1. Ingeniería

Supplementary Appendix
Supplementary materials and methods
Culture conditions
Freshly isolated primary CLL cells were cultured in RPMI 1640 medium (Gibco,
Grand Island, NY, USA), and MEC-1 cell line was cultured in Iscove's modified
Dulbecco's medium (IMDM; Gibco), both of which were supplemented with 10%
fetal bovine serum (FBS; HyClone, Logan, UT, USA), 2 mM L-glutamine, 100 U/mL
penicillin, and 100 g/mL streptomycin at 37 °C in a humidified 5% CO2 incubator.
Bone marrow stromal cells (BMSCs) were isolated from bone marrow
mononuclear cells of healthy volunteers. Briefly, bone marrow mononuclear cells
isolated by Ficoll-Hypaque density gradient separation were plated in 25 cm2 flasks
(Costar, Cambridge, MA, USA) at a concentration of 2 × 105 cells/cm2. The isolated
cells were cultured in Dulbecco’s Modified Eagle Medium containing 1.0 g/L glucose
(DMEM-LG; Gibco), with 10% FBS, 2 mM L-glutamine and antibiotics. After
72-hour incubation at 37°C in a humidified 5% CO2 incubator, non-adherent cells
were removed. When 70-80% confluent, the adherent cells were trypsinised and
expanded for 3-4 weeks. Once the third passage was reached, the phenotypes of
BMSCs were determined for positivity of CD73, CD90, CD105, and negativity of
CD34, CD45 by flow cytometric analysis. Then the BMSCs were harvested and
frozen routinely in 90% FBS and 10% dimethyl sulfoxide (DMSO; Solarbio, Beijing,
China).
Protein extraction and western blot analyses
After designated treatments, CLL cells were harvested, washed twice with cold
phosphate-buffered saline (PBS), and lysed in lysis buffer (Shenergy Biocolor,
Shanghai, China) with 1× final consentration of phosphatase inhibitor cocktail
(PhosSTOP; Roche, Mannheim, Germany). After incubation on ice for 30 minutes,
the cell lysate was centrifugated at 12000 g for 15 minutes at 4 °C. The total protein
concentrations of the samples were determined by the BCA assay (Shenergy
Biocolor). 20-30 g of total protein was loaded to 8-15% SDS/PAGE gel and
subjected to electrophoresis. Proteins were transferred onto a polyvinylidene fluoride
(PVDF) membrane (Millipore, Billerica, MA, USA), which was incubated with
blocking solution (5% nonfat dry milk in Tris-buffered saline containing 0.05%
Tween-20) for 1 hour and incubated with the recommended dilutions of indicated
antibodies overnight at 4 °C. The primary antibodies against phospho-STAT3
(Tyr705, D3A7), acetyl-STAT3 (Lysine 685), STAT3 (D3Z2G), Mcl-1 (D35A5),
Bcl-xL (54H6), Bcl-2 (50E3), caspase-3 (8G10), caspase-8 (1C12), caspase-9 (C9)
and PARP [specific to the full-length (116 kDa) and the cleaved form (89 kDa) of
PARP, 46D11] were obtained from Cell Signaling Technology (Danvers, MA, USA).
1
The expression level of GAPDH was used as the loading control for the western blots.
All primary antibodies were used at 1:1000 dilution. Anti-rabbit or anti-mouse
secondary antibodies conjugated to horseradish peroxidase (HRP) (ZSGB-BIO,
Beijing, China) were used at 1:10000 dilution. After incubation with
chemiluminescent HRP substrate (Millipore), the membrane was finally observed by
system LAS4000 mini (Fuji film, Tokyo, Japan) and analyzed by Multi Gauge
Version 3.0 software (Fuji film).
Flow cytometric analysis of efflux of Rhodamine 123
The measurement of Rhodamine 123 (Rho123) efflux was performed to evaluate
P-glycoprotein 1 (P-gp) activity. Generally, 1 × 106 cells were incubated with 5 M
Rho123 on ice in a 5% CO2 incubator for 1 hour, and then the medium was replaced
with Rho123-free medium with or without WP1066. Following incubation at 37 °C
for 1 hour, the medium was removed, and cells were washed two times with ice-cold
PBS. The fluorescent signal of Rho123 was measured by flow cytometry to evaluate
retention. Verapamil (VPL, 10 M), a P-gp inhibitor, was used as a positive control.
Real-time quantitative polymerase chain reaction (PCR)
Total RNA was extracted by TRIzol reagent (Takara, Dalian, China) from cells
following incubation with single drug or drug combination for 48 hours. Reverse
transcription to complementary DNA (cDNA) was conducted using PrimeScript RT
reagent kit with gDNA eraser (Takara). Amplification reactions were performed using
a SYBR Premix Ex Taq II kit (Takara) on a LightCycler 480 real-time PCR system
(Roche
Diagnostics,
Mannheim,
Germany).
Primer
sequences
were
5’-CCCATCATTGCAATAGCAGG-3’
(forward)
and
5’-GTTCAAACTTCTGCTCCTGA-3’
(reverse)
for
P-gp,
and
5'-TGACGTGGACATCCGCAAAG-3'
(forward)
and
5'-CTGGAAGGTGGACAGCGAGG-3' (reverse) for -actin. Isolation of RNA,
reverse transcription and real-time PCR were performed following the manufacturer's
instructions. Real-time PCR for each gene of each cDNA sample was assayed in
triplicate. Data were analyzed using the 2 Ct method with Light Cycler480 Gene
Scanning Version 1.5 Software (Roche Diagnostics).
2
Supplementary table
Table S1. Clinical characteristics of CLL patients
Patient
no.
Age,
years
Sex
Rai
stage
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
70
45
65
57
66
65
63
61
41
67
62
55
76
43
37
80
63
48
50
69
82
46
64
71
43
63
69
58
74
48
73
60
62
59
F
M
M
M
M
F
F
M
M
M
F
M
F
M
M
M
M
M
F
M
M
M
M
M
F
M
M
M
M
M
M
M
F
F
I
I
II
III
0
I
IV
0
I
II
II
II
0
III
0
II
I
I
II
IV
I
I
III
II
II
II
II
III
I
0
IV
II
0
I
Lymphocyte
count
(×109/L)
83.0
194.2
18.5
37.4
14.7
17.9
14.6
16.6
24.1
21.9
29.1
29.6
206.5
24.5
53.7
18.8
21.5
12.2
36.8
25.6
139.7
14.7
24.1
103.3
33.0
126.1
22.8
130.2
20.9
37.2
44.0
154.1
42.3
19.3
IgVH
status
ZAP70
CD38
Cytogenetic
abnormalities
ND
UM
M
UM
M
M
M
ND
ND
M
UM
M
M
UM
M
ND
UM
M
M
M
ND
ND
UM
M
M
UM
ND
M
M
M
UM
M
ND
M
ND
+
ND
+
ND
ND
+
+
ND
+
ND
ND
+
ND
ND
ND
+
ND
-
ND
+
ND
ND
+
+
+
ND
+
ND
ND
+
+
ND
ND
+
ND
-
ND
Normal
ND
ND
ND
Normal
Trisomy 12
ND
ND
13q-, Trisomy 12
ND
Normal
ND
11q13qND
Normal
Normal
Normal
13qND
ND
Normal
11qNormal
13qNormal
Normal
13q13q17p13qND
13q-
UM: unmutated; M, mutated; ND: not done or not determined. IgVH mutational status is based on on
98% cut-off value. Expressions of ZAP70 and CD38 are based on 20% cut-off value. Cytogenetic
abnormalities were investigated by FISH analysis for detecting del(11q), del(13q), del(17p) and
trisomy 12.
3
Supplementary figures
Fig. S1. Effects of HDAC inhibition on STAT3 phosphorylation and acetylation. CLL
cells were incubated for 30 minutes with the indicated concentrations of IL-6.
Different concentrations of SAHA were added to cultures 4-hour before the addition
of IL-6. Protein lysates were analyzed by western blotting using the antibodies
indicated. The representative cases used here were from patients CLL 6 (panel A),
CLL 10 (panel B).
4
Fig. S2. The dose dependence of WP1066 to reverse IL-6–induced SAHA resistance.
SAHA was added at 2.5 M, and IL-6 at 10 ng/mL. CLL cells were from patient CLL
9. (A) The inhibition of SAHA-induced apoptosis by IL-6 and the ability of increasing
concentrations of WP1066 to restore apoptosis induction after 48-hour incubation
were determined by western blot analysis of PARP cleavage. (B) Quantitative analysis
of A. Data are presented as mean plus standard deviation of triplicate experiments. *P
< 0.01 vs. normal control. (C) The percentage of morphologically identifiable
apoptotic cells. Data are presented as mean plus standard deviation. *P < 0.01 vs.
normal control.
5
Fig. S3. The WP1066 dose dependence of the blockade of IL-6–induced STAT3
phosphorylation in the presence of SAHA. The WP1066 dose dependence of the
blockade of IL-6–induced STAT3 phosphorylation in the presence of SAHA after
24-hour incubation was determined by western blot analysis. SAHA was added at 2.5
M, and IL-6 at 10 ng/mL. CLL cells were from patient CLL11 (panel A) and MEC-1
cell line (panel B)
6
Fig. S4. Effects of WP1066 on caspase activity in CLL cells. CLL cells from patient
CLL 34 and MEC-1 cell line were incubated with diluent DMSO or WP1066; and
after 24 hours, cell lysates were prepared and analyzed by western blotting with
anti-caspase-3, -8, -9 and GAPDH antibodies. Representative blots from triplicate
experiments are shown.
7
Fig. S5. Effects of WP1066 on the Rhodamine 123 (Rho123) efflux and mRNA level
of P-glycoprotein 1 (P-gp, MDR1) in MEC-1 cells. (A) The retention of Rho123 was
measured by flow cytometric analysis after the cells were treated with WP1066 or
verapamil (VPL) for 1 hour, respectively, as described in ‘‘supplementary materials
and methods”. (B) The relative fluorescence intensity of Rho123 retention was
calculated in the histogram. Data are presented as mean plus standard deviation of
triplicate experiments. *P < 0.05 vs. control; **P < 0.01 vs. control. (C) MEC-1 cells
were treated with diluent DMSO or WP1066 for 24 hours, followed by real-time PCR
analysis. The relative expression of P-gp mRNA was calculated in the histogram. Data
are presented as mean plus standard deviation of triplicate experiments. NS, not
significant vs. control.
8