Gonzales, Rayna J.

Doxorubicin (Dox), an anthracycline‐based chemotherapeutic agent, is highly effective at reducing recurrence and mortality in breast cancer patients. However, this anti‐cancer drug elicits dose‐dependent cardiotoxicity. Multiple mechanisms have been implicated in its pathogenesis, one of which involves the development and progression of inflammation mediated by activation of the TLR4/NFkappaB pathway. However, we recently demonstrated that chronic in vivo Dox treatment reduced expression of the NFkappaB downstream proinflammatory mediator, cyclooxygenase‐2 (COX‐2), in cerebral and peripheral vasculatures isolated from ovariectomized female rats. Therefore, in the current study we tested whether Dox similarly alters vascular proinflammatory enzyme expression in the human vasculature and whether this response is dose dependent. We further investigated whether Dox alters upstream mediators of COX‐2 expression by assessing levels of expression and activation of NFkappaB and an additional downstream gene product, TLR4. Primary human coronary vascular smooth muscle cells of female origin were studied at passage 6 and treated with vehicle or Dox (0.3, 1.0, and 5 μM). Following drug treatment, cells were isolated, homogenized, and lysates (whole cell; nuclear and cytosolic) analyzed for NFkappaBp65, COX‐2, and TLR4 protein levels via immunoblotting. Our results demonstrate that while total NFkappaB levels were unaffected following Dox, enhanced translocation of NFkappaB to the nucleus was observed. However, even in the presence of Dox‐induced NFkappa translocation, basal expression of its downstream mediator, COX‐2, was dose dependently attenuated. Similar to COX‐2, a trend for a decrease in TLR4 levels was observed. Dox had no effect on cell morphology or density, as visualized via light microscopy. In conclusion, although others have suggested the involvement of NFkappaB activation during the development and progression of Dox‐induced inflammation in cardiac muscle, our studies demonstrate a possible novel action for the anticancer agent targeting important downstream mediators of inflammation in the female vasculature. Future studies will evaluate the impact of Dox on COX‐2 at the transcriptional or translational level to determine the mode of molecular regulation. Support or Funding Information University of Arizona Sarver Heart Center (RG and MS); Mayo Clinic Dept. of Medicine Extramural Chair Award in collaboration with Arizona State University (SA)

Doxorubicin (DOX) is a highly effective chemotherapy agent. Its use is hampered however owing to severe dose‐dependent cardiovascular toxicity in cancer survivors. Multiple mechanisms have been implicated in the pathogenesis of DOX cardiotoxicity, one of which involves inflammation mediated by activation of the NFκB/TLR4/COX‐2 pathway in the heart. Knowledge regarding the toxic effects of DOX‐induced inflammation in other organ systems such as the brain has not been thoroughly investigated. Therefore, we explored the inflammatory potential of DOX by assessing TLR4 and COX‐2 levels in cortex and pial arteries isolated from ovariectomized (OVX) female Sprague‐Dawley rats. We hypothesized that DOX would promote inflammation by increasing COX‐2 expression along with expression of its upstream innate immune receptor, TLR4, both of which are under the transcriptional regulation of NFκB. OVX rats were treated with three, bi‐weekly, i.p. injections of DOX (4 mg/kg; cumulative dose 12 mg/kg) or vehicle (saline) and euthanized 5 days after the last dose. Tissues were isolated, snap frozen, homogenized, and analyzed for COX‐2, TLR4 and NFκBp65 levels using standard western blotting. Although COX‐2 is typically considered an inducible enzyme, it has been shown to be expressed under basal conditions. In cortex and pial arteries from vehicle‐treated rats, measurable levels of COX‐2 and TLR4 protein were detected. However, contrary to our hypothesis, levels of COX‐2 and TLR4 were decreased following DOX. In pial arteries, DOX elicited a reduction in both the COX‐2 72 kDa band 74 kDa band with the greatest reduction observed in the 72 kDa. The 72 kDa band corresponds to the partially glycosylated inactive form of enzyme, while the 74 kDa band has been shown to represent the fully glycosylated active form. Cytosolic levels of NFκBp65 levels were detected in brain and pial vessel lysate, however levels were not altered by DOX. Similar to brain and pial arteries, basal expression of COX‐2 and TLR4 were detected in left ventricle and DOX treatment attenuated expression. In conclusion, although others have suggested the involvement of the NFκB pathway during the development and progression of DOX‐induced cardiomyopathy, our studies demonstrate a possible novel action for the anticancer agent implicating anti‐inflammatory mechanisms, particularly in female cohorts with low circulating levels of gonadal hormones. Support or Funding Information Arizona State University Intramural Funds (SA and JD), Midwestern University Intramural Funds (CC), Sarver Heart Center (RG)