Trypsinization of cancer cells was performed using 0.05% Trypsin in PBS 1X (154 mM NaCl, 3.88 mM H2NaPO4 and 6.1 mM HNaPO4 pH 7.4) (Sigma-Aldrich, Madrid, Spain). elimination of tumor cells by macrophages in co-culture experiments . In addition, we demonstrated that PPRHs can act as pharmacological agents without causing hepatotoxicity or nephrotoxicity . The aim of the present study was to eliminate tumor cells by macrophages in co-culture experiments by decreasing both the levels of PD-1 in macrophages and those of PD-L1 in different cancer cells using PPRHs and to evaluate the involvement of apoptosis in this approach. Materials and methods Cell culture and PMA induced differentiation Prostate cancer PC3, melanoma M21, ovarian cancer HeLa, breast cancer SKBR3, and monocyte THP-1 cell lines were grown in Hams F-12 medium supplemented with 10% fetal bovine serum (both from Gibco, Barcelona, Spain) at 37C in a 5% CO2-controlled humidified atmosphere. Trypsinization of cancer cells was performed using 0.05% Trypsin in PBS 1X (154 mM NaCl, 3.88 mM H2NaPO4 and 6.1 mM HNaPO4 pH 7.4) (Sigma-Aldrich, Madrid, Spain). THP-1 monocytes grew on suspension. THP-1 cells were incubated with 2 ng/mL phorbol12-myristate 13-acetate (PMA) (Sigma-Aldrich, Madrid, Spain) for differentiation into macrophages. This concentration was chosen due to the patterns of pro-inflammatory cytokines and surface marker levels observed after three days of differentiation . We routinely checked THP-1 differentiation by monitoring their adhesion to the plate and changes in cell morphology. Design of PPRHs PPRHs were designed using The Triplex Oligonucleotide Target Sequence Search Software (http://utw10685.utweb.utexas.edu/tfo/, Austin, Texas, USA). PPRHs were synthesized as non-modified desalted oligodeoxynucleotides by Sigma-Aldrich (HaverHill, United Kingdom). Lyophilized PPRHs were resuspended in sterile Tris-EDTA buffer (1 mM EDTA and 10 mM Tris, pH 8.0) (Sigma-Aldrich, Madrid, Spain) and stored at ?20C until use. As a negative control, we Niranthin used a Watson-Crick hairpin (Hp-WC) that forms intramolecular WatsonCCrick bonds instead of reverse Hoogsteen bonds, and therefore the polypurine domain of the hairpin cannot bind to the polypyrimidine target sequence in the DNA. The sequences of the PPRHs and the negative control hairpin and their abbreviations are described in Fig 1. Open in a separate window Fig 1 PPRHs designed against and genes, as well as the negative control hairpin.Abbreviations are (i) Hp, hairpin; (ii) I, intron; (iii) Pr, promoter; (iv) E, exon. WC stands for the Watson-Crick negative control. Transfection of PPRHs Cells were plated in 6-well dishes. Transfection consisted in mixing 100 nM of PPRH with 10 M of the cationic liposome N-[1-(2,3-dioleoyloxy)propil]-N,N,N-trimethylammonium methylsufate (DOTAP) (Biontex, Mnchen, Germany) in a final volume of 200 L of culture medium. The mixture was incubated for 20 min at room temperature. Finally, the PPRH/liposome complex was added to the cells to realize a final volume of 1 mL. RNA extraction Total RNA was extracted from Personal computer3 and THP-1 cells 24 h and 48 h after transfection, respectively, using TRIzol (Existence Systems, Barcelona, Spain) following a manufacturers specifications. RNA was quantified by measuring its absorbance at 260 nm using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Barcelona, Spain). Reverse transcription cDNA was synthesized by reverse transcription inside a 20 Niranthin l reaction mixture comprising 1 g of total RNA, 125 ng of random hexamers (Roche, Madrid, Spain), 500 M of each dNTP (Panreac Applichem, Barcelona, Spain), 2 L of 10X buffer, 20 devices of RNAse inhibitor and 200 devices of Moloney murine leukemia disease reverse transcriptase (Last three from Lucigen, Wisconsin, USA). The reaction was incubated at 42C for 1 h. Real-time PCR The StepOnePlus Real-Time PCR Systems (Applied Biosystems, Barcelona, Spain) was used to perform these experiments. The primer sequences to determine mRNA levels were and and mRNA levels in Personal computer3 cells, Taqman probe (Assay ID: Hs00204257_m1) was used. Cyclophilin A Taqman probe (PPIA) (Assay ID: Hs04194521-s1) was used as endogenous control. The reaction contained 1x TaqMan Common PCR Master blend, 1x TaqMan probe (both from Applied Biosystems, Barcelona, Spain), 3 L of cDNA and H2O mQ to a final volume of 20 L. PCR cycling conditions were 10 min denaturation Mmp28 at 95C, followed by 40 cycles Niranthin of 15 s at 95C and Niranthin 1 min at 60C. The mRNA quantification was performed using the Ct method, where Ct is the threshold cycle that corresponds to the cycle where the amount of amplified mRNA reaches the threshold of fluorescence. Data were indicated as mRNA levels relative to the cells treated with the bad control Hp-WC. Western blot analyses Total protein extracts from Personal computer3 cells (90,000) were acquired 24 h after.
Trypsinization of cancer cells was performed using 0