Among the isoforms of heme oxygenases HO HO

Among the 3 isoforms of heme oxygenases (HO-1, HO-2, and HO-3), HO-1 expression is inducible, and it catalyzes the degradation of heme to biliverdin, carbon monoxide (CO) and free Fe. The Fe is then stored in ferritin, limiting its ability to participate as a catalyst through the Fenton reaction and produce cytotoxic free radicals. Both biliverdin and bilirubin are thought to play antioxidant roles. HO-1 is upregulated in response to a variety of stimuli, such as hypoxia, reactive oxygen species (ROS), viral infection, and stress, and appears to be protective in a variety of inflammatory disease conditions. Studies have demonstrated that induction of HO-1 suppresses apoptotic cell death through activation of mitogen-activated protein kinases (MAPK) pathways with involvement of CO. HO-1 is upregulated in different human cancers including PDAC. The inhibition of HO-1 and the chelation of Fe were associated with increased sensitivity and susceptibility of PDAC azidothymidine to chemotherapy in vivo. However, the impact of HO-1 expression in PDAC cells under hypoxic conditions has never been investigated. Therefore, we examined the mechanistic role of HO-1 in PDAC cell survival under hypoxia and evaluated the efficacy of targeting HO-1 in combination with gemcitabine. Our study showed that under hypoxic conditions, PDAC cells are more sensitive to treatment by gemcitabine in the presence of HO-1 inhibitors both in vitro and in vivo, suggesting a possible new therapeutic approach for the treatment of PDAC patients.
MATERIALS AND METHODS
Results
Discussion HO-1 is a rate-controlling enzyme of cellular heme catabolism. This microsomal enzyme acts on heme moieties to produce equimolar amounts of CO, Fe, and biliverdin that in turn is converted to bilirubin by biliverdin reductase. The protective effects of HO-1 are conferred by its ability to inhibit inflammation and oxidative stress. Previous studies have demonstrated that higher HO-1 levels play an important role in carcinogenesis and promote cellular growth, angiogenesis, and metastasis in several malignancies, including pancreatic cancer, renal cell carcinoma, and prostate cancer. When HO-1 over-expressing PDAC cells (Panc-1) were inoculated subcutaneously in severe combined immune deficient mice, tumor growth, angiogenesis, and metastasis were increased. Conversely, inhibiting HO-1 activity decreased tumor size and reduced metastasis. However, these studies did not consider the importance of the hypoxic microenvironment typical of PDAC. Among various factors, hypoxia plays a critical role in the aggressiveness of PDAC. Studies have demonstrated that hypoxia is responsible for tumor invasion, metastasis, and resistance to chemotherapeutics and radiotherapy in most solid tumors, including PDAC. Several studies have demonstrated that human PDACs are highly hypoxic, poorly perfused, and highly desmoplastic. Similarly, orthotopic implantation of human PDAC samples into mice showed intramural hypoxia. HIF-1α is highly expressed in PDAC tissues and is considered as the master regulator of cell adaptation to hypoxia by altering the transcriptome of tumor cells. Hypoxia is capable of reprogramming gene expression and mediates many of its effect by upregulating important genes including HO-1, which provides a survival advantage to the PDAC cells under stress conditions. Knockdown of HO-1 in the human PDAC cell lines Panc-1, MiaPaCa-2, SU8686, and COLO 357/FC led to pronounced proliferation inhibition. Our study indicates that the pharmacological inhibition of HO-1 suppresses PDAC cell proliferation, consistent with other studies., However, most of these studies were performed under normoxic conditions, and the specific relationships between HO-1 inhibition, hypoxia, and tumor response to chemotherapy and their mechanisms of action remain unclear. We have demonstrated that PDAC cells upregulated HO-1 expression under hypoxia, and inhibition of HO-1 by ZnPP or tin protoporphyrin (SnPP) under hypoxia further reduced the proliferation of PDAC cells as compared to normoxia. Additionally, inhibiting HO-1 sensitized PDAC cells to gemcitabine treatment under hypoxia. Combining the HO-1 inhibition with gemcitabine indicates that cells with higher native levels of HO-1, like Capan-1, were less responsive to combined therapy compared to cells with low HO-1 expression levels, like CD18/HPAF. These observations are mirrored in the tumors in our xenograft model. Tumors grown in vivo confirmed the hypoxic nature of PDAC lesions as demonstrated by increased expression of HIF-1α in tissue sections. HO-1 inhibitor enhanced gemcitabine effect in tumors when combined with gemcitabine in vivo as indicated by smaller tumor sizes and increased amounts of apoptotic cells. These results also suggest that the resistance of PDAC cells to anticancer agents is, in part, due to induction of HO-1. Therefore, inhibiting HO-1 may overcome the acquired resistance to chemotherapeutic agents.