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

Human ovarian carcinoma (MDAH2774), human pancreatic adenocarcinoma (Mia PaCa2), human breast carcinoma (MDA-MB231), murine breast carcinoma (EMT6) cell lines were purchased from ATCC (Manassas, VA, USA). Murine colon-26 (C-26), a poorly differentiated colon adenocarcinoma cell line was obtained from prof. Danuta Dus (Institute of Immunology and Experimental Medicine, Wroclaw, Poland). B16F10 murine melanoma cells were provided by Dr. M. Kubin (Wistar Institute, Philadelphia, PA). Cells were cultured in RPMI 1640 medium (C-26 and B16F10) (Invitrogen, Carlsbad, CA, USA) or DMEM (MDAH2774, Mia PaCa2, MDA-MB231 and EMT6) supplemented with 10% heat-inactivated fetal calf serum, antibiotics, 2-mercaptoethanol (50 μM) and L-glutamine (2 mM) (all from Invitrogen), hereafter referred to as culture medium.

Zinc (II) propoporphyrin IX (Zn(II)PPIX), a HO-1 inhibitor, was purchased from Frontier Scientific Europe Ltd. (Carnforth, Lancashire, United Kingdom) and was dissolved in dimethylsulfoxide (DMSO) (Sigma, St. Louis, USA) to the final stock concentration of 5 mM. Bilirubin (Sigma, St Louis, MO, USA) was dissolved in 0.1 N NaOH to the final stock concentration of 10 mM. Hemin (from Sigma) was dissolved in 0.1 N KOH to the final stock concentration of 10 mM. All the solutions were prepared in the dark right before adding to the cell cultures.

For Western blotting C-26 were treated with Zn(II)PPIX for 24, 48 or 72 h. After indicated times of culture cells were washed with PBS and lysed with radioimmunoprecipitation assay buffer (RIPA) containing Tris base 50 mM, NaCl 150 mM, NP-40 1%, sodium deoxycholate 0.25% and EDTA 1 mM supplemented with Complete^® protease inhibitor cocktail tablets (Roche Diagnostics, Mannheim, Germany). Protein concentration was measured using Bio-Rad Protein Assay (BioRad, Hercules, CA, USA). Equal amounts of whole cell proteins were separated on 12% SDS-polyacrylamide gel, transferred onto Protran^® nitrocellulose membranes (Schleicher and Schuell BioScience Inc., Keene, NH, USA), blocked with TBST [Tris buffered saline (pH 7.4) and 0.05% Tween 20] supplemented with 5% nonfat milk and 5% FBS. The following antibodies at 1:1000 dilution were used for the 24 h incubation: mouse monoclonal anti-α-tubulin (Promega Corporation, Madison, WI, USA), rabbit polyclonal anti-cleaved caspase 3 (Cell Signaling Technology, Beverly, MA, USA) and mouse monoclonal anti-cyclin D1 (Santa Cruz). After extensive washing with TBST, the membranes were incubated for 45 min with corresponding alkaline phosphatase-coupled secondary antibodies (from Jackson Immuno Research). The color reaction for alkaline phosphatase was developed using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Sigma).

The cytostatic and/or cytotoxic effects were measured using crystal violet staining as described [18]. Briefly, tumor cells were dispensed into 96-well plates (Sarstedt, Numbrecht, Germany) at a concentration of 5 × 10³ cells per well and allowed to attach overnight. The following day investigated agents were added at indicated concentrations. Cells were kept in dark for 48 or 72 h. After the incubation time the cells were rinsed with PBS and stained with 0.5% crystal violet in 2% ethanol for 10 min at room temperature. Plates were washed four times with tap water and the cells were lysed with 1% SDS solution. Absorbance was measured at 595 nm using an enzyme-linked immunosorbent assay reader (SLT Labinstrument GmbH, Salzburg, Austria), equipped with a 595 nm filter. Cytotoxicity was expressed as relative viability of tumor cells (% of control cultures incubated with medium only) and was calculated as follows: relative viability = (A_e - A_b) × 100/(A_c - A_b), where A_b is the background absorbance, A_e is experimental absorbance, and A_c is the absorbance of untreated controls.

MDAH2774 or Mia PaCa2 cells were plated at 2.5 × 10⁵ cells per 35-mm dish (Sarstedt). Four hours after seeding, Zn(II)PPIX was added at indicated concentrations. After 24 hours of incubation in dark, the cells were washed with PBS, trypsinized and seeded into 35-well plates in triplets at the concentration of 1 × 10³ cells per a dish. Fresh medium containing Zn(II)PPIX was added. The medium was removed daily for the 6 following days. After 14 day incubation in the dark, the cells were rinsed with PBS, fixed for 10 min in pure methanol and stained with 0.5% crystal violet in 2% ethanol for 10 min at room temperature. Then the plates were washed four times with tap water and air-dried. The images of the plates were made using the Olympus Camedia C750 Ultra Zoom digital camera.

For the cell cycle analysis, C-26 cells were seeded into 6-well plates (Sarstedt) at the concentrations 1 to 3 × 10⁵ cells per well and incubated in dark with 2.5 μM or 5 μM Zn(II)PPIX. The incubation time varied from 24 to 72 h. After the incubation, the cells were washed with PBS, trypsinized and centrifuged at 1000 rpm for 10 min in 4°C. The pellet was resuspended in 0.5 ml of PBS and injected under the surface of ice cold 70% ethanol for 24-h fixation in -20°C. At the day of analysis, the cells were washed from ethanol, stained with 5 μg/ml propidium iodide (PI, Becton Dickinson, Mountain View, CA, USA) at the presence of RNase A (Becton Dickinson) for 30 min at 37°C. For the analysis of apoptosis induction C-26 or Mia PaCa2 cells were seeded into 6-well plates (Sarstedt) at the concentrations 1 to 3 × 10⁵ cells per well and incubated in dark with 2.5 μM or 5 μM Zn(II)PPIX. The incubation time varied from 6 to 72 h. After the incubation, the cells were washed with PBS, trypsinized and centrifuged at 1000 rpm for 10 min in 4°C. The pellet was resuspended in 0.5 ml of Annexin-binding buffer (BioSource International, Camarillo, CA, USA) and then incubated for 15 min with 5 μl of Annexin V-FITC (BioSource). Finally, the cells were stained with 5 μg/ml PI. The cytofluorometric analysis was performed using FACSCalibur (Becton Dickinson). For single analysis 1 × 10⁴ cells were used. Data were collected at the wavelength of 580 nm (for PI) and 520 nm for FITC, and analysed with CELLQuest 1.2 software (Becton Dickinson).

Small interfering RNA against murine HO-1 was obtained by chemical synthesis from Dharmacon (Lafayette, CO, USA). The following sequence was used for targeting of murine HO-1: 5'-GCA-GAA-CCC-AGU-CUA-UGC-C-3'. For RNAi experiments 3,5 × 10⁵ C-26 cells were seeded into 6-well plates (Sarstedt). After 24 h cells were washed with Optimem™ medium (Invitrogen) and transfected with 100 nM siRNA using OligofectAMINE™ according to the manufacturer's protocol. 10 μM hemin was added to the appropriate groups 2 h befor RNAi procedure. After 8 h trasfection medium was replaced with full DMEM culture medium. 24 h after transfection cells were harvested and ROS generation analysis was performed (see below).

For determination of ROS generation, C-26 cells (3,5 × 10⁵ cells per well) were seeded into 6-well paltes (Sarstedt) and treated for 24 h with 2.5 μM Zn(II)PPIX and/or 50 μM biliubin or subjected to HO-1 RNAi (as described above). On the day of the analysis, cells were trypsynized, washed 3 times in ice-cold PBS and resuspended in 1 ml of PBS. One μl of CM-H₂DCFDA (Invitrogen, 5 mM DMSO solution) was added to each sample for 20 min incubation at 37°C. Then, cells were washed twice with ice-cold PBS and subjected to flow cytometry using FACSCalibur (Becton Dickinson). For single analysis 1 × 10⁴ cells were used. Data were collected at the wavelength of 520 nm and analysed with CELLQuest 1.2 software (Becton Dickinson).

For assessment of antitumor activity of Zn(II)PPIX in vivo, exponentially growing C-26 were harvested, re-suspended in PBS medium to the appropriate concentration, and injected at the dose of 1 × 10⁵ cells per mouse into the footpad of the right hind limb of experimental mice. Tumor cell viability measured by trypan blue exclusion was always above 95%. For in vivo treatment Zn(II)PPIX was dissolved in DMSO and further diluted in 0.9% NaCl to required concentrations. Final DMSO concentration was always less then 0.1%. Zn(II)PPIX was distributed intraperitoneally at doses from 12.5 to 50 mg per kg of body weight or orally at doses from 11 to 22 mg per kg of body weight. Control animals received 0.1% DMSO solution in 0.9% NaCl i.p. or orally.

For in vivo experiments evaluating the effectiveness of combine treatment using Zn(II)PPIX and chemotherapeutics, exponentially growing C-26, EMT6 and B16F10 cells were injected at the dose of 1 × 10⁵, 1 × 10⁵ and 1 × 10⁶ cells per mouse, respectively into the footpad of the right hind limb of experimental mice. Zn(II)PPIX treatment (50 mg/kg i.p.) was started on the day 7 after inoculation of tumor cells and continued for 7 consecutive days. First dose of HO-1 inhibitor was administered 1 h before each of the chemotherapeutics to eliminate any possible interactions (such as neutralization) between drugs. Cisplatin (Platidam, Pliva-Lachema, Cech Republic) at the dose of 7.5 mg/kg i.p., 5-FU (Fluorouracil 1000 Medac, Medac, Hamburg, Germany) – 50 mg/kg i.p., or doxorubicin (Adriblastina RD, Pharmacia Italia, Milan, Italy) – 7.5 mg/kg i.p. were administered at a single dose on the day 7^th after inoculation of tumor cells.

For in vivo experiments determining influence of HO-1 gene transfer on sensitivity of B16F10 melanoma cells to cisplatin treatment, exponentially growing B1 (HO-1 transfected) and B5E (empty plasmid transfected) cells were injected at the dose of 1 × 10⁶ cells per mouse into the footpad of the right hind limb of experimental mice. Cisplatin (Platidam, Pliva-Lachema) was administered i.p. at a single dose (2.5, 5.0 or 7.5 mg/kg) on the day 7^th after inoculation of tumor cells.

For in vivo experiments exponentially growing B1 and B5E cells were injected at a dose of 1 × 10⁶ cells per mouse into the footpad of the right hind limb of experimental mice. Cisplatin was administered i.p. at a single dose of 7.5 mg/kg on the 7^th day after inoculation of tumor cells. Zn(II)PPIX treatment (50 mg/kg i.p.) was started on the day 7 after inoculation of tumor cells and continued for 7 consecutive days. First dose of HO-1 inhibitor was administered 1 h before the chemotherapeutic.

In all the experiments mentioned above, local tumor growth was determined every second day as described previously [19] by the formula: tumor volume (mm³) = (longer diameter) × (shorter diameter)².

Data were calculated using Microsoft™ Excel 2003. Differences in in vitro cytotoxicity assays and tumor volume were analyzed for significance by Student's t test. Significance was defined as a two-sided P < 0.03.

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. HO-1 inhibitor exerted dose- and time-dependent cytostatic/cytotoxic effects as measured with crystal violet staining (Fig. 1). Similar effects were observed in MTT assay (not shown) and in a clonogenic assay performed with Mia PaCa2 and MDAH2774 cells (Fig. 2).

To get insight into the mechanism of cytostatic/cytotoxic effects induced by Zn(II)PPIX cell cycle analysis and induction of apoptosis were performed. Incubation of C-26 cells with Zn(II)PPIX for 48 or 72 h resulted in dose- and time-dependent reduction of cells in G1 phase of the cell cycle (Fig. 3A). This effect correlated with decreased cyclin D1 expression (Fig. 3B) and increased percentage of cells in sub-G1 phase (Fig. 3A). Propidium iodide and annexin V staining of C-26 cells incubated with 5 μM Zn(II)PPIX for 24–72 h indicated that the sub-G1 fraction of cells might represent apoptotic cells (Fig. 3C). The fraction of late apoptotic and necrotic cells increased from 10.9% in controls to 30.4% after 72 h incubation with Zn(II)PPIX. Western blotting analysis indicated that a 48-h incubation of C-26 cells leads to accumulation of cleaved (active) caspase-3 (Fig. 3D).

An influence of Zn(II)PPIX as well as knock-down of HO-1 expression with siRNA were used to get insight into the specificity of the Zn(II)PPIX-mediated effects. As shown in Fig. 3E siRNA against a murine HO-1 (moHO-1 siRNA) was effective in reducing the expression level of HO-1 in murine C-26 cells. A siRNA against a human gene (huHO-1 siRNA) was ineffective (Fig. 3E) as well as an irrelevant siRNA against enhanced green fluorescent protein (eGFP, not shown). Inhibition of HO-1 activity with Zn(II)PPIX resulted in increased ROS generation in C-26 cells (Fig. 3F). Similar effects were observed when HO-1 expression was knocked-down with a specific siRNA against HO-1 (Fig. 3G). The influence of Zn(II)PPIX on ROS formation was completely abrogated when C-26 were co-incubated with 50 μM bilirubin (Fig. 3F). Similarly, the influence of siRNA was significantly decreased by forced expression of HO-1 by pre-incubation of C-26 with 10 μM hemin (Fig. 3G).

BALB/c mice inoculated with C-26 cells were treated with Zn(II)PPIX for 7 consecutive days. HO-1 inhibitor was administered either intraperitoneally (i.p.) or per os and the tumor volume was monitored every second day, starting from day 7 after inoculation of tumor cells. Zn(II)PPIX exerted dose-dependent antitumor effects manifested by the retardation of tumor growth. A statistical significance was reached on days 17 and 19 for Zn(II)PPIX administered at a dose of 25 mg/kg either i.p. or orally (Fig. 4A and 4B). A stronger effect was observed when Zn(II)PPIX was administered i.p. at a dose of 50 mg/kg, where a statistically significant retardation of tumor growth was observed on days 13–19, as compared with controls (Fig. 4A).

B16F10 cells were transfected with HO-1 gene (B1 clones) or an empty control plasmid (B5E cells). Tumor cells were inoculated with mixtures of clones and their response to cisplatin treatment was investigated. Cisplatin was administered i.p. at three doses of 2.5, 5.0 or 7.5 mg/kg and the tumor growth was monitored every second day. While in control B5E tumors cisplatin administration led to a significant retardation of tumor growth, the B1 tumors were completely resistant to the chemotherapeutic (Fig. 5A and 5B). Administration of 50 mg/kg Zn(II)PIX alone produced significant retardation of tumor growth only in B5E tumor model (Fig. 5D), but was completely ineffective in B1 tumors (Fig. 5C). Furthermore, it did not restore cisplatin sensitivity in B1 tumors (Fig. 5C).

In further studies the influence of Zn(II)PPIX (50 mg/kg) on the in vivo antitumor effects of chemotherapeutics was evaluated. Three different cell lines syngeneic with BALB/c or C57Bl/6 mice were used, namely C-26, B16F10 melanoma and EMT6 breast adenocarcinoma. The following chemotherapeutics were used in these studies: 5-fluorouracil at a dose of 50 mg/kg (5-FU, for C-26), cisplatin at a dose of 5 mg/kg (for B16F10) and doxorubicin at a dose of 7,5 mg/kg (for EMT6 cells). Although in in vivo studies administration of Zn(II)PPIX (at a dose of 50 mg/kg) resulted in retardation of tumor growth (although in EMT6 tumors the effect was only modest) there was no further potentiation of the antitumor effects by concomitant administration of Zn(II)PPIX together with chemotherapeutics (Fig. 6A–C). Only for Zn(II)PPIX and 5-FU a slightly stronger effect was observed for the combination treatment, but the difference between the combination and single drug-treated tumors did not reach statistical significance (Fig. 5A). Remarkably, the combined administration of Zn(II)PPIX with either cisplatin or 5-FU resulted in significant weight loss (Fig. 6D and 6E). This effects was not observed in mice treated with Zn(II)PPIX and doxorubicin (Fig. 6F). No treatment-related mortality was observed in these experiments.