Effects of osteopontin inhibition on radiosensitivityof MDA-MB-231 breast cancer cells

The human breast cancer cell line MDA-MB-231 was grown as a monolayer in RPMI 1640 containing 25 mM HEPES and L-glutamine (Lonza, Walkersville, USA). The medium was supplemented with 10% fetal calf serum (FCS) (PAA, Cölbe, Germany), 1% pyruvate (Invitrogen, Karlsruhe, Germany), 185 units/ml penicillin (Invitrogen), and 185 μg/ml streptomycin (Invitrogen), and cells were cultured in a humidified atmosphere of 3% CO₂ at 37°C. All experiments were performed with cells in logarithmic growth phase.

Two double-stranded OPN siRNA oligonucleotides (Mix, OpnS) and a nonsense siRNA (negative control) were transfected using INTERFERin™ reagent as recommended by the manufacturer (Polyplus Transfection Illkirch, France). The cells (4-5*10⁵ cells) were plated overnight at 37°C, 3% CO₂ and then transfected with 100 nM of either nonsense non-targeting siRNA or target-specific siRNAs to knockdown OPN for 24 h and 72 h. The siRNA oligonucleotide sequences are shown in Table 1.

Furthermore, the cells were irradiated in tissue culture flasks (Greiner, Frickenhausen, Germany) at 2, 4 or 6 Gy 24 h after OPN siRNA transfection. Irradiation at 0 to 6 Gy was accomplished in logarithmically growing cultures with 6 MV photons and adequate bolus material on a SIEMENS ONCOR (Erlangen, Germany) linear accelerator at a dose rate of 2 Gy/min. Referring to the fractionated daily dose in therapy treatment and DMF₁₀-value of the MDA-MB-231 cell line, we have chosen a radiation dose of 2 Gy and 6 Gy, respectively. At 1 h and 48 h after irradiation, cells were processed for RNA and protein extraction, clonogenic assays (1 h) and migration and apoptosis assays (48 h).

Total RNA was isolated using the RNeasy^® Mini Kit as recommended by the manufacturer (Qiagen, Hilden, Germany). For hybridization, 1 μg of RNA was incubated with random primers (150 ng/μL) at 70°C for 10 min followed by addition of 5× first strand buffer, 0.1 M DTT, 2.5 mM dNTPs and SuperScript™ II reverse transcriptase (200 U/μl) (Invitrogen). The reaction conditions were: 20°C for 10 min, 42°C for 80 min and 95°C for 10 min.

All qRT-PCR reactions were performed on a Rotorgene RG-6000 (LTF, Wasserburg, Germany) using the QuantiTect SYBRGreen PCR Kit (Qiagen). For each PCR reaction, 1 μl of cDNA was added to SYBRGreen Quantitect 2×, PCR primers (20 μM) and aqua bidest in a total volume of 15 μl. As a negative control, we used a no-template reaction. The primers used are cited in Table 2. HPRT (hypoxanthineguanine phosphoribosyltransferase) served as a housekeeping gene and for control of cDNA integrity. PCR conditions were: 95°C for 15 min followed by 40 cycles of denaturation for 30 s at 95°C, hybridization for 30 s at 60°C, extension for 30 s at 72°C, a final step for 30 s at 60°C and a melting curve program (65-95°C with a heating rate of 0.2°C/s). RNA was isolated as well as cDNA was generated and quantified from three independent experiments.

The cells were lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 200 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.25% desoxycholate, 1:100 phosphatase inhibitor, 1:100 proteinase inhibitor) followed by ultrasonic homogenization. The conditioned medium (serum-free RPMI) was harvested after 24 h and 48 h and spun at 1,300 rpm for 10 min to remove cell debris. The supernatant was concentrated using Amicon^® Ultra Centrifugal Filters (Millipore, Billerica, MA, USA) with a 3 kDa cut-off.

Equal amounts of protein (15-20 μg/lane) were electrophoresed on 4-12% Bis-Tris gradient gels (Invitrogen) under reducing conditions and transferred to PDVF membrane (Millipore GmbH, Schwalbach, Germany). The membrane was blocked with 10% non-fat milk in TBST (50 mM NaCl, 30 mM Tris-HCl pH 8.0, 0.1% Tween) for 1 h and probed with polyclonal rabbit anti-human OPN (1:2,000, 0-17, IBL, Hamburg, Germany), rabbit anti-human cleaved PARP (poly-(ADP-ribose)-polymerase) (Asp214) (1:2,000, Cell Signaling, Danvers, MA, USA) and mouse anti-β-actin (1:5,000, Sigma, Steinheim, Germany) at 4°C overnight. The membrane was washed three times with TBST buffer for 7 min followed by incubation with HRP-conjugated secondary antibodies (DAKO, Hamburg, Germany) diluted 1:5,000 in TBST containing 10% non-fat milk for 1 h at room temperature. After further washing steps (three times with TBST buffer and one time with TBS), the immunocomplexes were visualized by ECL or ECL Plus Blotting Detection System (Amersham, Freiburg, Germany). We analyzed the conditioned medium of two independent experiments and the protein data of three independent experiments.

The cells were trypsinized 1 h after irradiation, and different numbers of cells (100-10,000), depending on treatment and irradiation dose, were seeded into 25-cm² cell culture flasks. The cells were cultured in RPMI supplemented with 10% FCS in a humidified atmosphere of 3% CO₂ at 37°C. The cells were incubated for two weeks and then fixed with paraformaldehyde (Sigma), and colony formation (colonies of ≥50 cells) was visualized by staining with 10% Giemsa solution (Sigma). The number of colonies was counted to determine the survival fraction (SF), determined as the ratio of number of colonies formed by irradiated cells to the number of colonies formed by non-irradiated cells. The enhancement factor was determined as the ratio of the survival fraction of OPN siRNA-treated cells to nonsense siRNA-treated control cells. The DMF₁₀ is the radiation dose that characterizes an effect at the survival level of 10% of the colonies. The data represent at least three independent experiments.

Cell migration was assessed using modified Boyden chambers [19]. Cells (2.0*10⁴) were suspended in 300 μl of RPMI without FCS and were added to the upper chamber (membrane filter with 8 μm pore size), and the bottom chamber was filled with 1 ml of RPMI supplemented with 20% FCS as chemoattractant. The assay was incubated at 37°C in a humidified atmosphere containing 3% CO₂ for at least 16 h. Non-migrating cells on the upper side of the transwell inserts were removed. The migrated cells on the bottom side of the membrane filter were trypsinized and counted with CASY^® DT (Schärfe System GmbH, Reutlingen, Germany). The data represent at least three independent experiments.

Furthermore, we used a wound scratch assay to determine the migration of MDA-MB-231 cells after transfection with OPN siRNA. Cells were grown in 6-well culture plates [19] in RPMI culture medium containing 10% FCS and cultured to 100% confluence. A uniform cell-free area was created by scratching a confluent monolayer with a 200 μl pipette tip. To determine the migration of MDA-MB-231 cells, the wound closure was observed at different time points. The wound scratch assay was also performed in three independent experiments.

For quantitative determination of the rate of apoptosis, we analyzed suspended cells and the corresponding supernatant. The cells were fixed with 80% ethanol (Merck, Darmstadt, Germany) and centrifuged on microscope slides at 1000 g for 5 min. After staining with DAPI solution (4,6-diamidino-2-phenylindole dihydrochloride) (Serva, Heidelberg, Germany) and washing with PBS, the cells were covered with ProLong^® Gold antifade reagent (Invitrogen). The rate of apoptosis was quantified with a fluorescent microscope at 200× magnification (MC 100 Spot, Zeiss universal microscope, Jena, Germany) by counting 500 cells in separate visual fields (described in [20]). The data represent the results of at least three independent experiments.

At 24 h and 72 h after transfection, the OPN mRNA level in cells treated with OPN-specific siRNAs (Mix, OpnS) was approximately 20% compared to that in cells treated with control siRNA (nonsense siRNA) (Fig. 1A.). We further studied the OPN mRNA level after treatment with OPN-specific siRNAs and additional irradiation. We found that irradiation alone had no effect on OPN mRNA levels. However, after irradiation at 2 Gy in both Mix and OpnS transfected cells, OPN mRNA levels were found to be reduced to 30% compared to cells treated with control siRNA (Fig. 1A.). These effects could be seen at 24 h as well as 72 h after transfection in combination with irradiation at 2, 4 or 6 Gy (data not shown).

Western blot analysis was used to determine the effects of OPN knockdown on the OPN protein level. Transfection with either Mix or OpnS resulted in a clear decrease in the extracellular OPN protein level (Fig. 1B.). However, a decreased intracellular OPN protein level after siRNA transfection was only partially detectable (Fig. 1C.). Furthermore, our experiments demonstrated that the OPN protein level is reduced in control cells transfected with nonsense siRNA after irradiation at 2 Gy compared to non-irradiated cells. The irradiation-induced inhibition of OPN protein expression was also detected in cells transfected with OPN siRNAs (Fig.1C.).

We determined the effects of OPN siRNA and irradiation on the migration rate of MDA-MB-231 cells with the Boyden chamber assay and scratch assay. Cells transfected with siRNA targeting OPN showed reduced migration rates compared to control cells (control and nonsense siRNA). Transfection with Mix resulted in a decreased migration rate to 40% (p = 0.09), whereas the migration rate of cells transfected with OpnS was less than 62% (p = 0.15) compared to the migration rate of cells treated with control siRNA (Fig. 2B.). Similarly, we found a reduced migration rate after transfection with OPN siRNA using the scratch assay (Fig. 2A.). Furthermore, we demonstrated that irradiation at 2 Gy to 6 Gy had no effect on the migration rate (data not shown). However, combination of OPN siRNA transfection and irradiation at 2 Gy resulted in a significant inhibition of migration. After incubation with Mix and 2 Gy irradiation, migration was reduced to 32% (p = 0.03). Additionally, transfection with OpnS and irradiation at 2 Gy attenuated the migration rate to 40% (p = 0.03). Using Western blot analysis, we examined PARP cleavage as an indicator for the induction of apoptosis. However, 24 h after incubation with OPN siRNA, we could not detect any PARP cleavage products using Mix or OpnS. Moreover, Fig. 3A. shows a distinctive accumulation of the PARP cleavage product (89 kDa) 72 h after transfection with siRNA OpnS. However, only OpnS, not Mix, induced apoptosis (Fig. 3A. and 3B.). In addition, we examined the morphology of the cell nuclei to quantify the rate of apoptosis by the use of DAPI staining. The results observed in Western blot analyses were supported by the findings of the quantitative assay. After incubation with OpnS, the apoptosis rate increased from 0.3% to 1.7% (p = 0.04), whereas transfection with Mix had no effect on apoptosis. We found that irradiation alone at 2 Gy did not significantly increase apoptosis in MDA-MB-231 cells (Fig. 3B.). Nevertheless, the combination of OpnS and irradiation at 2 Gy resulted in a significant increase in apoptosis rate to 4% (p = 0.0001). In contrast to that, incubation with Mix and irradiation at 2 Gy had no effect on apoptosis.

We demonstrated that incubation with siRNA OpnS is more effective to reduce the clonogenic survival of MDA-MB-231 cells than incubation with siRNA Mix. In particular, we found that transfection with OpnS significantly decreased the clonogenic survival to 42% (p = 0.008) (Fig. 4A.). In contrast, transfection with Mix was ineffective at reducing the clonogenic survival (82%) (p = 0.4).

Irradiation of MDA-MB-231 cells at 2 Gy reduced the clonogenic survival to 60% (SF₂ = 0.60) (data not shown). The combination of treatment with OpnS siRNA and irradiation also reduced the clonogenic survival as compared to single siRNA treatment. Incubation with OpnS, and additional irradiation at 2 Gy significantly decreased the clonogenic survival to 30% (p < 0.001). Furthermore, with higher irradiation dose transfection with OpnS resulted in a weak radiosensitization with a DMF₁₀ of 1.1 and an enhancement factor of 1.5 at 6 Gy (p = 0.09) (Fig. 4B.).