Zinc protoporphyrin IX, a heme oxygenase-1 inhibitor, demonstrates potent antitumor effects but is unable to potentiate antitumor effects of chemotherapeutics in mice - PubMed (original) (raw)

doi: 10.1186/1471-2407-8-197.

Marek Bugajski, Magdalena Winiarska, Jacek Bil, Angelika Szokalska, Pawel Salwa, Tadeusz Issat, Halina Was, Alicja Jozkowicz, Jozef Dulak, Tomasz Stoklosa, Jakub Golab

Affiliations

Zinc protoporphyrin IX, a heme oxygenase-1 inhibitor, demonstrates potent antitumor effects but is unable to potentiate antitumor effects of chemotherapeutics in mice

Dominika Nowis et al. BMC Cancer. 2008.

Abstract

Background: 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. 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.

Methods: Cytostatic/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.

Results: Zn(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.

Conclusion: 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.

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Figures

Figure 1

Figure 1

Zn(II)PPIX exerts cystostatic/cytotoxic effects against tumor cells. Tumor cells were incubated with serial dilutions of Zn(II)PPIX for 48 or 72 hours. The cytostatic and/or cytotoxic effects of treatment were measured with a standard crystal violet staining assay. Bars represent means ± SD. *P < 0.05 (two way Student's _t_-test) in comparison with controls.

Figure 2

Figure 2

The influence of Zn(II)PPIX on formation of tumor cell colonies. For the clonogenic assay, MDAH2774 or Mia PaCa2 cells were plated at a concentration of 1 × 103 cells/dish. Medium containing Zn(II)PPIX was replaced daily for 1–6 consecutive days. On day 14 after PDT, the plates were fixed with methanol and stained with crystal violet.

Figure 3

Figure 3

Zn(II)PPIX induces apoptosis and increases formation of reactive oxygen species. C-26 cells were grown in Petri dishes for 24 hours before addition of Zn(II)PPIX for the indicated time. [A] Cell cycle analysis was performed by flow cytometry analysis using ethanol-fixed, propidium iodide-stained cells. [B and D] Western blotting for expression of cyclin D1, cleaved caspase 3 or tubulin as a loading control was performed. [C] Induction of apoptosis was evaluated by staining of cells with propidium iodide and annexin V. [E] HO-1 silencing was evaluated with Western blotting. Controls represent expression of HO-1 in hemin-treated C-26 cells. HuHO-1 siRNA is a negative control targeting xenogeneic (human) HO-1 gene, muHO-1 siRNA is a murine sequence that knocks-down HO-1 expression in C-26 cells. [F and G] production of reactive oxygen species was evaluated with flow cytometry by measuring CM-H2DCFDA fluorescence in comparison with controls.

Figure 4

Figure 4

Zn(II)PPIX exerts antitumor effects against C-26 adencarcinomas in mice. BALB/c mice were inoculated with 1 × 105 of C-26 cells. On day 7 after inoculation of tumor cells mice were treated with Zn(II)PPIX administered i.p. or p.o. for 7 consecutive days [A and B]. Graphs show the influence of the treatment on the growth of C-26 tumors in mice. *P < 0.05 (two way Student's _t_-test) in comparison with controls.

Figure 5

Figure 5

Antitumor effect of cisplatin or the combined treatment with Zn(II)PPIX and/or cisplatin in mice transplanted with control or HO-1 overexpressing B16F10 tumors. C57Bl/6 mice were inoculated with 1 × 106 of HO-1 overexpressing (B1) or mock-transfected (B5E) cells. On day 7 after inoculation of tumor cells mice were treated with cisplatin at a dose of 2.5, 5.0 or 7.5 mg/kg administered i.p. [A and B] or with cisplatin (5 mg/kg in a single i.p. injection) and 50 mg/kg of Zn(II)PPIX administered i.p. for 7 consecutive days [C and D]. Graphs show the influence of the treatment on the growth of melanoma in mice. *P < 0.05 (two way Student's _t_-test) in comparison with controls.

Figure 6

Figure 6

Antitumor effect of the combined treatment with Zn(II)PPIX and/or chemotherapeutics. Mice were inoculated with 1 × 105 of C-26 or EMT6 cells or with 1 × 106 of B16F10 cells. On day 7 after inoculation of tumor cells mice received a single dose of chemotherapeutics: 50 mg/kg of 5-FU [A, D], 5 mg/kg of cisplatin [B, E] or 7.5 mg/kg of doxorubicin [C, F]. Zn(II)PPIX was administered i.p. for 7 consecutive days at a dose of 50 mg/kg [A-F]. Graphs show the influence of the treatment on the growth of tumors in mice [A-C] or on the change in mice weight as compared with initial weight. *P < 0.05 (two way Student's _t_-test) in comparison all other groups.

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