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Supplementary Information
Materials and Methods
RNA extraction, reverse transcription, and real-time reverse transcription-PCR. Total
RNA from cells and primary tumor material was extracted using Trizol reagent (Invitrogen,
Carlsbad, CA) according to the manufacturer's instructions. The extracted RNA was
pretreated with RNase-free DNase, and 2 μg RNA was used for cDNA synthesis with random
hexamers. For PCR amplification of SPHK1, an initial amplification using SPHK1 primers
was done with a denaturation step at 95°C for 10 min, followed by 28 cycles of denaturation at
95°C for 1 min, primer annealing at 55°C for 30 s, and primer extension at 72°C for 30s.
Upon completion of the cycling steps, a final extension at 72°C for 5 min was done before the
reaction was stored at 4°C. Real-time PCR was applied for quantifying the folds of change of
SPHK1 mRNA relative to NSCLC cells and transformed NSCLC cell lines, and among 8
paired primary NSCLC (T) and adjacent non-tumor lung tissue (ANT) from the same patient.
Primers were designed with Primer Express v 2.0 (Applied Biosystems) and sequences of the
primers were: SPHK1 forward primer, 5'-CTTGCAGCTCTTCCGGAGTC-3'; SPHK1 reverse
primer, 5'-GCTCAGTGAGCA TCAGCGTG-3'; c-IAP1 forward primer, 5'-GCTCAGTAACT
GGGAACCAAA-3'; c-IAP1 reverse primer, 5'-ATCATTGCGACCCACATAATA-3'; c-IAP2
forward primer, 5'-GATGTT TCAGATCTACCAGTG-3'; c-IAP2 reverse primer, 5'-GAAAT
GTACGAACTGTACCCT-3'; Bcl-xl forward primer, 5'-TCCTTGTCTACGCTTTCCACG-3';
Bcl-xl reverse primer, 5'-GG TCGCATTGTGGCCTTT-3'; TRAF1 forward primer, 5'- GCCC
TTCCGGAACAAGGTC -3'; TRAF1 reverse primer, 5'- CGTCAATGGCGTGCTCAC -3';
A2O forward primer, 5'-CTGCCCAGGAATGCTACAGATAC-3'; A2O reverse primer, 5'-C
AGGGTCACCAAGG GTACAAA-3'. β-actin forward primer, 5'- CTCCATCCTGGCCTC
GCTGT -3'; β-actin reverse primer, 5'- GCTGTCACCTTCACCGTTCC -3'. Expression data
2
were normalized to the housekeeping geneβ-actin as a loading control.
Cell survival assay. Cells were plated in six-well plates at a density of 1×103 cells/well and
incubated for 24h, followed by doxorubicin treatment (0.1, 0.2 or 0.4 μM) or Docetaxel
treatment (1, 5 or 10 nM) and further incubation for 24 h. Numbers of surviving cells were
determined by the trypan blue exclusion method.
TUNEL assay. The DeadEndTM Fluorometric TUNEL System (Promega, Madison, WI) was
used for TUNEL assay according to the manufacturer's instruction. Briefly, cells treated with
Doxorubicin or Docetaxel, or vehicle control, were washed once with cold PBS followed by
fixation in freshly prepared 4% formaldehyde solution in PBS (pH 7.4) for 25 min at 4°C.
The fixed slides were washed with PBS for 5 min, followed by permeabilizzion with 0.2%
Triton X-100 in PBS for 5 min. After a 5-min rinse with PBS, and cells were covered with
100 µL Equilibration Buffer for 5 min, followed by a 60-min incubation with 2 x SSC at 37°C
to terminate the reaction and a 5-min PBS wash. The samples were then stained in the dark
with 1µg/ml propidium iodide (PI) solution for 15 min. After a final wash with H2O for 5
min at ambient temperature and air-dry, samples were immediately analyzed under a
fluorescence microscope using a standard fluorescein filter set to view the green fluorescence
of fluorescein at 520 nm, the red fluorescence of PI at 620nm, and the blue DAPI at 460nm.
Annexin-V binding assay. The ApopNexinTM FITC Apoptosis Detection Kit (Millipore,
Lake Placid, NY) was used to quantitatively examine apoptotic cells according to
manufacturer’s instruction. Cells were treated with Doxorubicin or Docetaxel in multi-well
plates and subjected to a 6-h incubation, followed by washes with PBS and then with the
Annexin-V binding solution. Subsequently, 150 μl of an Annexin-V antibody in Binding
Buffer was added to each culture well and incubated for 15 min, followed by addition of 1.5 μl
of PI at 1 mg/ml and a further incubation for 5 min. After washing with the Annexin-V
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Binding Buffer, positive Annexin-V staining was visualized under a fluorescence microscope
equipped with a filter for fluorescein isothiocyanate (excitation: 490 nm, emission: 525 nm),
and PI staining was assessed with the filter for Texas red (excitation: 570 nm, emission: 610
nm).
Pharmacological Inhibitors. The inhibitors were dissolved in Me2SO (cell culture grade;
Sigma) and used in the following concentrations: LY294002 (10 μM; specific inhibitor of the
p110 catalytic subunit of PI3K), Akt inhibitor X (10 μM; specific inhibitor of Akt) or JSH-23
(30μM, NF-κB Activation Inhibitor II) or SK1-I (Sphingosine Kinase Inhibitor) (EMD, La
Jolla, CA).
Cell viability assay. Cells (1×104) were seeded in 96-well plates and cultured for 24 h,
followed by exposure to various concentrations of SK1-I for 48 h. Subsequently, 20 μl of MTT
reagent (Genview, Houston, TX) dissolved in phosphate-buffered saline (PBS) (pH 7.4) at a
concentration of 5 mg/ml was added to each well and incubated for additional 4 h. The
MTT-formazan crystals formed were dissolved in 150 μl DMSO (Sangon Biotech, Shanghai,
China), and the absorbance was measured at 570 nm with a reference wavelength of 630 nm
using a microplate reader. Cell growth inhibition was determined using the following formula
according to a previously published method: growth inhibition (%) = (1-OD of treated
cells/OD of control cells) ×100% (1). The half maximal inhibitory concentration (IC50) was
calculated by the Bliss’s software and the data were analyzed by SPSS.
Measurement of S1P Levels in Cellular Assays. S1P levels were analyzed using a S1P
competitive ELISA kit (Echelon Biosciences, Salt Lake City, UT) according to the
manufacturer’s instructions. In brief, cells were serum starved for 24h and lysed in 400μL of
Lysis Buffer. Diluted cell lysate (1:10 in delipidized human sera) were analyzed with the
Echelon S1P ELISA with anti-S1P antibody, and the absorbance was measured at 450 nm
using a microplate reader.
4
Reference
1. Xie G, Zhu X, Li Q, et al. SZ-685C, a marine anthraquinone, is a potent inducer of apoptosis with anticancer activity by suppression of the Akt/FOXO pathway. Br J Pharmacol 2010; 159:689-97.
5
Supplementary Figure legends
Supplementary Figure 1. The expression of SPHK1 is elevated in NSCLC. (A)
Expression of SPHK1 mRNA in two normal human lung epithelial cells (NLECs) and cultured
NSCLC cell lines (A549, HLAMP, PAa, H-1299 and 95D). Expression levels were
normalized forβ-actin. Error bars represent standard deviations (SD) calculated from three
parallel experiments. (B) Real time-PCR analysis of SPHK1 expression in 8 primary NSCLC
(T) and lung adjacent non-cancerous tissues (ANT) paired from the same patient. β-actin was
used as a loading control. (C) Quantification of SPHK1 protein in each of the primary
NSCLC (T) and lung adjacent non-cancerous tissues (ANT) paired from the same patient by
Western blotting.
Supplementary Figure 2. Dysregulation of SPHK1 altered the apoptotic sensitivity of
NSCLC cells. (A) Left panel, A549-Vector, A549-SPHK1, A549-shRNA-Vector and
A549-SPHK1-shRNA cells (2x106) were injected s.c. in nude mice. When the mean tumor
volume reached 50 mm3, mice were injected i.p. with Docetaxel (10mg/kg) every 2 days for 21
days. Right panel, mean tumor weights. (B) Left panel, A549-vector cells (2x106) were
injected in the right oxter of 15 nude mice. When the mean tumor volume reached 50mm3,
mice were randomized into three groups (n= 5) and injected i.p. with vehicle (DMSO),
Docetaxel (10mg/kg) or SK1-I (50mg/kg) every 2 days for 17 days. Right panel, mean tumor
weights. (C) The cytotoxicity of SK1-I was examined in A549 and 95D NSCLC cells. MTT
assay indicated that the IC50 of SK1-I in A549 cells is 35.19 μM and in 95D cells, is 28.52
μM.
Supplementary Figure 3. (A) Vector- or SPHK1- or SPHK1RNAi-transduced A549 and
95D cells treated by Docetaxel (1nM, 5nM,10nM) or doxorubicin (0.1μM, 0.2μM, 0.4μM) for
6
24 h were counted for viable cells using trypan blue exclusion assay. Results are expressed as
percentages of total cells. (B) Quantification of TUNEL staining of vector- or
SPHK1-transduced A549 and 95D cells after doxorubicin treatment (0.2μM) for 24 hr.
Numbers of cells were counted from 10 random fields. **, P < 0.01. (C) Quantification of
Annexin V+/PI- positive vector- or SPHK1RNAi-transduced A549 and 95D cells after
doxorubicin treatment (0.2μM) for 6 hr. Numbers of cells were counted from 10 random
fields. **, P < 0.01. (D) Western analysis of proteolytic cleavage of pro-caspase-3 and PARP
in vector- or SPHK1- or SPHK1RNAi-transduced A549 and 95D cells after doxorubicin
treatment (0.2μM) for 24 hr. α-tubulin was used as a loading control. Data was obtained from
three independent experiments with similar results.
Supplementary Figure 4. Real-time PCR analysis of the expression of NF-κB-targets,
including Bcl-xL, c-IAP1, c-IAP2, TRAF1 and A2O in vector- or SPHK1- or SPHK1
RNAi-transduced A549 and 95D cells. Expression ofβ-actin was used as a control of gene
expression.
Supplementary Figure 5. The cellular S1P level was examined in SPHK transduced cells
and SPHK1 knockdown cells. Data was obtained from three independent experiments with
similar results. *, P < 0.05.
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Supplementary Table
Supplementary Table S1. Clinicopathologic characteristics of patient samples and expression
of SPHK1 in NSCLC
No. (%)
Gender
Male 164 (75.2)
Female 54 (24.8)
Clinical Stage
I 71 (32.6)
II 47 (21.6)
III 78 (35.8)
IV 22 (10.1)
T classification
I 24 (11.0)
II 125 (57.3)
III 51 (23.4)
IV 18 (8.3)
N classification
0 105 (48.2)
1 41 (18.8)
2 68 (31.2)
3 4 (1.8)
Distant metastasis
No 196 (89.9)
Yes 22 (10.1)
Vital status (at follow-up)
Alive 114 (52.8)
Death due to lung cancer cause 104 (47.2)
Expression of SPHK1
Negative 19 (8.7)
Positive 199 (91.3)
Low expression 98 (45.0)
High expression 120 (55.0)
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Supplementary Table S2. Correlation between the clinical pathologic features and
expressions of SPHK1
Characteristics SPHK1
P-value
Low or None High
Gender Male 73 91
0.819
Female 25 29
Pathology
Squamous cell carcinoma 37 45
0.286
Adenocarcinoma 38 53
Adenosquamous carcinoma 20 22
Other NSCLC 3 0
Clinical Stage
I 40 31
0.019 II 19 28
III 33 45
IV 6 16
T classification
I 11 13
0.022 II 66 59
III 17 34
IV 4 14
N classification
N0 58 47
0.010 N1 18 23
N2 21 47
N3 1 3
Distant metastasis No 93 103
0.027
Yes 5 17
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Supplementary Table S3. Univariate and multivariate analysis of different prognostic
parameters in patients with lung cancer by Cox-regression analysis
Univariate analysis Multivariate analysis
No.
patients P SE HR
95%
confidence
interval
P SE HR
95%
confidence
interval
T classification
I 24
II 125 0.048 0.468 2.526 1.010-6.319 0.004 0.477 3.950 1.549-10.068
III 51 0.001 0.483 4.904 1.904-12.633 0.000 0.487 5.745 2.214-14.909
IV 18 0.003 0.533 4.895 1.721-13.918 0.05 0.537 4.533 1.583-12.984
N classification
N0 105
N1 41 0.328 0.276 1.310 0.762-2.251 0.478 0.277 1.218 0.707-2.097
N2 68 0.000 0.225 2.802 1.803-4.355 0.000 0.231 2.656 1.688-4.181
N3 4 0.201 0.731 2.546 0.608-10.662 0.499 0.748 1.658 0.382-7.186
Distant
metastasis
No 196
Yes 22 0.236 0.321 1.463 0.7779-2.745 0.611 0.332 0.845 0.441-1.618
SPHK1
Low 98
High 120 0.000 0.221 3.533 2.290-5.451 0.000 0.241 3.570 2.226-5.728
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Supplementary Table S4 Categorical variable Codings
Categorical Variable Codingsb,c,d,e
Frequency (1) (2) (3)
Ta
1 24 0 0 0
2 125 1 0 0
3 51 0 1 0
4 18 0 0 1
Na
0 105 0 0 0
1 41 1 0 0
2 68 0 1 0
3 4 0 0 1
Ma 0 196 0
1 22 1
SPHK1a 1 98 0
2 120 1
a. Indicator Parameter Coding; b. Category variable: t; c. Category variable: n; d. Category variable: m;
e. Category variable: SPHK1
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