Page 1 of 40
Accepted Preprint first posted on 10 October 2017 as Manuscript ERC-17-0246
The induction of myeloma cell death and DNA damage by tetrac, a thyroid hormone derivative
Keren Cohen1-3, Uri Abadi1,3, Aleck Hercbergs4, Paul J Davis5, Martin Ellis1,3 and Osnat Ashur-Fabian1-3 1
Translational Hemato-Oncology Laboratory, The Hematology Institute and Blood
Bank, Meir Medical Center, Kfar-Saba, Israel; 2 Department of Human Molecular Genetics and Biochemistry; 3 Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 4 Radiation Oncology, Cleveland Clinic, Cleveland, OH, USA; 5 Department of Medicine, Albany Medical College, Albany, NY, USA
Correspondence: Dr. Osnat Ashur-Fabian, Translational Hemato-Oncology Laboratory, The Hematology Institute and Blood Bank, Meir Medical Center, Tchernichovsky 59, Kfar-Saba 6997801, Israel. E-mail: [email protected]
Short title: THE CYTOCIDAL EFFECTS OF TETRAC IN MM
Keywords: Integrin, myeloma, thyroid hormone derivatives Word counts: 5221 Abstract: 194
1
Copyright © 2017 by the Society for Endocrinology.
Page 2 of 40
Abstract Multiple myeloma (MM) is a plasma cell malignancy in which involvement of the thyroid hormone-integrin αvβ3 pathway was shown and pharmacologic inhibition of this pathway is a rational approach to disease management. A thyroid hormone derivative, tetraiodothyroacetic acid (tetrac), which inhibits Lthyroxine (T4) and 3,5,3’-triiodo-L-thyronine (T3) binding to αvβ3 integrin, was studied in five MM cell lines and primary bone marrow (BM) MM cells. Tetrac inhibited MM cell proliferation (absolute cell number/viability) and induced caspase-dependent apoptosis (annexin-V/PI and cell cycle). Activation of caspase-9 and caspase-3 was further demonstrated. Moreover, DNA damage markers, ataxiatelangiectasia mutated (ATM) kinase, poly ADP-ribose polymerase (PARP-1) and histone γH2AX, were induced by tetrac. The various tetrac-initiated effects were attenuated by Arg-Gly-Asp (RGD) peptide, suggesting integrin involvement. Primary BM mononuclear cells were harvested from MM patients (n = 39) at various disease stages. Tetrac induced apoptosis (12/17 samples) and sensitized the cytotoxic action of bortezomib (6/9 samples). Lastly, expression of plasma membrane integrin αvβ3 was shown not only in the malignant plasma clone, but also in other cell populations within the BM samples (n=25). Tetrac is anti-proliferative and pro-apoptotic in MM cells may offer a therapeutic approach for this disease.
2
Page 3 of 40
Introduction
1
A large number of epidemiological and human studies have demonstrated an
2
association between thyroid hormone and cancer. In patients with elevations of serum
3
thyroid hormone, an increased risk of cancer compared with euthyroid patients has
4
been reported (Hellevik, et al. 2009 ; Ness, et al. 2000
5
). An association between
thyroid dysfunction and the risk of development of multiple myeloma (MM), a plasma
6
cell (PC) malignancy accounting for more than 13% of hematological malignancies,
7
has also been suggested (Dalamaga, et al. 2008). Another body of epidemiological and
8
clinical evidence suggests improved survival in individuals with a variety of cancers
9
who also have hypothyroidism (Baldazzi, et al. 2010; Cristofanilli, et al. 2005 ;
10
Hercbergs, et al. 2010; Illouz, et al. 2009; Nelson, et al. 2006 ; Schmidinger, et al. 2010
11
). A few years ago, a binding site for the thyroid hormones L-thyroxine (T4) and
12
3,5,3’-triiodo-L-thyronine (T3) was described on αvβ3 integrin (Bergh, et al. 2005).
13
This integrin is overexpressed in an array of cancer types (Desgrosellier and Cheresh
14
2010), including MM (Ria, et al. 2002; Tucci, et al. 2009; Vacca, et al. 2001). The
15
affinity of the receptor is higher for T4 than for T3 (Bergh et al. 2005), and binding of
16
T4 occurs at physiological free T4 levels. The cell surface receptor binding site for
17
thyroid hormones on integrin αvβ3 is located in close proximity to the well-
18
investigated Arg-Gly-Asp (RGD) recognition site. This region of αvβ3 contains
19
receptors for other small molecules in addition to thyroid hormone that do not contain
20
an RGD sequence and thus are "non-RGD" binding sites. This thyroid hormone
21
binding site has been studied by crystallography and mathematical modeling of the
22
thyroid hormone-binding kinetics (Aghajanova, et al. 2011). The site has no structural
23
homologies with the nuclear thyroid hormone receptors (TRs) that mediate genomic
24
actions of the hormone. The description of a cell surface receptor binding site that is
25
1
Page 4 of 40
unrelated to conventional nuclear TRs has served to define the contributions of non-
26
genomic actions of thyroid hormones to cancer. Upon binding of either hormone to the
27
integrin, numerous effects are exerted by activating several signaling pathways (Davis,
28
et al. 2011). Although the thyroid hormone-integrin axis has been shown to be present
29
in an array of tumor types, our group suggested a role for this pathway in a
30
hematological malignancy, multiple myeloma. This was confirmed by showing an
31
increase in cell proliferation and activation of MAPK by T3/T4 via the αvβ3 integrin
32
(Cohen, et al. 2011). In this current work, we have characterized the effects of an
33
antagonist at the hormone receptor site on the integrin, tetraiodothyroacetic acid
34
(tetrac). Tetrac is a naturally-occurring thyroid hormone derivative with low-potency
35
thyromimetic activity inside the cell at TRs, but that selectively blocks binding of T4
36
and T3 to the thyroid hormone receptor site on αvβ3 (Davis et al. 2011; Davis, et al.
37
2016). This agent was shown by in vitro and animal models to reduce cancer cell
38
proliferation, cell migration and angiogenesis (Mousa, et al. 2008; Mousa, et al. 2012;
39
Rebbaa, et al. 2008; Yalcin, et al. 2009), to induce apoptosis in tumor cells (Glinskii, et
40
al. 2009; Yalcin, et al. 2010) and to foster DNA double-strand breaks (Hercbergs, et al.
41
2011). Tetrac has also been shown to chemosensitize several cancer cells that express
42
chemoresistance (Rebbaa et al. 2008). Despite this extensive research in solid tumor
43
models, tetrac has never been studied as a potential therapeutic agent in multiple
44
myeloma.
45 46
2
Page 5 of 40
Methods and materials
47
Cell lines and primary BM cells
48
MM cell lines: CAG and ARK (established at the Arkansas Cancer Research Center),
49
RPMI-8226 (CCL 155, ATCC; Rockville, MD, USA), U266 (TIB-196, ATCC; Rockville, 50 MD, USA) and ARP-1. All cell lines were negative for mycoplasma contamination. Bone 51 marrow (BM) aspirates were obtained upon written consent from a total of 40 patients at
52
various disease stages (clinical data is presented in Supplementary Table I).
53
Additionally, one normal BM sample from healthy subject was collected. All bone
54
marrow aspirates were obtained from patients treated at the Meir Medical Center after
55
approval by an Institutional Review Board (IRB number #180-2010). BM mononuclear
56
cells were isolated by Ficoll-Paque gradient centrifugation according to the
57
manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO, USA). The different
58
mononuclear cell populations were characterized and immunophenotyping was performed 59 by flow-cytometry. Cells were grown in RPMI 1640 medium supplemented with 10%
60
heat-inactivated FCS, penicillin and streptomycin antibiotics and the passage number for
61
the experiments described was up to 20.
62
Reagents and chemicals
63
T3, T4 and tetrac (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in DMSO to
64
100mM and further dissolved in 1 mM KOH-propylene glycol, which also was used as
65
a vehicle control [final concentration of 0.04N KOH with 0.4% polyethylene glycol
66
(vol/vol)]. RGD and Arg-Gly-Glu (RGE) peptides (Sigma-Aldrich, St. Louis, MO,
67
USA) were dissolved at 100 mM in PBS. Bortezomib (dissolved in saline to 2.6µM)
68
was obtained from the oncology pharmacy at Meir Medical Center. The volume of
69
solvent added to each well has been kept constant for all conditions. Pan-caspase
70
3
Page 6 of 40
inhibitor, Z-VAD-FMK was purchased from Enzo life sciences (NY, USA).
71
Monoclonal antibody against αvβ3 integrin (LM609, PE/FITC-conjugated) was from
72
Chemicon International (Harrow, UK). APC-CD138 antibodies (Miltenyi Biotec,
73
Bergisch Gladbach, Germany), FITC-CD45 and PE-CD38 antibodies (BioLegend, San
74
Diego, CA, USA). Proliferating cell nuclear antigen (PCNA) antibody (Santa Cruz
75
Biotechnology, Santa Cruz, CA, USA). Antibodies against total and cleaved caspase-9,
76
caspase-3, PARP-1 and tubulin were from Cell Signaling (Danvers, MA, USA). Anti-
77
AIF was from EMD Millipore (Billerica, MA, USA). Anti-pATM (phospho-serine
78
1981) was from Epitomics (Burlingame, CA, USA) and anti-pγH2AX (phospho-serine
79
139) from Chemicon International. (Billerica, MA, USA). All antibodies were
80
appropriately validated.
81
WST-1 proliferation assay
82
WST-1 reagent (Roche, Indianapolis, IN, USA; 10% final concentration) was
83
incubated according to the manufacturer's instructions as previously described (Cohen,
84
et al. 2015).
85
Flow cytometry (MACSQuant, Miltenyi Biotec, Bergisch Gladbach, Germany)
86
Absolute cell number: Cells were harvested in a fixed volume and counted. Cell cycle:
87
Following permeabilization and fixation by incubation with 70% ethanol (20 min,
88
-20 °C), the cells were stained with DNA propidium iodide (PI) (50 µg/mL) for 15 min
89
at room temperature, along with RNAse A (10 µg/mL) (Sigma-Aldrich, St. Louis, MO,
90
USA) and cell cycle analysis was carried out by flow-cytometry. Apoptosis /necrosis:
91
Apoptotic cell death was measured by annexin-V/PI assay (BioVision Inc., Milpitas,
92
CA, USA) as previously described (Shinderman-Maman, et al. 2016).
93
Immunophenotype: Immunophenotyping was performed in BM-derived mononuclear
94
4
Page 7 of 40
cells were by using FITC-CD45, PE-CD38 (data not shown) and APC-CD138
95
antibodies. αvβ3 expression: Cell lines and primary BM cells were harvested in RPMI
96
1640 medium and labeled with 10 µg/mL FITC-αvβ3 or PE-αvβ3 antibody (LM609).
97
RNA extraction and cDNA synthesis
98
RNA was extracted using a NucleoSpine RNA II kit (Macherey-Nagel, Duren,
99
Germany) according to the manufacturer's instructions and eluted in 40 µ L RNase-free
100
water, as previously described(Cohen et al. 2015). RNA concentration and purity were
101
measured using a NanoDrop™ 1000 Spectrophotometer (Thermo Scientific,
102
Wilmington, DE, USA). 200 ng RNA were reverse transcribed using a High Capacity
103
cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA), according to
104
the manufacturer's instructions.
105
Real-time PCR
106
Complementary DNA (cDNA) was analyzed for the following apoptotic genes:
107
Apoptotic protease activating factor-1 (apaf1, accession number Nm_013229),
108
caspase-3 (accession number Nm_004346), p53 upregulated modulator of apoptosis
109
(PUMA, accession number Nm_014417), NOXA (accession number Nm_021127) and
110
FAS (accession number Nm_000043), by 7500 Fast Real-Time PCR system (Applied
111
Biosystems, Carlsbad, CA, USA), using Applied Biosystems Fast Sybr Green Mix (cat
112
# 4385612). Results, normalized to beta actin (accession number Nm_001101), were
113
calculated as -fold change, using the comparative threshold cycle method (2-∆∆CT)
114
relative to control cells (i.e., controls were assigned a value of 1 by definition). Primers
115
(Hylabs, Israel) were designed as published before (Cohen et al. 2011) in different
116
exons in order to eliminate DNA contamination, using Primer-Express software
117
(Applied-Biosystems). Different bioinformatics tools, including BLAST and alignment
118
5
Page 8 of 40
PCR analysis were used to design primers suitable for the amplification of fragments
119
from the candidate genes. Primers sets were: Apaf1: F TGCGCTGCTCTGCCTTCT,
120
R: CCATGGGTAGCAGCTCCTTCT. Caspase 3: F:
121
CAGACAGTGGTGTTGATGATGAC, R: TCGCCAAGAATAATAACCAGGTG.
122
Puma: F: GAAGAGCAAATGAGCCAAACG, R: GGAGCAACCGGCAAACG.
123
Noxa: F: CGGAGATGCCTGGGAAGAA, R:
124
CCAAATCTCCTGAGTTGAGTAGCA. Fas: F:
125
CCCTCCTACCTCTGGTTCTTACG, R: TGATGTCAGTCACTTGGGCATT. Beta
126
actin: F: CCTGGCACCCAGCACAAT, R: GCCGATCCACACGGAGTACT.
127
Western blotting
128
Equal numbers of cells (106 cells/6-well plate) were used for total protein extraction
129
quantified by BCA Protein assay (Thermo Scientific, Wilmington, DE, USA). The
130
proteins (30 µg) were separated on 10-12.5% polyacrylamide gels, transferred to
131
PVDF membranes and analyzed by western blot, using primary antibodies and
132
appropriate second HRP-conjugated antibody (Jackson Immuno-Research
133
Laboratories, West Grove, PA, USA). Immunoreactive proteins were detected by
134
chemiluminescence reagents (Biological Industries, Israel). Alpha tubulin or beta actin
135
were used for normalization. Band intensity was visualized using LAS-3000
136
(FujiFilm, Japan) and quantified by Multi-gauge v3.0 software and normalized to
137
protein amount.
138
Inhibition of caspase-dependent apoptosis
139
MM cells were treated in the presence/absence of 50 µM pan-caspase inhibitor, Z-
140
VAD-FMK, dissolved in DMSO.
141
6
Page 9 of 40
Statistical analysis
142
Mean ± standard deviation was calculated for each value. In order to compare the
143
means for pairs of groups (treatment versus control), Student’s unpaired t-test was
144
used. A value of p
Accepted Preprint first posted on 10 October 2017 as Manuscript ERC-17-0246
The induction of myeloma cell death and DNA damage by tetrac, a thyroid hormone derivative
Keren Cohen1-3, Uri Abadi1,3, Aleck Hercbergs4, Paul J Davis5, Martin Ellis1,3 and Osnat Ashur-Fabian1-3 1
Translational Hemato-Oncology Laboratory, The Hematology Institute and Blood
Bank, Meir Medical Center, Kfar-Saba, Israel; 2 Department of Human Molecular Genetics and Biochemistry; 3 Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 4 Radiation Oncology, Cleveland Clinic, Cleveland, OH, USA; 5 Department of Medicine, Albany Medical College, Albany, NY, USA
Correspondence: Dr. Osnat Ashur-Fabian, Translational Hemato-Oncology Laboratory, The Hematology Institute and Blood Bank, Meir Medical Center, Tchernichovsky 59, Kfar-Saba 6997801, Israel. E-mail: [email protected]
Short title: THE CYTOCIDAL EFFECTS OF TETRAC IN MM
Keywords: Integrin, myeloma, thyroid hormone derivatives Word counts: 5221 Abstract: 194
1
Copyright © 2017 by the Society for Endocrinology.
Page 2 of 40
Abstract Multiple myeloma (MM) is a plasma cell malignancy in which involvement of the thyroid hormone-integrin αvβ3 pathway was shown and pharmacologic inhibition of this pathway is a rational approach to disease management. A thyroid hormone derivative, tetraiodothyroacetic acid (tetrac), which inhibits Lthyroxine (T4) and 3,5,3’-triiodo-L-thyronine (T3) binding to αvβ3 integrin, was studied in five MM cell lines and primary bone marrow (BM) MM cells. Tetrac inhibited MM cell proliferation (absolute cell number/viability) and induced caspase-dependent apoptosis (annexin-V/PI and cell cycle). Activation of caspase-9 and caspase-3 was further demonstrated. Moreover, DNA damage markers, ataxiatelangiectasia mutated (ATM) kinase, poly ADP-ribose polymerase (PARP-1) and histone γH2AX, were induced by tetrac. The various tetrac-initiated effects were attenuated by Arg-Gly-Asp (RGD) peptide, suggesting integrin involvement. Primary BM mononuclear cells were harvested from MM patients (n = 39) at various disease stages. Tetrac induced apoptosis (12/17 samples) and sensitized the cytotoxic action of bortezomib (6/9 samples). Lastly, expression of plasma membrane integrin αvβ3 was shown not only in the malignant plasma clone, but also in other cell populations within the BM samples (n=25). Tetrac is anti-proliferative and pro-apoptotic in MM cells may offer a therapeutic approach for this disease.
2
Page 3 of 40
Introduction
1
A large number of epidemiological and human studies have demonstrated an
2
association between thyroid hormone and cancer. In patients with elevations of serum
3
thyroid hormone, an increased risk of cancer compared with euthyroid patients has
4
been reported (Hellevik, et al. 2009 ; Ness, et al. 2000
5
). An association between
thyroid dysfunction and the risk of development of multiple myeloma (MM), a plasma
6
cell (PC) malignancy accounting for more than 13% of hematological malignancies,
7
has also been suggested (Dalamaga, et al. 2008). Another body of epidemiological and
8
clinical evidence suggests improved survival in individuals with a variety of cancers
9
who also have hypothyroidism (Baldazzi, et al. 2010; Cristofanilli, et al. 2005 ;
10
Hercbergs, et al. 2010; Illouz, et al. 2009; Nelson, et al. 2006 ; Schmidinger, et al. 2010
11
). A few years ago, a binding site for the thyroid hormones L-thyroxine (T4) and
12
3,5,3’-triiodo-L-thyronine (T3) was described on αvβ3 integrin (Bergh, et al. 2005).
13
This integrin is overexpressed in an array of cancer types (Desgrosellier and Cheresh
14
2010), including MM (Ria, et al. 2002; Tucci, et al. 2009; Vacca, et al. 2001). The
15
affinity of the receptor is higher for T4 than for T3 (Bergh et al. 2005), and binding of
16
T4 occurs at physiological free T4 levels. The cell surface receptor binding site for
17
thyroid hormones on integrin αvβ3 is located in close proximity to the well-
18
investigated Arg-Gly-Asp (RGD) recognition site. This region of αvβ3 contains
19
receptors for other small molecules in addition to thyroid hormone that do not contain
20
an RGD sequence and thus are "non-RGD" binding sites. This thyroid hormone
21
binding site has been studied by crystallography and mathematical modeling of the
22
thyroid hormone-binding kinetics (Aghajanova, et al. 2011). The site has no structural
23
homologies with the nuclear thyroid hormone receptors (TRs) that mediate genomic
24
actions of the hormone. The description of a cell surface receptor binding site that is
25
1
Page 4 of 40
unrelated to conventional nuclear TRs has served to define the contributions of non-
26
genomic actions of thyroid hormones to cancer. Upon binding of either hormone to the
27
integrin, numerous effects are exerted by activating several signaling pathways (Davis,
28
et al. 2011). Although the thyroid hormone-integrin axis has been shown to be present
29
in an array of tumor types, our group suggested a role for this pathway in a
30
hematological malignancy, multiple myeloma. This was confirmed by showing an
31
increase in cell proliferation and activation of MAPK by T3/T4 via the αvβ3 integrin
32
(Cohen, et al. 2011). In this current work, we have characterized the effects of an
33
antagonist at the hormone receptor site on the integrin, tetraiodothyroacetic acid
34
(tetrac). Tetrac is a naturally-occurring thyroid hormone derivative with low-potency
35
thyromimetic activity inside the cell at TRs, but that selectively blocks binding of T4
36
and T3 to the thyroid hormone receptor site on αvβ3 (Davis et al. 2011; Davis, et al.
37
2016). This agent was shown by in vitro and animal models to reduce cancer cell
38
proliferation, cell migration and angiogenesis (Mousa, et al. 2008; Mousa, et al. 2012;
39
Rebbaa, et al. 2008; Yalcin, et al. 2009), to induce apoptosis in tumor cells (Glinskii, et
40
al. 2009; Yalcin, et al. 2010) and to foster DNA double-strand breaks (Hercbergs, et al.
41
2011). Tetrac has also been shown to chemosensitize several cancer cells that express
42
chemoresistance (Rebbaa et al. 2008). Despite this extensive research in solid tumor
43
models, tetrac has never been studied as a potential therapeutic agent in multiple
44
myeloma.
45 46
2
Page 5 of 40
Methods and materials
47
Cell lines and primary BM cells
48
MM cell lines: CAG and ARK (established at the Arkansas Cancer Research Center),
49
RPMI-8226 (CCL 155, ATCC; Rockville, MD, USA), U266 (TIB-196, ATCC; Rockville, 50 MD, USA) and ARP-1. All cell lines were negative for mycoplasma contamination. Bone 51 marrow (BM) aspirates were obtained upon written consent from a total of 40 patients at
52
various disease stages (clinical data is presented in Supplementary Table I).
53
Additionally, one normal BM sample from healthy subject was collected. All bone
54
marrow aspirates were obtained from patients treated at the Meir Medical Center after
55
approval by an Institutional Review Board (IRB number #180-2010). BM mononuclear
56
cells were isolated by Ficoll-Paque gradient centrifugation according to the
57
manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO, USA). The different
58
mononuclear cell populations were characterized and immunophenotyping was performed 59 by flow-cytometry. Cells were grown in RPMI 1640 medium supplemented with 10%
60
heat-inactivated FCS, penicillin and streptomycin antibiotics and the passage number for
61
the experiments described was up to 20.
62
Reagents and chemicals
63
T3, T4 and tetrac (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in DMSO to
64
100mM and further dissolved in 1 mM KOH-propylene glycol, which also was used as
65
a vehicle control [final concentration of 0.04N KOH with 0.4% polyethylene glycol
66
(vol/vol)]. RGD and Arg-Gly-Glu (RGE) peptides (Sigma-Aldrich, St. Louis, MO,
67
USA) were dissolved at 100 mM in PBS. Bortezomib (dissolved in saline to 2.6µM)
68
was obtained from the oncology pharmacy at Meir Medical Center. The volume of
69
solvent added to each well has been kept constant for all conditions. Pan-caspase
70
3
Page 6 of 40
inhibitor, Z-VAD-FMK was purchased from Enzo life sciences (NY, USA).
71
Monoclonal antibody against αvβ3 integrin (LM609, PE/FITC-conjugated) was from
72
Chemicon International (Harrow, UK). APC-CD138 antibodies (Miltenyi Biotec,
73
Bergisch Gladbach, Germany), FITC-CD45 and PE-CD38 antibodies (BioLegend, San
74
Diego, CA, USA). Proliferating cell nuclear antigen (PCNA) antibody (Santa Cruz
75
Biotechnology, Santa Cruz, CA, USA). Antibodies against total and cleaved caspase-9,
76
caspase-3, PARP-1 and tubulin were from Cell Signaling (Danvers, MA, USA). Anti-
77
AIF was from EMD Millipore (Billerica, MA, USA). Anti-pATM (phospho-serine
78
1981) was from Epitomics (Burlingame, CA, USA) and anti-pγH2AX (phospho-serine
79
139) from Chemicon International. (Billerica, MA, USA). All antibodies were
80
appropriately validated.
81
WST-1 proliferation assay
82
WST-1 reagent (Roche, Indianapolis, IN, USA; 10% final concentration) was
83
incubated according to the manufacturer's instructions as previously described (Cohen,
84
et al. 2015).
85
Flow cytometry (MACSQuant, Miltenyi Biotec, Bergisch Gladbach, Germany)
86
Absolute cell number: Cells were harvested in a fixed volume and counted. Cell cycle:
87
Following permeabilization and fixation by incubation with 70% ethanol (20 min,
88
-20 °C), the cells were stained with DNA propidium iodide (PI) (50 µg/mL) for 15 min
89
at room temperature, along with RNAse A (10 µg/mL) (Sigma-Aldrich, St. Louis, MO,
90
USA) and cell cycle analysis was carried out by flow-cytometry. Apoptosis /necrosis:
91
Apoptotic cell death was measured by annexin-V/PI assay (BioVision Inc., Milpitas,
92
CA, USA) as previously described (Shinderman-Maman, et al. 2016).
93
Immunophenotype: Immunophenotyping was performed in BM-derived mononuclear
94
4
Page 7 of 40
cells were by using FITC-CD45, PE-CD38 (data not shown) and APC-CD138
95
antibodies. αvβ3 expression: Cell lines and primary BM cells were harvested in RPMI
96
1640 medium and labeled with 10 µg/mL FITC-αvβ3 or PE-αvβ3 antibody (LM609).
97
RNA extraction and cDNA synthesis
98
RNA was extracted using a NucleoSpine RNA II kit (Macherey-Nagel, Duren,
99
Germany) according to the manufacturer's instructions and eluted in 40 µ L RNase-free
100
water, as previously described(Cohen et al. 2015). RNA concentration and purity were
101
measured using a NanoDrop™ 1000 Spectrophotometer (Thermo Scientific,
102
Wilmington, DE, USA). 200 ng RNA were reverse transcribed using a High Capacity
103
cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA), according to
104
the manufacturer's instructions.
105
Real-time PCR
106
Complementary DNA (cDNA) was analyzed for the following apoptotic genes:
107
Apoptotic protease activating factor-1 (apaf1, accession number Nm_013229),
108
caspase-3 (accession number Nm_004346), p53 upregulated modulator of apoptosis
109
(PUMA, accession number Nm_014417), NOXA (accession number Nm_021127) and
110
FAS (accession number Nm_000043), by 7500 Fast Real-Time PCR system (Applied
111
Biosystems, Carlsbad, CA, USA), using Applied Biosystems Fast Sybr Green Mix (cat
112
# 4385612). Results, normalized to beta actin (accession number Nm_001101), were
113
calculated as -fold change, using the comparative threshold cycle method (2-∆∆CT)
114
relative to control cells (i.e., controls were assigned a value of 1 by definition). Primers
115
(Hylabs, Israel) were designed as published before (Cohen et al. 2011) in different
116
exons in order to eliminate DNA contamination, using Primer-Express software
117
(Applied-Biosystems). Different bioinformatics tools, including BLAST and alignment
118
5
Page 8 of 40
PCR analysis were used to design primers suitable for the amplification of fragments
119
from the candidate genes. Primers sets were: Apaf1: F TGCGCTGCTCTGCCTTCT,
120
R: CCATGGGTAGCAGCTCCTTCT. Caspase 3: F:
121
CAGACAGTGGTGTTGATGATGAC, R: TCGCCAAGAATAATAACCAGGTG.
122
Puma: F: GAAGAGCAAATGAGCCAAACG, R: GGAGCAACCGGCAAACG.
123
Noxa: F: CGGAGATGCCTGGGAAGAA, R:
124
CCAAATCTCCTGAGTTGAGTAGCA. Fas: F:
125
CCCTCCTACCTCTGGTTCTTACG, R: TGATGTCAGTCACTTGGGCATT. Beta
126
actin: F: CCTGGCACCCAGCACAAT, R: GCCGATCCACACGGAGTACT.
127
Western blotting
128
Equal numbers of cells (106 cells/6-well plate) were used for total protein extraction
129
quantified by BCA Protein assay (Thermo Scientific, Wilmington, DE, USA). The
130
proteins (30 µg) were separated on 10-12.5% polyacrylamide gels, transferred to
131
PVDF membranes and analyzed by western blot, using primary antibodies and
132
appropriate second HRP-conjugated antibody (Jackson Immuno-Research
133
Laboratories, West Grove, PA, USA). Immunoreactive proteins were detected by
134
chemiluminescence reagents (Biological Industries, Israel). Alpha tubulin or beta actin
135
were used for normalization. Band intensity was visualized using LAS-3000
136
(FujiFilm, Japan) and quantified by Multi-gauge v3.0 software and normalized to
137
protein amount.
138
Inhibition of caspase-dependent apoptosis
139
MM cells were treated in the presence/absence of 50 µM pan-caspase inhibitor, Z-
140
VAD-FMK, dissolved in DMSO.
141
6
Page 9 of 40
Statistical analysis
142
Mean ± standard deviation was calculated for each value. In order to compare the
143
means for pairs of groups (treatment versus control), Student’s unpaired t-test was
144
used. A value of p
