Cell culture and chemicals
Human MM cell lines were maintained at 37 °C in a 5% CO2 humidified incubator in either RPMI-1640 medium (MSTO-221H, NCI-H2452) or Dulbecco’s Modified Eagle’s Medium (Ist-Mes-2) supplemented with 10% fetal bovine serum (FBS), glutamine (2 mM), sodium pyruvate and antibiotics (0.02 IU/mL-1 penicillin and 0.02 mg/mL-1 streptomycin).
Curcumin C3 complex® (C3) and Bioperine® (BP) were provided from Sabinsa (Sabinsa Corporation, NJ, USA). Curcumin and Bioperine (CBP) stock solution contains 20 mM C3 complex® and 26 nM of Bioperine® in DMSO. This means that in all the experiments C3 is added to BP in the ratio 100:1 in weight (100 g of C3 for 1 g of BP).
In vitro uptake of curcumin C3 complex®
MSTO-221H cells were seeded at a density of 2 × 105 cells/well in six-well plates, and exposed to C3 with the concentration of 20 μM for 10′. For nuclear counterstain Hoechst 33342 (Invitrogen, Thermofisher, Waltham, MA, USA) was added in the culture medium. Since curcumin exhibits autofluorescence when excited at 455 nm and emits at 540 nm , uptake of the molecule was monitored under fluorescent microscope (DMI8, Leica, Instruments, Germany) using GFP filter with 20x magnification.
Cell proliferation assay
To evaluate the effect of curcumin or CBP on cell proliferation, approximately 1 × 104 cells/well in 48-well plates were plated and treated with 5, 10 and 20 μM for 24, 48 or 72 h. Cells were fixed with 3.7% formaldehyde for 10 min, washed with PBS and stained with 0.5% crystal violet for 10 min. A microplate reader (Cytation3 ASHI, BioTek, VT, USA) was then used to measure the absorbance at 595 nm. All the experiments were performed in triplicate. Data are expressed as the mean ± SD.
Colony formation assay
Cells were seeded at a density of 500 cells/well in six-well plates and incubated for 7 days. Then, cells were treated with 20 μM for 24 h before replacing the media. Cells were grown for additional 7 days and then colonies were stained with crystal violet and counted. Representative plates were captured using scanner (Epson Stylus Photo, PX 650). All the experiments were performed in triplicate. Data are expressed as the mean ± SD.
Wound healing assays
For wound healing assay, approximately 3 × 104 cells/well were plated in six-well plates. After overnight incubation, wounds were created using a 200 μl pipette tip. Cells were treated with 20 μM of CBP for 24, 48 or 72 h. Representative plates were photographed using phase contrast microscope (DMI8, Leica, Instruments, Germany). The gap was photographed and measured using Image J software. All the experiments were performed in triplicate. Data are expressed as the mean ± SD.
The ability of cells to invade into the matrix and migrate towards was analysed in vitro using 24-well inserts with a pore size of 8 μm (Falcon, Corning NY) coated with Matrigel (BD Biosciences, Franklin Lakes, NJ) according to the manufacturer’s guidelines. Approximately 2 × 104 cells were seeded in 250 μL of serum free medium with 20 μM of CBP in the upper surface of chamber; the lower chamber was filled with 750 μL of medium with 10% FBS. After 24 h of treatment, non-invasive cells remaining in upper chamber were removed by PBS washing. Invasive cells that had penetrated the Matrigel (the lower surface) were fixed with 3.7% formaldehyde for 10 min, washed with methanol for 20 min and stained with 0.5% crystal violet for 10 min, and then counted. Cells that invaded the lower surface of the filters were surveyed under a microscope at 10× magnifications, and five fields were randomly selected. Representative plates were photographed using bright field microscope (DMI8, Leica, Instruments, Germany).
All the experiments were performed in triplicate. Data are expressed as the mean ± SD.
Approximately 7,5 × 105 cells/well were plated in 100 mm plates. After overnight incubation, cells were treated with 20 μM of CBP for 24, 48 or 72 h and stained with propidium iodide and annexin V (BD Biosciences, Franklin Lakes, NJ), according to the manufacturer’s protocols. Flow cytometry was performed using a FACSCanto TM flow cytometry system (Becton Dickinson, San Jose, CA). All the experiments were performed in triplicate. Data are expressed as the mean ± SD.
RNA extraction and q-PCR
RNA from treated or untreated cells was extracted using Trizol (Thermo Fisher Scientific, MA USA) following manufacturer’s instructions. 200 ng of total RNA from each sample were retro-transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystem, Thermo Fisher Scientific, MA USA). qPCR reactions were performed by means of a 7900 HT Real Time PCR (Applied Biosystem) using gene specific primers for the following selected genes:
BAX: Forward 5′-TTTGCTTCAGGGTTTCATCCA-3′: Reverse 5′- CTCCATGTTACTGTCCAGTTCGT-3′; BCL-2: Forward 5′-GTTCCCTTTCCTTCCATCC-3′; Reverse 5′-TAGCCAGTCCAGAGGTGAG-3′; FAS: Forward 5′-CCCTCCTACCTCTGGTTCTTACG-3′; Reverse 5’TCAGTCACTTGGGCATTAACACTTT-3′; FASL: Forward 5′-CCTGAAAAAAAGGAGCTGAGGAA-3′; Reverse 5′-GGCATGGACCTTGAGTTGGA-3′; GAPDH: Forward 5′-CAAGGCTGTGGGCAAGGT-3′; Reverse 5′-GGAAGGCCATGCCAGTGA-3:
Primers were designed at exon-exon junctions using Primer express 2.0 (Applied Biosystems). Target expression level was performed as previously described  using GAPDH as housekeeping gene. All the experiments were performed in triplicate. Data are expressed as the mean ± SD.
Protein extracts were prepared as previously described . For each lane, 20 μg of total cell lysates were separated in 4–15% Tris–glycine gels (Bio-Rad Laboratories, Inc., CA, USA) at 100 V. Proteins were then transferred to PVDF membranes (Biorad Laboratories, Inc., CA, USA), probed with the specific primary antibodies, followed by secondary antibodies conjugated with horseradish peroxidase according manufacturer’s indications. Primary antibodies used for western blot include p53 (Cell Signaling, #2524) PARP (Cell Signaling, #9542), BCL-2 (Abcam, ab182858), BAX (Santa Cruz Biotechnology, sc-493), FAS (Abcam, ab133619) and Cytochrome c (Abcam, ab133504). β-Actin (Cell Signaling, #3700) was used as loading control. All the antibodies were used at working concentration indicated by manufacturers. Protein bands were detected by Clarity western ECL (Bio-Rad Laboratories, Inc., CA, USA) and quantified with ImageJ software. All the experiments were performed in triplicate. Data are expressed as the mean ± SD.
Cytochrome c release
To determine the cytochrome c release from mitochondria to cytosol, cytosolic fractions were isolated resuspending 1 × 106 cells in 100 μl of ice-cold plasma membrane permeabilization buffer (200 μg/ml digitonin, 80 mM KCl in PBS). After 5 min incubation on ice, lysates were centrifuged at 800 X g for 5 min at 4 °C, and the supernatants (cytosolic fraction) were then collected. Protein fractions were separated, transferred and probed with and Cytochrome c primary antibodies as described in the previous section.
Mesothelioma xenograft tumor model
Xenograft mouse model of MM was induced by dorsal injection of human mesothelioma cells (MSTO-211H) as previously described .
Male CD1 Nude mice were purchased from Charles River Laboratories Italia (Calco, Italy) and housed in the animal Facility IRRC-Neuromed in accordance with protocols approved by the IRRC-Neuromed Animal Care Review Board and by Ministry of Health. In vivo experiments were conducted according to EU directive 2010/63/EU for animal experiments.
MSTO-211H cells (2.5 × 106) were suspended in 0.2 ml serum-free DMEM medium and inoculated subcutaneously (s.c.) in the right flank of each mouse aged 5 weeks. 10 days after inoculation, when the tumours became visible, 8 mice for each experimental group were randomly assigned into control (DMSO) or treated groups (CBP or cisplatin) and treatments were administered by intra-peritoneal injection (i.p.).
To analyse anticancer properties of CBP, mice were daily treated with CBP (40 mg/kg) for 4 weeks or with Cisplatin (3.3 mg/kg) only for the first 3 days. Mice treated with vehicle alone (DMSO, daily for 4 weeks) were used as control.
Each tumor was measured weekly using a calliper; tumour’s size was assessed by using the formula: (long axis × short axis × short axis)/2.
The mice were sacrificed after 4-week treatments.
Histology and immunohistochemistry
For histology, staining with hematoxylin/eosin and hematoxylin/Van Gieson were used. For immunohistochemistry, tissue sections were heated twice in a microwave oven for 5 min each at 700 W in citrate buffer (pH 6) and then processed with the standard streptavidin-biotin-immunoperoxidase method (DAKO Universal Kit, DAKO Corp., Carpinteria, CA, USA). Mouse monoclonal anti-human Ki67 (MIB-1 clone) and anti-CD31 (M0823 clone) antibodies from DAKO were used at a 1:100 dilution. Diaminobenzidine was used as the final chromogen, and hematoxylin as the nuclear counterstain. Negative control experiments for each tissue section were performed in the absence of the primary antibody. Positive controls, included in each experiment, consisted of tissue previously shown to express the antigen of interest. Two observers (S.C and A.B.), blinded to treatment conditions, evaluated the staining pattern of the proteins separately and quantitated the protein expression in each specimen by scanning the entire section and estimating the number of vessels or positive cells at the high-power-field 10 × 20. The level of concordance, expressed as the percentage of agreement between the observers, was 95%. In the remaining specimens, the score was obtained after collegial revision and agreement. The U-Mann Whitney test was used to assess relationship between ordinal data. Two-tailed p-value was considered significant when ≤0.05.
TUNEL reaction was performed using the peroxidase-based Apoptag kit (Oncor, Gaithersburg, MD, USA). The experiment was repeated on at least two different sections for each specimen. Fifty random fields (250X) per section were analysed (6 mm2). The level of concordance, expressed as the percentage of agreement between the two observers (SC and AB), was 100%. The U-Mann Whitney test was used to assess relationship between ordinal data. Two-tailed p-value was considered significant when ≤0.05.
Analysis was performed using Graph Pad Prism 6.0 (GraphPad Software, San Diego, CA, USA). Significance was evaluated using One-Way ANOVA with Bonferroni post hoc test for multiple comparisons or a Student’s t-test. p-value ≤0.05 was considered statistically significant.