Abstract
BackgroundHO-1 participates in the degradation of heme. Its products can exert unique cytoprotective effects. Numerous tumors express high levels of HO-1 indicating that this enzyme might be a potential therapeutic target. In this study we decided to evaluate potential cytostatic/cytotoxic effects of zinc protoporphyrin IX (Zn(II)PPIX), a selective HO-1 inhibitor and to evaluate its antitumor activity in combination with chemotherapeutics.MethodsCytostatic/cytotoxic effects of Zn(II)PPIX were evaluated with crystal violet staining and clonogenic assay. Western blotting was used for the evaluation of protein expression. Flow cytometry was used to evaluate the influence of Zn(II)PPIX on the induction of apoptosis and generation of reactive oxygen species. Knock-down of HO-1 expression was achieved with siRNA. Antitumor effects of Zn(II)PPIX alone or in combination with chemotherapeutics were measured in transplantation tumor models.ResultsZn(II)PPIX induced significant accumulation of reactive oxygen species in tumor cells. This effect was partly reversed by administration of exogenous bilirubin. Moreover, Zn(II)PPIX exerted potent cytostatic/cytotoxic effects against human and murine tumor cell lines. Despite a significant time and dose-dependent decrease in cyclin D expression in Zn(II)PPIX-treated cells no accumulation of tumor cells in G1 phase of the cell cycle was observed. However, incubation of C-26 cells with Zn(II)PPIX increased the percentage of cells in sub-G1 phase of the cells cycle. Flow cytometry studies with propidium iodide and annexin V staining as well as detection of cleaved caspase 3 by Western blotting revealed that Zn(II)PPIX can induce apoptosis of tumor cells. B16F10 melanoma cells overexpressing HO-1 and transplanted into syngeneic mice were resistant to either Zn(II)PPIX or antitumor effects of cisplatin. Zn(II)PPIX was unable to potentiate antitumor effects of 5-fluorouracil, cisplatin or doxorubicin in three different tumor models, but significantly potentiated toxicity of 5-FU and cisplatin.ConclusionInhibition of HO-1 exerts antitumor effects but should not be used to potentiate antitumor effects of cancer chemotherapeutics unless procedures of selective tumor targeting of HO-1 inhibitors are developed.
Highlights
Heme oxygenase (HO)-1 participates in the degradation of heme
Inhibition of HO-1 exerts antitumor effects but should not be used to potentiate antitumor effects of cancer chemotherapeutics unless procedures of selective tumor targeting of HO-1 inhibitors are developed
Zn(II)PPIX induces potent cytostatic/cytotoxic effects against murine and human tumor cells Four different cell lines of murine (C-26, colon adenocarcinoma) and human (Mia PaCa2, a pancreatic cancer, MDAH2774, ovarian carcinoma, and MDA-MB231, breast carcinoma) origin were incubated with increasing concentrations of Zn(II)PPIX for 48 and/or 72 hours
Summary
HO-1 participates in the degradation of heme. Its products can exert unique cytoprotective effects. Numerous tumors express high levels of HO-1 indicating that this enzyme might be a potential therapeutic target. All HO products exert pleiotropic effects including numerous cytoprotective responses [3]. CO exerts strong antiapoptotic and anti-inflammatory effects through induction of soluble guanylyl cyclase. It suppresses production of tumor necrosis factor (TNF), interleukin-1β (IL-1β) and CCL4 chemokine (macrophage inflammatory protein-1β), but up-regulates synthesis of anti-inflammatory IL-10 [5]. Free iron (Fe2+) despite participation in Fenton reaction that leads to formation of highly reactive hydroxyl radicals, activates Fe-ATPase, a transporter that removes intracellular iron, as well as induces expression of ferritin heavy chains which sequester free iron and exert specific cytoprotective roles [6]
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