed after individual treatments.
• The progression of EAC-tumor was accompanied by a gradual decrease in hemoglobin content, erythrocytic count and bone marrow cellularity, a gradual increase in leukocytes and thrombocytes and reversal of the lymphoid-myeloid ratio in the differential leukocytic count.
• Cisplatin treatment caused an inhibitory effect on peripheral blood components and bone marrow cellularity.
• Daily treatment with either thalidomide or captopril exhibited a depressive effect on leukocytes with reduction in granulocytes percentage on 3rd week and a subsequent increase in lymphocytes percentage.
• Although a reduction in RBCs count and Hb level was observed in all treatment groups, cisplatin had the most depressive effect on RBCs count and Hb level on day 7.
• A significant reduction in platelets count after combined treatment of cisplatin with thalidomide was detected on days 7 and 14.
• The combination of cisplatin with either rofecoxib or captopril produced a significant reduction in the percentage of erythroid progenitors and bone marrow on day 7.
• Treatment with cisplatin produced the most depressive effect on the percentage of lymphoid progenitors as well as bone marrow cellularity on day 7.
• A significant reduction in bone marrow cellularity in cisplatin/thalidomide-treated group was detected on day 21 as compared to the EAC-control.
Conclusion:
It is concluded that thalidomide, rofecoxib, or captopril exerts an antitumorigenic effect on EAC solid tumor in Swiss mice. Thalidomide and rofecoxib proved to have an antiangiogenic effect in EAC-model. The data also indicate that EAC is a VEGF-producing tumor and VEGF is essential for initial but not continued in vivo growth of EAC cells. Thalidomide and rofecoxib proved to exert their antitumor and antiangiogenic effects through the inhibition of VEGF, while the antitumor effect of captopril might be mediated through a different mechanism. It is also concluded that thalidomide, rofecoxib, and captopril had lower hematopoietic toxicities in comparison with cisplatin in the current EAC-model. The results suggest a beneficial role of antiangiogenic agents as an adjuvant treatment to chemotherapy.
Chemotherapy has been the cornerstone of cancer treatment for several decades. However, chemotherapeutic agents have considerable nonspecific toxicities. Myelosuppression is the most common dose-limiting side effect of cytotoxic agents. Moreover, development of resistance to treatment is common. Recent years have seen the advent of a new generation of agents that directly target either the malignant cell itself or cells supporting tumor growth.
Strategies targeting tumor angiogenesis have been a focus of intense research over the past decade, following observations that the growth and metastatic spread of tumors are dependent on their development of a vascular supply. This research effort has led to the isolation of an array of factors that mediate both positive and negative regulation of angiogenesis, with the most pivotal positive regulator being VEGF.
In the current study, the effects of thalidomide, rofecoxib and captopril on tumor growth, angiogenesis, VEGF and hematotoxicity either alone or in combination with cisplatin were investigated.
The objectives were:
1. To evaluate the antitumor effects of thalidomide, rofecoxib and captopril alone or in combination with cisplatin using EAC cells implanted as a solid tumor in the right flank of female Swiss albino mice. The following parameters were assessed:
• The tumor volume
• Tumor growth time (TGT) and tumor growth delay time (TGDT).
• Percentage survival of animals.
• Mean survival time (MST).
• Percentage increased life span (%ILS).
2. To assess the angiostatic activities of thalidomide, rofecoxib and captopril alone or in combination with cisplatin on EAC-bearing female Swiss albino mice. The percentage of angiogenesis was assessed according to the method of Lee et al. (1990).
3. To measure the time course levels of VEGF both in plasma and tumor tissue. VEGF was measured before and after single or combined therapy as a possible mechanism through which thalidomide, rofecoxib and captopril achieved their antiangiogenic effects.
4. To evaluate the hematotoxicity profile of thalidomide, rofecoxib and captopril in EAC-bearing female Swiss albino mice. The modulatory effects of these drugs on cisplatin-induced hematotoxicity were also assessed.
Antitumor activity:
Eighty female Swiss albino mice were used in this experiment. Each mouse was inoculated s.c. in the right flank with 2.5x106 EAC cells in 0.1 ml saline. Twenty-four hours after tumor inoculation, the animals were randomly divided into 8 groups, 10 animals each.
The treatment regimens were as follows:
1. Group I: animals that received saline and served as a control group.
2. Group II: animals that received cisplatin (i.p.) at a dose of 2 mg/kg.
3. Group III: animals that received thalidomide (i.p.) at a dose of 100 mg/kg.
4. Group IV: animals that received rofecoxib (p.o.) at a dose of 20 mg/kg.
5. Group V: animals that received captopril (p.o.) at a dose of 50 mg/kg.
6. Group VI: animals that received a combination therapy of cisplatin (2 mg/kg, i.p.) and thalidomide (100 mg/kg, i.p.).
7. Group VI: animals that received a combination therapy of cisplatin (2 mg/kg, i.p.) and rofecoxib (20 mg/kg, p.o.).
8. Group VII: animals that received a combination therapy of cisplatin (2 mg/kg, i.p.) and captopril (50 mg/kg, p. o.).
All treatments (except cisplatin) were given for 21 consecutive days starting 24 hours after tumor cells inoculation. Cisplatin was given for 3 consecutive days (Wagner et al., 1998) starting 24 hours after tumor cells inoculation. The tumor volume for each animal was measured every other day along the experiment. Tumor growth time (TGT) and tumor growth delay time (TGDT) were calculated. Animals were monitored and the mortality was recorded daily along the study period (100 days) to calculate the percentage survival of animals, mean survival time (MST) and percentage increased life span (%ILS).
Angiostatic activity:
Eighty female Swiss albino mice were used. Each mouse was inoculated i.d. at 4 sites bilaterally on the lower ventral side (after shaving this area) with 100 μl EAC suspension (2.5x106 cells/ 0.1 ml) on each site. Animals were randomly divided into 8 groups. The treatment was initiated 24 hours after tumor inoculation, which was designated day (0). Animals were given the drug regimens, mentioned under the antitumor experiment section, for 3 consecutive days. The degree of angiogenesis was assessed by measuring the tumor vascular volume on day 3.
Angiogenic marker (VEGF) and hematotoxicity:
One hundred and sixty eight female Swiss albino mice were inoculated s.c. at 2 sites bilaterally on the lower ventral side (after shaving this area) with 100 μl EAC suspension (2.5x106 cells/ 0.1 ml) on each site. Animals were randomly divided into 8 main groups, 21 mice each, and given the same treatment regimens as in the antitumor experiment section. Each group was further subdivided into 3 subgroups, A, B and C, 7 mice each. These subgroups were sacrificed on days 7, 14 and 21, respectively. Plasma levels of VEGF were determined using ELISA. Immunohistochemistry was used to determine VEGF levels in tumor tissues. Complete blood count and bone marrow examination were done.
Date were collected, tabulated, described and analyzed by:
1. One way analysis of variance (ANOVA) followed by Post Hoc test (Bonferroni ) for multiple comparison for tumor volume, TGT, TGDT, MST, % of angiogenesis, plasma VEGF levels, blood count and bone marrow cellularity.
2. Chi-square test was used to analyze the effect of different treatments on survival and tissue VEGF pattern.
The following results were obtained from the 1st experiment:
• Individual treatments with thalidomide, rofecoxib or captopril produced a significant reduction in tumor volume as compared to the control group.
• Their depressing effect on tumor volume tended to be enhanced progressively from the individual treatment to the combinations with cisplatin.
• The treatment with cisplatin, thalidomide and their combination could expand the life span of animals for 86, 96 and 100 days, respectively.
The following results were obtained from the 2nd experiment:
• Thalidomide and rofecoxib significantly inhibited the angiogenesis compared to the control group.
• The combination of cisplatin with thalidomide or rofecoxib further inhibited the angiogenesis significantly compared to the control group as well as cisplatin-treated group.
• In the contrast, a significant inhibitory effect of captopril or cisplatin on angiogenesis was not evident. However, the combination of captopril and cisplatin could produce a significant inhibition of angiogenesis.
The following results were obtained from the 3rd experiment:
• Individual treatments with thalidomide or rofecoxib and their combination with cisplatin produced a significant reduction in plasma levels of VEGF on day 7.
• Treatment with captopril, either alone or in combination with cisplatin, failed to produce a significant decrease in plasma levels of VEGF neither from the control nor cisplatin.
• Comparing the plasma levels of VEGF on days 14 and 21 post inoculation, one-way ANOVA showed a non-significant effect among all groups.
• EACs in the control group showed a strong expression of VEGF (VEGF-rich tumors).
• Individual treatments with thalidomide, rofecoxib or captopril could reduce the percentage of VEGF-rich tumors to reach 42.9%, 57.1% and 66.6% of the control, respectively.
• The combination of cisplatin with thalidomide, rofecoxib or captopril has produced a further reduction in the percentage of VEGF-rich tumors to reach 28.6%, 42.9% and 57.1%, respectively.
• A significant reduction in tumor weight was observed after individual treatments.
• The progression of EAC-tumor was accompanied by a gradual decrease in hemoglobin content, erythrocytic count and bone marrow cellularity, a gradual increase in leukocytes and thrombocytes and reversal of the lymphoid-myeloid ratio in the differential leukocytic count.
• Cisplatin treatment caused an inhibitory effect on peripheral blood components and bone marrow cellularity.
• Daily treatment with either thalidomide or captopril exhibited a depressive effect on leukocytes with reduction in granulocytes percentage on 3rd week and a subsequent increase in lymphocytes percentage.
• Although a reduction in RBCs count and Hb level was observed in all treatment groups, cisplatin had the most depressive effect on RBCs count and Hb level on day 7.
• A significant reduction in platelets count after combined treatment of cisplatin with thalidomide was detected on days 7 and 14.
• The combination of cisplatin with either rofecoxib or captopril produced a significant reduction in the percentage of erythroid progenitors and bone marrow on day 7.
• Treatment with cisplatin produced the most depressive effect on the percentage of lymphoid progenitors as well as bone marrow cellularity on day 7.
• A significant reduction in bone marrow cellularity in cisplatin/thalidomide-treated group was detected on day 21 as compared to the EAC-control.
Conclusion:
It is concluded that thalidomide, rofecoxib, or captopril exerts an antitumorigenic effect on EAC solid tumor in Swiss mice. Thalidomide and rofecoxib proved to have an antiangiogenic effect in EAC-model. The data also indicate that EAC is a VEGF-producing tumor and VEGF is essential for initial but not continued in vivo growth of EAC cells. Thalidomide and rofecoxib proved to exert their antitumor and antiangiogenic effects through the inhibition of VEGF, while the antitumor effect of captopril might be mediated through a different mechanism. It is also concluded that thalidomide, rofecoxib, and captopril had lower hematopoietic toxicities in comparison with cisplatin in the current EAC-model. The results suggest a beneficial role of antiangiogenic agents as an adjuvant treatment to chemotherapy.