Supplementary Materials and Methods
Cells and cell culture
Human cancer cell lines (SUM159, A549, U87MG) and human endothelial cells
(HUVEC) were purchased from the American Type Culture Collection. A549 and
U87MG cells were cultured in DMEM medium with 10% fetal bovine serum (FBS),
SUM159 was maintained in Ham's F-12 medium with 5% FBS, 5 μg/ml insulin and 1
μg/ml hydrocortisone, and HUVEC cells were cultured in M199 medium with 20% FBS,
20 μg/ml ECGS, and 50 μg/ml heparin. All media were supplemented with 100 units/ml
penicillin, 100 μg/ml streptomycin, and 2 mM glutamine. All cells were cultured in a
humidified atmosphere of 95% air and 5% CO2 at 37C.
Western blotting
Samples separated by SDS-PAGE were transferred to nitrocellulose membranes. After
being blocked in 5% bovine serum albumin (w/v) at room temperature for 1 hr, the
membranes were rinsed and incubated at 4°C overnight with primary anti-ARF6 antibody
(1:1000). The membranes were then washed and incubated with secondary antibody
(1:2000) at room temperature for 1 hr, developed with chemiluminescence ECL reagent
(LumiGold, SignaGen) and exposed to Hyperfilm MP (GE Healthcare).
Preparation of DM-PIT-1-loaded micelles
1,2-Disteratoyl-sn-glycero-3-phosphoethanolamine-N-[Methoxy(polyethyleneglycol)-2000] (PEG-PE) was supplied in chloroform (10 mg/ml) and stored at -80°C. A
stock solution of DM-PIT-1 (0.5 mg/ml) was prepared by dissolving 20 mg DM-PIT-1 in
40 ml acetonitrile and stored at 4°C. 2 ml of DM-PIT-1 from stock solution was added to
2.5 ml chloroform solution of PEG-PE. The organic solvents were removed by the rotary
evaporation to form a thin film of drug/micelle mixture. This film was further dried under
high vacuum overnight to remove any remaining traces of the solvent. To form micelles,
the film was rehydrated in a 10 mM HEPES buffer, pH 7.4 and sonicated for 5 min. The
non-incorporated, precipitated DM-PIT-1 was removed by filtration through a 0.22 μm
filter (Fisher Scientific). The amount of DM-PIT-1 was determined by isocratic reversephase HPLC (Hitachi, Elite La Chrome) equipped with photodiode-array detector (L2455). The chromatographic separation was performed on a C-18 column (5 μm, 4.6×250
mm, Hichrom, CA). Isocratic elution was performed with a mobile phase consisted of
acetonitrile and water (70:30) containing 0.1% formic acid (v/v). The flow rate was 1
ml/min and the total run time was 10 min. Sample injection (10 or 20 μl) was performed
with autosampler (Model L-2200, Hitachi). DM-PIT-1 was detected by the UV
absorbance at 320 nm.
Drug content loading (%) was calculated as the weight of DM-PIT-1 in micelles
divided by the PEG-PE weight which was used in micelle preparation.
The mean size of micellar preparations was measured by the dynamic light
scattering (DLS) with a scattering angle of 90° at 25°C using a N4 Plus Submicron
Particle System (Coulter Corporation, Miami). The micelle suspensions were diluted with
a 10 mM HBS, pH 7.4 until a concentration providing a light scattering intensity of 5×104
to 1×106 count was achieved. The measurements were done in triplicate.
Immunohistochemistry
Tumor tissues were fixed in phosphate-buffered formalin, embed in paraffin, cut in 4
μm thickness, and applied to slides. The slides were deparaffinized in xylenes using three
changes for 5 min each, and hydrated gradually through graded alcohols: 100% ethanol
twice for 10 min each, 95% ethanol twice for 10 min each, and then deionized water for 1
min with stirring. For antigen unmasking, slides were placed in a container, covered with
10 mM sodium citrate buffer, pH 6.0, and heated in a convection steamer for 1 hr. The
slides were washed in deionized water three times for 2 min each, blocked with 5%
normal goat blocking serum for 30 min, incubated with goat anti-CD31 primary antibody
for 1 hr, and incubated with an Alexa Fluor 488-conjugated secondary antibody for 30
min, and then incubated with Hoechst for 10 min. The slides were analyzed and
photographed using a fluorescent microscope.
Supplementary Figure Legends
Figure S1. PIT-1 inhibits lamellipodia formation
(A,B) PITs inhibit lamellipodia formation in cancer cells. SUM159 cells were serum
starved and treated with compounds for 2 hr, followed by PDGF (100 ng/ml) stimulation
for 15 min. Then cells were stained and analyzed using fluorescent microscopy. The
representative images are shown (A, the stress fibers and lamellipodia are indicated by
arrowheads). The number of cells with significant lamellipodia was counted in five
random fields and the percentage was calculated (B).
(C) PITs induces cellular morphological alterations. SUM159 cells were incubated with
compounds for 2 hr, followed by staining and analysis using fluorescent microscopy. The
representative fluorescent images are shown.
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P<0.01 compared with control group, **P<0.01 compared with PDGF group.
Figure S2. PIT-1 inhibits cancer cell migration and invasion at non-cytotoxic
concentrations
(A) PIT-1 inhibits migration of different cancer cell lines in a transwell assay.
(B) PIT-1 treatment under the migration and invasion experimental conditions has no
significant effect on the viability of SUM159 cells evaluated using ATP assay.
(C) PIT-1 at 6.25 and 12.5 μM has no significant effect on the viability of SUM159 cells
in 48 hr evaluated using ATP assay.
(D) PIT-3 and PIT-4 have much weaker inhibition on cancer cell migration in a wound
healing assay.
(E) PIT-1 inhibits invasion of different cancer cell lines through the matrigel-coated
transwell membrane.
(F) PIT-1 treatment under the migration experimental conditions has no significant effect
on HUVEC cell survival evaluated using ATP assay.
(G) DM-PIT-1 exhibits a stronger inhibition on cancer cell migration both in a transwell
and wound healing assay. SUM159 cells were treated with PIT-1 or DM-PIT-1 (3.125100 μM) for 8 hr, and the inhibition of cell migration was analyzed as described above.
(H) PIT-1 synergizes with SecinH3 in suppressing cancer cell migration. Wounds were
generated in SUM159 cell monolayer, followed by treatment with PIT-1 (12.5, 25 μM) or
SecinH3 (12.5, 25 μM) or PIT-1 plus SecinH3 for 8 hr. The width of wounded cell
monolayer was measured in five random fields and the inhibition was calculated.
(I) Akt inhibitor VIII has much weaker inhibition on cancer cell migration compared with
PIT-1 and LY294002 in a wound healing assay.
(J) Akt inhibitor VIII and LY294002, but not PIT-1 significantly inhibit Akt
phosphorylation at concentrations used in wound healing assay.
Figure S3. PIT-2, but not PIT-1i-1 inhibits tube formation with HUVEC
(A-C) HUVEC cells were seeded on matrigel and treated with PIT-2 (A) or PIT-1i-1 (B)
at 3.125-100 μM for 8 hr. The representative images are shown (A,B), and the number of
closed tubes was counted in five random fields from each well, and the inhibition was
calculated (C).
(D) The body weights of mice after 5-day administration of DM-PIT-1-M or plain
micelles show no significant changes in B16-F10 pulmonary metastasis experiment.
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P>0.05 compared with control group.
(E) PITs inhibit Rac1 activation. SUM159 cells were serum starved and treated with
compounds for 2 hr, followed by stimulation with 100 ng/ml PDGF for 15 min. Rac1
activation assay was performed as described in the Methods section.