U.S. patent application number 14/622230 was filed with the patent office on 2015-07-09 for method of potentiating an antitumor immune response.
The applicant listed for this patent is National Tsing Hua University. Invention is credited to Guan-Yu CHEN, Yu-Chen HU, Hsing-Yu TUAN.
Application Number | 20150190423 14/622230 |
Document ID | / |
Family ID | 52005659 |
Filed Date | 2015-07-09 |
United States Patent
Application |
20150190423 |
Kind Code |
A1 |
HU; Yu-Chen ; et
al. |
July 9, 2015 |
METHOD OF POTENTIATING AN ANTITUMOR IMMUNE RESPONSE
Abstract
A method of inducing autophagy in a cell is achieved by
contacting the cell with graphene oxide (GO) in an amount effective
to induce autophagy in the cell, wherein the cell expresses at
least one of TLR-4 (Toll-like receptor 4) and TLR-9 (Toll-like
receptor 9). Differences between autophagy triggered by GO and
other conventional agonists such as rapamycin have been observed.
GO may activate autophagy in some cells that may not be triggered
by rapamycin. The cell reveals no apparent apoptosis after
treatment of the graphene oxide. A method of method of potentiating
an antitumor immune response is also herein provided.
Inventors: |
HU; Yu-Chen; (Hsinchu,
TW) ; CHEN; Guan-Yu; (Hsinchu, TW) ; TUAN;
Hsing-Yu; (Hsinchu, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
National Tsing Hua University |
Hsinchu |
|
TW |
|
|
Family ID: |
52005659 |
Appl. No.: |
14/622230 |
Filed: |
February 13, 2015 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13913716 |
Jun 10, 2013 |
|
|
|
14622230 |
|
|
|
|
Current U.S.
Class: |
424/489 ;
424/278.1 |
Current CPC
Class: |
A61K 31/194 20130101;
A61K 9/14 20130101; A61K 33/00 20130101; A61K 9/0019 20130101 |
International
Class: |
A61K 33/00 20060101
A61K033/00; A61K 9/00 20060101 A61K009/00; A61K 9/14 20060101
A61K009/14 |
Claims
1. A method of potentiating an antitumor immune response in a
subject, comprising: administering an effective amount of graphene
oxide to the subject.
2. The method as claimed in claim 1, wherein the subject is
diagnosed with tumor cells expressing both of TLR-4 and TLR-9.
3. The method as claimed in claim 1, wherein the antitumor immune
response comprises cell death, apoptosis, autophagy induction and
infiltration of immune cells within a tumor.
4. The method as claimed in claim 1, wherein the particle sizes of
the graphene oxide range from 100 nm to 3 .mu.m.
5. The method as claimed in claim 1, wherein the particle sizes of
the graphene oxide range from 100-800 nm.
6. The method as claimed in claim 1, wherein the concentration of
the graphene oxide is greater than or equal to 5 .mu.M.
7. The method as claimed in claim 1, wherein the concentration of
the graphene oxide is greater than or equal to 100 .mu.M.
8. The method as claimed in claim 1, wherein the graphene oxide is
an active ingredient.
9. The method as claimed in claim 1, wherein the graphene oxide is
administered via intratumoral injection.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a method of inducing
autophagy in a cell, particularly to a method of inducing autophagy
in a cell by activating Toll-like receptors.
[0003] 2. Description of the Prior Art
[0004] Graphene and its oxidized form, graphene oxide (GO), have
drawn intense attention in recent years for biological and medical
applications. The surface of GO contains hydrophilic
oxygen-containing functional groups (i.e. hydroxyl, epoxyl and
carboxyl tails) on the basal plane and edges, rendering GO amenable
to stable dispersion in water and functionalization. These
attributes have prompted the use of GO for bioimaging, cellular
probing, cellular growth and differentiation, gene and drug
delivery and photothermal therapy. These burgeoning applications in
biomedicine entail the need to evaluate the in vitro and in vivo
safety of GO.
[0005] Autophagy is a process that degrades intracellular
components in response to stressful conditions (e.g. starvation and
infection) and is linked to cellular processes as diverse as cell
survival, cell death, pathogen clearance and antigen presentation.
Autophagy involves the formation of double-membraned vesicles
termed autophagosomes, which sequester cytoplasm and organelles and
then fuse with lysosomes to form autolysosomes, thus degrading the
contents of the vacuole. Autophagy is negatively controlled by mTOR
(mammalian target of rapamycin) complex 1 (mTORC1) and inhibition
of mTORC1 kinase activity initiates the formation of autophagosome
that comprises a complex consisting of Beclin 1 and other factors.
The autophagosome formation also involves the conversion of
microtubule-associated protein light chain 3 (LC3-I) to the
lipidated form LC3-II, consequently conversion from LC3-I to LC3-II
is a common indicator of autophagy.
[0006] Toll-like receptors (TLRs) are important receptors for the
detection of microbial antigens and subsequent induction of innate
immune responses. Among the TLRs, TLR2 recognizes bacterial
lipoproteins while TLR3 detects virus-derived dsRNA. TLR4
recognizes lipopolysaccharides (LPS) and TLR5 recognizes bacterial
flagellin. TLR7 mediates recognition of viral ssRNA while TLR9
senses unmethylated DNA with CpG motifs derived from bacteria and
viruses. Upon engagement with cognate ligands, the TLRs transduce
signals by first recruiting adaptor proteins including myeloid
differentiating factor 88 (MyD88) and TIR domain-containing adaptor
inducing IFN-beta (TRIF), followed by activation of downstream
signaling proteins such as TRAF6 and NF-.kappa.B, eventually
resulting in various cellular responses including secretion of
cytokines and interferons (IFNs).
[0007] The connection between autophagy and TLRs was discovered in
2007 as it was found that TLRs signaling in macrophages links the
autophagy pathway to phagocytosis and TLR4 stimulation enhances the
autophagic elimination of phagocytosed mycobacteria in macrophages.
Ensuing studies further reported that TLR2, TLR3 and TLR7 play
roles in autophagy induction. To date the precise mechanisms
regulating the TLRs-elicited autophagy remain to be established
although agonists stimulating TLR2, TLR3, TLR4 and TLR7 were shown
to trigger autophagy.
SUMMARY OF THE INVENTION
[0008] The present invention is directed to provide a new mechanism
by which cells respond to nanomaterials and underscores the
importance of future safety evaluation of nanomaterials.
[0009] According to an embodiment, A method of inducing autophagy
in a cell is achieved by contacting the cell with graphene oxide
(GO) in an amount effective to induce autophagy in the cell,
wherein the cell expresses at least one of TLR-4 (Toll-like
receptor 4) and TLR-9 (Toll-like receptor 9).
[0010] According to another embodiment, a method of activating a
Toll-like receptor in a cell is achieved by contacting the cell
with graphene oxide in an amount effective to activate a at least
one of TLR-2 (Toll-like receptor 2), TLR-4 (Toll-like receptor 4),
TLR-7 (Toll-like receptor 7) and TLR-9 (Toll-like receptor 9) in
the cell, whereby at least one of TLR-2, TLR-4, TLR-7 and TLR-9 are
activated in the cell.
[0011] Other advantages of the present invention will become
apparent from the following descriptions taken in conjunction with
the accompanying drawings wherein certain embodiments of the
present invention are set forth by way of illustration and
examples.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The foregoing aspects and many of the accompanying
advantages of this invention will become more readily appreciated
as the same becomes better understood by reference to the following
detailed descriptions, when taken in conjunction with the
accompanying drawings, wherein:
[0013] FIG. 1A is a diagram illustrating characteristic peaks of
oxygen-containing groups for both large and small GO;
[0014] FIG. 1B-1C are Atomic force microscopy (AFM) images showing
significant difference of lateral dimensions between large and
small GO;
[0015] FIG. 1D is a diagram illustrating hydrodynamic diameters of
large and small GO;
[0016] FIG. 2A is a microscopy illustrating the formation of small
vacuoles inside the cells induced by GO at 24 h
post-incubation;
[0017] FIG. 2B-2C are transmission electron microscopy
demonstrating that GO5 evoked the appearance of some autophagic
vacuoles (AV) while GO100 and LPS triggered more prominent AV;
[0018] FIG. 3A are Immunofluorescence microscopy illustrating
Beclin 1 and LC3 activation of GO5 and GO100;
[0019] FIG. 3B are Quantitative analysis of immunofluorescence
micrographs showing cells with LC3+ dots;
[0020] FIG. 3C are Western blot illustrating the expression of
Beclin 1, LC3-I and LC3-II;
[0021] FIG. 4A-4E are ELISA analyses depicting that treatment of
macrophage cells with small GO at 100 .mu.g/ml significantly
induced the production of IL-2, IL-10, IFN-.gamma. and TNF-.alpha.
but not IFN-.beta. when compared with the untreated cells;
[0022] FIG. 5A are RT-PCR analyses illustrating upregulated the
transcription of TLRs;
[0023] FIG. 5B illustrates expression of TLRs by immunofluorescence
labeling coupled with flow cytometry;
[0024] FIG. 5C-5D are immunofluorescence microscopy illustrating
upregulation of TLR4 and TLR9;
[0025] FIG. 6A-6B are flow cytometry outcomes illustrating
upregulated the expression of MyD88 and TRAF6;
[0026] FIG. 6C-6D are immunofluorescence microscopy illustrating
upregulated the expression of MyD88 and TRAF6;
[0027] FIG. 6E is immunofluorescence microscopy illustrating the
activation and nuclear translocation of phosphorylated
NF-.kappa.B;
[0028] FIG. 7A-7B are RT-PCR outcomes of macrophage cells treated
with siRNA specific for TLR4, TLR9, MyD88, TRIF or TRAF6;
[0029] FIG. 7C is ELISA analysis depicting that silencing TLR4,
TLR9, MyD88 and TRAF6 attenuated the IFN-.gamma. and TNF-.alpha.
expression;
[0030] FIG. 7D is Immunofluorescence microscopy further
illustrating that silencing TLR4, TLR9, TRIF, MyD88 and TRAF6
abolished the GO-induced formation of Beclin 1 aggregates;
[0031] FIG. 7E-7F are immunofluorescence microscopy and
quantitative analysis illustrating inhibition of GO-induced LC3+
aggregates also occurred after silencing TLR4, TLR9, TRIF, MyD88
and TRAF6;
[0032] FIG. 7G is Western blot illustrating that suppression of
Beclin 1 expression and LC3-I conversion to LC3-II by gene
silencing;
[0033] FIG. 8A is immunofluorescence microscopy illustrating
responsiveness of cancer cells to GO;
[0034] FIG. 8B is transmission electron microscopy (TEM) revealing
the formation of autophagic vacuoles and engulfment of GO
nanosheets;
[0035] FIG. 8C is a diagram illustrating GO-induced autophagy is
dose-dependent in CT26 cells;
[0036] FIG. 8D is a diagram illustrating cell viability of GO50 and
GO100 treated cell by MTT assays;
[0037] FIG. 8E is a diagram illustrating that no apparent apoptosis
or necrosis in CT26 cells even for GO50 and GO100 by PE Annexin V
apoptosis analysis;
[0038] FIG. 8F is immunofluorescence microscopy illustrating
responsiveness of cancer cells to GO;
[0039] FIG. 9A is a diagram illustrating that GO50 significantly
provoked the production of TNF-.alpha. and IL-1.beta. when compared
with the untreated cells;
[0040] FIG. 9B is immunofluorescence microscopy demonstrating that
GO50, but not rapamycin, simultaneously activated TLR-4, TLR-9,
MyD88 and TRAF6 and enhanced the phosphorylation of NF-.kappa.B and
IRF7;
[0041] FIG. 9C is a diagram illustrating GO was taken up by CT26
cells via phagocytosis;
[0042] FIG. 9D is a diagram illustrating that rapamycin suppressed
the phosphorylation of Akt and mTOR and GO50 enhanced the
phosphorylation of Akt and mTOR;
[0043] FIG. 9E is a diagram illustrating that the phosphorylation
of Akt and mTOR was barely altered regardless of TLR-4/9 knockdown
prior to GO50 treatment;
[0044] FIG. 9F-9G are immunofluorescence microscopy and diagram
illustrating that siTLR-4 and siTLR-9 significantly mitigated the
GO-induced activation of LC3 and Beclin 1;
[0045] FIG. 10A is a diagram illustrating transfected cells with
siRNA for atg5 and atg7 to knockdown the GO-induced autophagy;
[0046] FIG. 10B-10D are diagrams illustrating inhibition of
GO-induced autophagy by siATG5 or siATG7 neither abolished the
GO-induced production of TNF-.alpha. and IL-1.beta. (FIG. 10B) nor
affected the expression of TLR-4 and TLR-9 upon GO50 treatment
(FIG. 10C-10D);
[0047] FIG. 11A-11B are diagrams illustrating that GO alone
significantly suppressed the tumor progression without considerably
compromising the body weight;
[0048] FIG. 11C is a Live/Dead assay diagram illustrating analysis
of the tumor sections 5 days after GO injection;
[0049] FIG. 11D is a diagram illustrating that no apparent
apoptosis or necrosis was observed in GO50;
[0050] FIG. 11E is a diagram illustrating LC3.sup.+ aggregation in
GO50; and
[0051] FIG. 11F is a diagram illustrating that GO50 remarkably
potentiated the infiltration of macrophage, dendritic cells (DCs),
CD4.sup.+ and CD8.sup.+ T cells into the tumor bed.
DESCRIPTION OF THE PREFERRED EMBODIMENT
[0052] The present invention demonstrated that treatment of cells
with GO simultaneously triggers autophagy and mainly
TLR4/TLR9-regulated inflammatory responses.
[0053] In one embodiment, the particle sizes of the graphene oxide
may range from 100 nm to 3 .mu.m, preferably 100 nm to 800 nm and
average around 450 nm. The concentration of the graphene oxide may
be greater than or equal to 5 .mu.M, preferably greater than or
equal to 50 .mu.M or 100 .mu.M.
[0054] Autophagy triggered by GO have been observed in various
types of cells such as cancer cells and immune cells. In one
preferred embodiment, cancer cells may include an ovarian cancer
cell (SKOV3), a brain can cell (ALTS1C1), a prostate cancer cell
(Tramp C1), a cervical cancer cell (HeLa), a lung cancer cell
(A549), a liver cancer cell (Mahlavu) or a colon cancer cell
(CT26). Immune cells may include primary immune cells such as
macrophages.
[0055] In addition, differences between autophagy triggered by GO
and other conventional agonists such as rapamycin have been
observed. GO may activate autophagy in some cells that may not be
triggered by rapamycin. Some cells are likely damaged by rapamycin
in comparison to GO treatment. The cell reveals no apparent
apoptosis or necrosis after treatment of the graphene oxide.
Furthermore, the autophagy induced by GO may be more than 40% of
the cell. In one preferred embodiment, autophagy may be induced in
essentially 80% or more of the cell.
[0056] The autophagy presented by the present invention was at
least partly regulated by the TLRs pathway. Very importantly, TLRs
are well known detectors for various biological molecules, but
their sensing of non-living nanomaterials such as GO has yet to be
reported. Neither has any study documented that nanomaterials can
induce autophagy via the regulation of TLRs. This present invention
thus presents a new mechanism by which cells respond to
nanomaterials and underscores the importance of future safety
evaluation of nanomaterials.
[0057] The detailed explanation of the present invention is
described as follows. The described preferred embodiments are
presented for purposes of illustrations and description, and they
are not intended to limit the scope of the present invention.
[0058] Reference of Chen et al. (Biomaterials 33 (2012) 6559-6569,
hence abbreviated as Reference) is herein incorporated by reference
in its entirety.
Preparation and Characterization of GO
[0059] Large GO with a size of .apprxeq.2.4 .mu.m was prepared from
natural graphite (Bay Carbon, SP-1, average particle size
.apprxeq.30 .mu.m) by the modified Hummers method as described
previously [20] and dispersed in water. The solution was
centrifuged (7,200.times.g for 5 min) to remove unexfoliated GO and
byproducts and centrifuged again (400.times.g for 15 min) to remove
broken fragments and debris. The pellet was dried under vacuum
overnight to yield the large GO, weighed on a Sartorius SE2
ultra-micro balance with 0.1 .mu.g resolution and dissolved in
deionized water to a final concentration of 250 .mu.g/ml. Small GO
with a size of .apprxeq.350 .mu.m was prepared via tip sonication
(Misonix Sonicator 3000) of the large GO solution in an ice bath at
a power of 30 W for 1 h, filtered through a 0.45 .mu.m syringe
filter (Sartorius Stedim Biotech) and dried under vacuum overnight.
The small GO was weighed and dissolved in water to a desired
concentration.
[0060] The surface morphology of GO was characterized with an
atomic force microscope (AFM, XE-70, Park System) in tapping mode
using the aluminum coating silicon probe (frequency 300 kHz, spring
constants 40 N/m, scanning rate 1 Hz), under ambient conditions and
scanning line of 512. High-resolution X-ray photoelectron
spectroscopy (HRXPS) and attenuated total reflectance HRXPS were
performed on a Kratos Axis Ultra DLD using a focused monochromatic
Al X-ray source (1486.6 eV). The Fourier transform infrared
(ATR-FTIR) spectra of GO were recorded using a Perkin-Elmer
Spectrum RXI FTIR spectrometer with 2 cm.sup.-1 resolution and 32
scans, and the background was collected in the absence of samples.
The size distribution of GO was characterized by using Dynamic
Light Scattering (380 ZLS, Nicomp, USA) from Particle Sizing
Systems at room temperature.
Cell Culture and Treatment with GO
[0061] The mouse macrophage cell line RAW264.7 was maintained in
Dulbecco's modified Eagles medium (DMEM, Gibco) containing 10%
fetal bovine serum (FBS, Gibco) and subcultured upon 70-80%
confluency. For GO treatment, the cells were seeded to 6-well
plates (3.times.10.sup.5 cells/cm.sup.2) overnight and cultured
using the medium supplemented with GO at final concentrations of 5
or 100 .mu.g/ml for 24 h. In parallel, the cells were treated with
LPS (10 .mu.g/ml, Sigma) for 24 h as the positive control. After
the treatment, the cell morphology and vacuoles were observed under
the phase contrast microscope.
Transmission Electron Microscopy (TEM)
[0062] The cells were harvested, centrifuged (215.times.g, 10 min),
washed with cold PBS and fixed with 2.5% glutaraldehyde (in 0.2 M
sodium cacodylate, pH 7.4). The samples were then fixed in 1%
OsO.sub.4 for 1 h at 4.degree. C., dehydrated with increasing
concentrations of ethanol, embedded in spur resin and sectioned.
The ultrathin sections were stained with uranyl acetate and
observed under the TEM.
Immunofluorescence Microscopy
[0063] The cells were fixed and permeabilized as described
previously [4], followed by extensive washing and primary antibody
staining (1:100 dilution) for 1 h at 4.degree. C. in the dark. The
primary antibody was specific for LC3 (4108, Cell Signaling
Technology), Beclin 1 (ab55878, Abcam), TLR4 (14-9924,
eBioscience), TLR9 (ab17236, abcam), MyD88 (ab2068, abcam), TRAF6
(ab33915, abcam), phosphorylated NF-.kappa.B (3033, Cell Signaling
Technology) or IRF3 (sc-15991, Santa Cruz Biotechnology). After
washing, the cells were incubated with the goat anti-mouse antibody
conjugated with Alexa 488 (for TLR9, Invitrogen), goat anti-rabbit
antibody conjugated with Alexa 488 (for LC3, MyD88, TRAF6 and
NF-.kappa.B, Invitrogen) or donkey anti-goat IgG conjugated with
Dylight 488 (for IRF3, Jackson ImmunoResearch) for 1 h at 4.degree.
C. in the dark. After washing, the cells were counterstained with
4,6-diamidino-2-phenylindole (DAPI, Vector Labs) and visualized
with a confocal microscope (Nikon TE2000 equipped with the confocal
upgrade laser kit). Fifty to one hundred cells in the images for
LC3 were counted for quantification of LC3+ cells.
ELISA and Western Blot
[0064] At 24 h post-treatment, the supernatant was collected from
the GO-treated cell culture and analyzed using ELISA kits specific
for mouse IL-2, IL-10, TNF-.alpha., IFN-.beta. and IFN-.gamma.. The
cells were lysed for Western blot using primary antibodies (1:1000
dilution) specific for LC3, Beclin 1 or .beta.-actin (A-2066,
Sigma) and the secondary antibody was HRP-conjugated IgG (1:5000
dilution, Amersham Biosciences). The images were developed using
the GeneGnome HR scanner (Syngene).
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
[0065] Total RNA was extracted from the cells using the
NucleoSpin.RTM. RNA II purification kit (Clontech) and reverse
transcribed to cDNA using the MMLV Reverse Transcriptase 1st-Strand
cDNA Synthesis Kit (Epicentre Biotechnologies). The RT-PCR
reactions were performed using Taq DNA polymerase (Promega) in the
P.times.2 Thermal Cycler (Thermo Electron) under the condition of
30 s at 95.degree. C., 45 s at 60.degree. C. and 30 s at 72.degree.
C., and the amplicons were subjected to 2% agarose gel
electrophoresis. For TLRs transcription analysis, the cDNA was
amplified using the Murine TLR RT-Primers (Invivogen).
Flow Cytometry
[0066] The cells were fixed and permeabilized with 4% formaldehyde
and 0.5% Tween-20. After washing, the cells were incubated with the
primary antibody (1:100 dilution) for 1 h at 4.degree. C. in the
dark. For TLR2 and TLR4 detection, the primary antibody was Alexa
488-conjugated MAb specific for mouse TLR2 (53-9024, eBioscience)
or PE-conjugated MAb specific for mouse TLR4 (12-9924,
eBioscience). For TLR7, TLR9, MyD88 and TRAF6 detection, the cells
were incubated with the primary antibody specific for mouse TLR7
(ab45371, abcam), TLR9 (ab17236, abcam), MyD88 (ab2068, Abcam) or
TRAF6 (ab33915, Abcam) and then incubated with Alexa 488-conjugated
goat anti-rabbit (for TLR7, MyD88 and TRAF6) or goat anti-mouse
(for TLR9) IgG for 1 h at 4.degree. C. in the dark. After washing,
the cells were collected for flow cytometry (FACSCalibur, BD
Biosciences) analyses.
Gene Knockdown by Small Interfering RNA (siRNA)
[0067] To knockdown specific genes, macrophages cells were
transfected with 5 .mu.g of scramble siRNA (SC-36869, Santa Cruz
Biotechnology) or siRNA specific for TLR4, TLR9, MyD88, TRAF6 or
TRIF (Santa Cruz Biotechnology). At 48 h post-transfection, cells
were treated with GO or LPS as described above. The supernatant was
collected 24 h later for ELISA and the cells were harvested for
immunofluorescence microscopy and Western blot. Statistical
analysis
[0068] All data represented the mean.+-.standard deviation of at
least 3 independent culture experiments. The data were
statistically analyzed by one-way ANOVA. p<0.05 was considered
significant.
Example 1
Preparation and Characterization of Large and Small GO
Nanosheets
[0069] Large-size GO was prepared from natural graphite by the
modified Hummers method while small-size GO was obtained by
sonicating large GO into smaller pieces via tip sonication. Atomic
force microscopy (AFM) images showed significant difference of
lateral dimensions between large and small GO (FIG. 1B-1C). The
thicknesses of both large and small GO measured .apprxeq.1.0-1.2
nm, which agreed with the GO thickness reported previously and
indicated the formation of single-layer GO. The GO was thicker than
graphene (.apprxeq.0.34 nm) due to the surface functional groups.
The effective hydrodynamic diameters of large and small GO were
.apprxeq.2.4 .mu.m and .apprxeq.350 nm, respectively, as measured
by Dynamic Light Scattering (FIG. 1D). The surface states of large
and small GO were identical as demonstrated by high-resolution C 1
s XPS spectra (not illustrated), in which the 4 peaks centering at
285, 286.4, 287.1 and 289.0 eV corresponded to C.dbd.C/C.dbd.C in
the non-oxygenated aromatic rings, C--O (epoxy and alkoxy),
C.dbd.O, and O.dbd.C--O groups, respectively. The FTIR spectra
(FIG. 1A) also delineated the same characteristic peaks of
oxygen-containing groups for both large and small GO.
Example 2
GO Nanosheets Induced Autophagy in a Dose-Dependent Manner
[0070] To examine how GO nanosheets influenced the macrophage,
RAW264.7 cells were incubated with small GO at either 5 .mu.g/ml
(designated as GO5 group) or 100 .mu.g/ml (designated as GO100
group) for 24 h. In comparison with the untreated control, GO5
induced the formation of small vacuoles inside the cells at 24 h
post-incubation (FIG. 2A) but did not cause apparent cell death
(Fig. S1A of reference). Increasing the small GO concentration to
100 .mu.g/ml (GO100 group) gave rise to more evident vacuole
formation (FIG. 2A) and significant cell death (Fig. S1A of
reference), which were also observed in the LPS-treated cells (10
.mu.g/ml). However, GO treatment did not elicit discernible
apoptosis as illustrated by TUNEL assays (Fig. S1B of reference).
Similar vacuoles were also observed in the cells treated with large
GO (Fig. S2A of reference).
[0071] Since the GO-induced vacuoles were observed in cells treated
with LPS, a ligand that induces both autophagy and TLR pathway, we
surmised that GO triggered autophagy. Indeed, the transmission
electron microscopy demonstrated that GO5 evoked the appearance of
some autophagic vacuoles (AV) while GO100 and LPS triggered more
prominent AV (FIG. 2B). Notably, electron-dense materials within
the AV were scarcely present in the LPS group but were abundant in
the GO100 group (FIG. 2C), presumably due to the sequestered GO
nanosheets.
[0072] Beclin 1 and LC3 are two key proteins associated with the
autophagy pathway and are common indicators of autophagy induction.
LC3 is normally present diffusely in the cytosol but upon autophagy
is converted from LC3-I (18 kD) to LC3-II (16 kD), accumulates on
the autophagosome membrane and appears as dots. Immunofluorescence
microscopy for Beclin 1 and LC3 (FIG. 3A) showed that GO5 and GO100
provoked the appearance of many green dots, which were also
observed in the LPS-treated cells but not in the untreated cells.
The formation of such large aggregate dots similarly occurred in
macrophages treated with large GO (100 .mu.g/ml, Fig. S2B of
reference) or treated with dsDNA, and took place in stem cells
treated with quantum dots. Quantitative analysis of
immunofluorescence micrographs (FIG. 3B) verified that GO100
triggered a significantly higher percentage of cells with LC3+ dots
than the untreated, GO5 and LPS groups. Besides, pre-treatment of
cells with the autophagy inhibitor 3-methyl adenine (3-MA)
diminished the GO100-triggered formation of LC3+ aggregate dots
(Fig. S3 of reference). Furthermore, Western blot (FIG. 3C) not
only attested that small GO provoked the expression of both Beclin
1 and LC3, but also revealed the emergence of LC3-II, thus
confirming the LC3 ligation to autophagosome. These data altogether
proved the induction of autophagy in macrophages by large and small
GO in a concentration-dependent manner.
GO Treatment of Macrophage Elicited the Cytokine Expression and
TLR4/TLR9 Signaling
[0073] Since the interplay between autophagy and TLRs signaling was
recently revealed, we were inspired to explore whether GO elicited
TLRs-associated inflammatory responses. ELISA analysis (FIG. 4A-4E)
depicted that treatment of macrophage cells with small GO at 100
.mu.g/ml significantly induced the production of IL-2, IL-10,
IFN-.gamma. and TNF-.alpha. when compared with the untreated cells
Such cytokine response was GO concentration-dependent and concurred
with the cytokine secretion triggered by LPS. However, treatments
of macrophage with small GO at 5 and 100 .mu.g/ml did not elicit
the secretion of IFN-.beta..
[0074] Conversely, GO5 and GO100 evidently upregulated the
transcription of TLR9 but barely triggered other TLRs genes, as
depicted by RT-PCR analyses (FIG. 5A). Since TLR2, TLR4 and TLR7
also induce autophagy, we further assayed the upregulation of these
TLRs in addition to TLR9, by immunofluorescence labeling coupled
with flow cytometry. FIG. 5B reveals that GO100 only marginally
induced the expression of TLR2 and TLR7, but pronouncedly
upregulated the TLR4 and TLR9 expression. The upregulation of TLR4
and TLR9 by GO100 was further confirmed by immunofluorescence
microscopy (FIG. 5C-5D). Treatment of macrophage cells with large
GO at 100 .mu.g/ml likewise provoked remarkable upregulation of
TLR4 and TLR9 (Fig. S4 of reference).
[0075] Since only the expression of TLR4 and TLR9 was markedly
elicited by GO, we next examined the roles of TLR4 and TLR9
pathways on the inflammatory response. The TLR4 pathway signals
through either TRIF or MyD88. The TRIF-dependent pathway results in
activation and nuclear translocation of IRF3, thereby triggering
the secretion of IFN-.alpha./.beta.. However, GO5 and GO100 neither
evoked nuclear translocation of IRF3 (Fig. S5 of reference) nor
elicited IFN-.beta. expression (FIG. 4), thus indicating the
dispensable role of IRF3.
[0076] Conversely, TLR4 signaling through MyD88 leads to the
formation of MyD88/IRAK4/TRAF6 signalsome, nuclear translocation of
phosphorylated NF-.kappa.B and subsequent cytokine expression. TLR9
stimulation recruits MyD88 and results in the formation of
MyD88/IRAK4/TRAF6/TRAF3 complex, which relays signals either
through IRF7 for IFN-.alpha./.beta. secretion, or through
NF-.kappa.B for cytokine expression. As demonstrated by the flow
cytometry (FIG. 6A-6B) and immunofluorescence microscopy (FIG.
6C-6D), GO100 upregulated the expression of MyD88 and TRAF6 and
formation of aggregates indicative of signalosome complex.
Concomitantly, GO100 led to the activation and nuclear
translocation of phosphorylated NF-.kappa.B (FIG. 6E). In
conjunction with the cytokine expression downstream of NF-.kappa.B
signaling (FIG. 4), FIGS. 5 and 6 collectively suggested that GO100
activated the TLR4 and TLR9 signaling cascades.
Inhibition of TLR4/TLR9 Pathways Mitigated the GO-Induced Cytokine
Response and Autophagy
[0077] To confirm the roles of individual signaling mediators on
the cytokine response, the macrophage cells were treated with siRNA
specific for TLR4, TLR9, MyD88, TRIF or TRAF6. Following the
silencing as confirmed by RT-PCR (FIG. 7A-7B), the macrophages
cells were incubated with GO100 as in FIG. 4. ELISA analysis (FIG.
7C) depicted that silencing TLR4, TLR9, MyD88 and TRAF6 attenuated
the IFN-.gamma. and TNF-.alpha. expression with statistical
significance (p<0.05) when compared with the control treated
with scramble siRNA, thereby attesting the roles of TLR4, TLR9 and
their downstream MyD88-dependent pathway in the GO-triggered
inflammatory response. In contrast, silencing TRIF did not
significantly diminish the IFN-.gamma. and TNF-.alpha. expression,
nor was IFN-.beta. expression attenuated by silencing these genes
(FIG. 7C), thereby suggesting the dispensable role of
TRIF-dependent pathway in the GO-triggered innate responses.
[0078] Immunofluorescence microscopy (FIG. 7D) further illustrated
that silencing TLR4, TLR9, TRIF, MyD88 and TRAF6 abolished the
GO-induced formation of Beclin 1 aggregates. Similar inhibition of
GO-induced LC3+ aggregates also occurred after silencing TLR4,
TLR9, TRIF, MyD88 and TRAF6, as confirmed by immunofluorescence
microscopy (FIG. 7E) and quantitative analysis (FIG. 7F). The
Western blot (FIG. 7G) further confirmed that silencing these genes
suppressed Beclin 1 expression and LC3-I conversion to LC3-II.
These data altogether indicated that blockade of TLR4, TLR9 and
their downstream MyD88- and TRIF-dependent signaling could abrogate
the GO-induced autophagy.
Example 3
GO Induces Autophagy in Different Cancer Cells in a Dose-Dependent
Manner
[0079] To assess the responsiveness of cancer cells to GO, cells of
different cancer types including human ovarian carcinoma (SKOV-3),
murine astrocytoma (ALTS1C1), murine colon carcinoma (CT26) and
murine prostate adenocarcinoma (TRAMP-C1) were cultured in medium
supplemented with GO nanosheets (thickness<2 nm, lateral size
.apprxeq.450 nm in mean diameter). Immunofluorescence microscopy
revealed that GO at a concentration of 50 .mu.g/ml (GO50 group)
only induced evident autophagy in CT26 cells after 18 h (FIG. 8A),
as judged from the appearance of LC3.sup.+ punctate dots (which
indicates the formation of autophagosomes and hence autophagy).
Quantitative analysis of micrographs attested a significantly
higher percentage of CT26 cells containing LC3+ dots than other
cancer cells, indicating that the GO-induced autophagy is cell
type-dependent. Transmission electron microscopy (TEM) further
revealed the formation of autophagic vacuoles and engulfment of GO
nanosheets (FIG. 8B) while immunofluorescence microscopy
illustrated the activation of Beclin1 and p62, thereby confirming
the GO-induced autophagy at 50 .mu.g/ml in CT26 cells.
[0080] The GO-induced autophagy was also dose-dependent in CT26
cells (FIG. 8C). GO at 100 .mu.g/ml (GO100) also elicited apparent
autophagy in such cancer cells as SKOV-3, A549, mahlavu and HeLa
(FIG. 8F). In comparison with untreated cells, GO50 and GO100
resulted in reduced cell viability as judged from MTT assays (FIG.
8D), yet PE Annexin V apoptosis analysis (FIG. 8E) and
mitochondrial membrane potential analysis (not illustrated)
revealed no apparent apoptosis or necrosis in CT26 cells even for
GO50 and GO100.
GO Activates TLR-4/9 Pathways in CT26 Cells.
[0081] Owing to the findings that GO provokes both TLR-4 and TLR-9
signaling pathways in macrophage cells in vitro, we surmised that
GO also triggered TLR-4/9 cascades and their downstream cytokine
(e.g. TNF-.alpha. and IL-1.beta.) production in CT26 cells. Indeed,
GO50 significantly provoked the production of TNF-.alpha. and
IL-1.beta. when compared with the untreated cells (FIG. 9A). Flow
cytometry (not illustrated) and immunofluorescence microscopy (FIG.
9B) demonstrated that GO50, but not rapamycin, simultaneously
activated TLR-4, TLR-9, MyD88 and TRAF6 and enhanced the
phosphorylation of NF-.kappa.B and IRF7. Since MyD88, TRAF6 and
NF-.kappa.B are signaling mediators shared by TLR-4 and TLR-9
pathways while IRF7 mediates the TLR-9 cascade, these data proved
the elicitation of TLR-4 and TLR-9 pathways by GO50. However,
neither GO5 nor rapamycin apparently activated both pathways.
GO was Phagocytosed by CT26 Cells in a Way Related to TLR-4/9
Signaling.
[0082] TLR-4 is a receptor on the cell surface whereas TLR-9 is
produced in ER and translocates to endosome. To explore how GO
entered the cells to engage TLR-9, we treated CT26 cells with
FITC-conjugated beads as a marker of phagocytosis. Comparison of
the cells treated with beads only with the cells co-treated with
beads and GO50 (FIG. 9C) revealed that GO was taken up by CT26
cells via phagocytosis. To explore whether the phagocytosis was
associated with TLRs signaling, CT26 cells were transfected with
small interfering RNA (siRNA) specific for tlr4 (siTLR-4) or tlr9
(siTLR-9), which downregulated the expression of TLR-4 and TLR-9,
respectively, and significantly attenuated the GO-induced
production of TNF-.alpha. and IL-1.beta.. siTLR-4 and siTLR-9 also
markedly abrogated the phagocytosis of GO50 (FIG. 9C), suggesting
that TLR-4 and TLR-9 played a role in the uptake of GO.
GO-Induced TLR-4/9 Cascades were Independent of the mTOR
Pathway.
[0083] mTOR is a negative autophagy regulator, and repressing the
phosphorylation of mTOR and its upstream Akt can elicit autophagy.
Indeed, rapamycin suppressed the phosphorylation of Akt and mTOR
(FIG. 9D) and induced autophagy (FIG. 8C). However, GO50 enhanced
the phosphorylation of Akt and mTOR (FIG. 9D), suggesting that the
GO-induced autophagy proceeded through a pathway unrelated to mTOR.
Conversely, the phosphorylation of Akt and mTOR was barely altered
regardless of TLR-4/9 knockdown prior to GO50 treatment (FIG. 9E),
indicating that the GO-induced TLR-4/9 pathways were independent of
the mTOR pathway.
GO-Induced TLR4/9 Signaling was Upstream of the GO-Induced
Autophagy.
[0084] The interplay between autophagy and immunity has drawn
intensive attention in recent years. It was shown that TLR-4
signaling can activate autophagy in a way dependent on ATG5 and
Beclin 1. Oppositely, it was also suggested that autophagy
regulates the activation of TLRs pathways. To elucidate the
crosstalk between the GO-induced TLR-4/9 signaling and autophagy,
CT26 cells were transfected with siTLR-4 or siTLR-9, followed by
GO50 treatment. Compared with the scrambled siRNA, siTLR-4 and
siTLR-9 significantly mitigated the GO-induced activation of LC3
and Beclin 1 (FIG. 9F-9G), proving that TLR-4/9 regulated the
GO-induced autophagy.
[0085] To evaluate whether the opposite was true, we transfected
cells with siRNA for atg5 and atg7 (genes essential for autophagy
induction) to knockdown the GO-induced autophagy (FIG. 10A). The
inhibition of GO-induced autophagy by siATG5 or siATG7 neither
abolished the GO-induced production of TNF-.alpha. and IL-1.beta.
(FIG. 10B) nor affected the expression of TLR-4 and TLR-9 upon GO50
treatment (FIG. 10C-10D), thus autophagy did not regulate the
GO-induced TLR-4/9 signaling. These data collectively attested that
GO-activated TLR-4/9 signaling was upstream of autophagy.
[0086] Beclin 1 is inactivated by the inhibitory interaction with
TAB2/3, Bcl-2 and Bcl-xL in the usual state and TLR signaling can
release Beclin 1 from the inhibitory molecules, enhance the
interaction between Beclin 1 and MyD88, while activated TRAF6
stimulates Beclin 1 to initiate autophagy. Since GO50 induced
TLR-4/9 and downstream signaling effectors MyD88 and TRAF6, and
concurrently activated LC3, Beclin 1 and the ensuing autophagy, we
propose that GO engagement of TLR-4/9 activates MyD88/TRAF6 and
induces autophagy through the activation of Beclin 1 and LC3, in a
way independent of the mTOR pathway.
GO Injection Suppressed Tumor Formation, Enhanced Cell Death,
Autophagy and Immune Cell Infiltration
[0087] To assess the potential of GO-induced autophagy in cancer
therapy, CT26 cells were injected subcutaneously into BALB/c mice,
followed by intratumoral injections of PBS or GO at day 0 (when the
tumor volume reached .apprxeq.30-40 mm.sup.3) and day 8. In
comparison with PBS, GO alone significantly suppressed the tumor
progression (FIG. 11A) without considerably compromising the body
weight (FIG. 11B). Analysis of the tumor sections 5 days after GO
injection revealed pronounced cell death as confirmed by Live/Dead
assay (FIG. 11C). However, no apparent apoptosis was observed at
day 5 (FIG. 11D). GO alone also induced autophagy as evidenced by
the considerable LC3.sup.+ aggregation (FIG. 11E). Strikingly, GO50
remarkably potentiated the infiltration of macrophage, dendritic
cells (DCs), CD4.sup.+ and CD8.sup.+ T cells into the tumor bed
(FIG. 11F). Analysis of the tumor sections at the endpoint revealed
significant cell death, apoptosis, autophagy induction and
infiltration of immune cells within the tumors. The concurrent
induction of autophagy and enhanced immune cell infiltration
indicate that GO alone is sufficient to potentiate the antitumor
immune responses.
[0088] While the invention can be subject to various modifications
and alternative forms, a specific example thereof has been shown in
the drawings and is herein described in detail. It should be
understood, however, that the invention is not to be limited to the
particular form disclosed, but on the contrary, the invention is to
cover all modifications, equivalents, and alternatives falling
within the spirit and scope of the appended claims.
* * * * *