All animals in these groups had to be sacrificed due to large tumor size (1,000 mm3). The 6-week course of weekly doxorubicin followed by zoledronic acid caused a decrease in mean tumor volume from 191.4 �� 36.95 mm3 to 123.5 �� 8.5 mm3 (p > 0.001 compared with control or single agents). Following withdrawal of sequential treatment, tumor volume did not increase over the next 107 days (mean tumor volume was 115.7 http://www.selleckchem.com/
�� 60.9 mm3 on day 62 and 93.6 �� 47.3 mm3 on day 169). No significant differences in tumor volume were observed between animals treated with doxorubicin then zoledronic acid weekly for the duration of the experimental protocol, and those whose sequential treatment was stopped after 6 weeks. Animals treated sequentially with doxorubicin followed by zoledronic acid exhibited significant increases in survival; 70% of animals receiving continuous treatment and 60% of animals whose treatment was withdrawn after 6 weeks survived the entire duration of the experiment (169 days) (Fig. 1b). Tumors did not reach maximum volume in either of the sequential treatment groups; however, 7 animals were sacrificed as a result of skin blistering on the tumor surface. With this exception, all mice appeared healthy, and no significant weight loss was detected throughout the course of the study. To elucidate the molecular mechanisms by which sequential treatment reduced tumor growth, we used Bleomycin manufacturer
pathway-specific microarrays to examine alterations in the expression of 112 apoptosis-related genes in tumors isolated from animals following a 6-week course of treatment. Genes that showed at least a 2-fold change in expression in tumors isolated from animals treated with doxorubicin followed by zoledronic acid compared to controls (saline or the single agents) were clustered using GEAsuite software, and Pathway Architect software was subsequently used to link altered genes to specific pathways. This analytical method identified increased expression of 5 proapoptotic genes (p53, Bax, CRADD, FADD and Caspase 2) and a decrease in the antiapoptotic gene bcl-2, in tumors from animals receiving doxorubicin followed by zoledronic acid, DAPT
compared to saline or single-agent treatment. The changes in gene expression identified in the microarray analysis were subsequently confirmed by real-time quantitative RT-PCR (Table 1). Expression of Bcl-2 was decreased in tumors from animals receiving doxorubicin followed by zoledronic acid compared with controls (p < 0.001) and expression of proapoptotic Caspase 2, CRADD, FADD bax and p53 were increased, compared with all other treatment groups (p < 0.005). Caspase 2, FADD and Bax expression were also increased following administration of doxorubicin and zoledronic acid at the same time, compared with control or single agent treatment (p < 0.05).