Figure legends
Figure 1 . IR-780 specifically targets tumor tissue and locates
to cancer cell mitochondria. (A) Nude mice with T24 subcutaneous tumor
xenografts and C57 BL/6 mice with MB49 subcutaneous transplanted tumors
were subjected to a single-dose intraperitoneal administration of IR-780
at 1 mg/kg (n=5). The dissected organs at 24 h after injection were
subjected to near-infrared fluorescence (NIRF) imaging.
(B)
The fluorescence intensity of dissected organs and tumors in (A). (C)
Tumor and the normal tissue from patients with MIBC were subjected to
NIRF imaging after incubation with IR-780 at 10 μM for 2 h
(n=5).
(D) The fluorescence intensity of (C). (D) Colocalization of IR-780 with
a mitochondria-specific tracker (MitoTracker Green) in T24 and MB49
bladder cancer cell lines was imaged using a confocal microscope; the
HK2 and SV-HUC-1 normal cell lines were used as controls. The nuclei
were stained with Hoechst 33258. Scale bar = 25
μm.
(*, p<0.05).
Figure
2 .
The
anti-tumor
effect of IR-780 and influence on the mitochondrial activities of T24
cells. (A) T24, 5637, TCCSUP, and MB49
bladder
cancer cell viability were measured using a CCK-8 kit
after
treatment with various doses of IR-780 for 48 h. (B) T24 cells were
treated with IR-780 for
24
h and harvested for detection of apoptosis using flow cytometry. (C)
Mitochondrial ROS levels, (D) ATP production, (E) mitochondrial membrane
potential, and (F) Mitochondrial complex I activity were detected after
treatment with IR-780 for 24 h. (G) Western blot detection of
recombinant human DNUFS1 protein treated with IR-780 in vitro. (*,
p<0.05).
Figure 3 . HBO promotes IR-780 antitumor efficacy in vitro. (A)
Different bladder cancer cell viability was tested after treatment with
IR-780 with or without HBO for 48 h. (B) Image of colony formation
by
T24 cells after treatment with 0 or 7.5 μM IR-780 with or without HBO
for 6 h before seeding. (C) The colony formation efficiency of T24 cells
at 14 days after seeding. (D) T24 cells were treated with 0 or 7.5 μM
IR-780 with or without HBO for 24 h and harvested for detection of
apoptosis using flow cytometry. (E) Analysis of variance statistics of
the cytometry data derived from samples presented in (D). (F) The
morphology of T24 cells after treatment with 0 or 7.5 μM IR-780 combined
or not with HBO was observed by inverted microscopy. Scale bar = 25 μm.
(G) Expression of apoptosis-related proteins in T24 cells. Cleaved PARP,
caspase-3, c-caspase-9, and cytochrome c levels were tested by western
blotting. (H) Viability of T24 cells treated with IR-780+HBO in the
presence of Z-VAD (an apoptosis inhibitor). (*, p<0.05).
Figure 4 . The antitumor efficacy of IR-780+HBO in vivo.
Treatment was carried out by intraperitoneal injection every two days
for a total of 5 treatments. The IR-780+HBO and IR-780 groups were
treated with 3 mg/kg IR-780. (A)
The
transplanted tumor size was monitored every 3 days using a sliding
caliper. After excision from the mice,
the
transplanted tumor were photographed (B) and weighed (C). Scale bar = 1
cm. (D) The body weights of mice were measured every 3 days before
excision. (E) Long-term observation of tumor volume after mice were
treated with IR-780+HBO or DDP. Images of the mouse transplanted tumor
at day 28 are shown in (F). Scale bar = 1 cm. (G) H&E staining in major
organs of mice. Scale bar = 50 μm. (*, p<0.05).
Figure 5 . HBO promotes uptake of IR-780 in cancer cells by
increasing
the plasma membrane potential.
T24
cells were treated with the indicated doses of IR-780 with or without
HBO and imaged using a fluorescence microscope (A), and fluorescence
intensity was measured using flow cytometry (B) immediately after the
completion of HBO. Scale bar = 25
μm.
(C)
The
fluorescence spectrum of 7.5 μM IR-780 in cell culture medium combined
or not with HBO with 740 nm using an excitation wavelength from
750~900 nm to scan their emission spectra. (D) T24 cells
were treated with 0 or 7.5
μM
IR-780 with or without HBO for 24 h, and NDUFS1 was detected by western
blotting. (E) T24 cells were treated with pre-IR-780 (IR-780 was removed
after incubation for 30 minutes)+HBO and stained with DiBAC4 to detect
the plasma membrane potential by flow cytometry. (F) Plasma membrane
potential was tested after cell incubation different doses of
K+ medium for 1 h. Cells were pre-incubated for 1 h
with the indicated doses of K+ medium followed by
2
μM IR-780 for 10 min, and the fluorescence intensity of IR-780 was
examined (G, H). Scale bar = 50 μm. After 2 μM IR-780 treatment for
30min, the cells were incubated with the indicated doses of
K+ medium and treated with HBO, and the fluorescence
intensity of IR-780 was examined (I). (J) T24 cells were treated with
IR-780, pre-IR-780 +HBO, or IR-780+HBO, and cell viability was measured.
(*, p<0.05).
Figure 6 . IR-780+HBO induces excessive mitochondrial ROS
production. (A)
Fluorescence
image of the ROS probe DCFDA by T24 cells after treatment with 0 or 7.5
μM IR-780 with or without HBO. Scale bar = 100 μm.
(B)
DCFDA fluorescence intensity was measured using flow cytometry. (C)
Fluorescence images of T24 cells labeled with DCFDA and
mitoSOX
after treatment with IR-780+HBO. Scale bar = 25 μm. Mitochondrial ROS
production detected using mitoSOX (D). (E) IR-780+HBO-induced cellular
mitochondrial ROS levels were positively correlated with cell death. (F)
Mitochondrial ROS levels were detected after treatment with IR-780+HBO
in the presence of DPI (an NADPH inhibitor), MitoQ (a mitochondrial ROS
inhibitor) or NAC, and then cell viability was detected (G). (H) Western
blot detection of apoptosis induction by IR-780+HBO in the presence or
absence of MitoQ or NAC. T24 cells were treated with 7.5 μM IR-780 with
or without the indicated concentrations of
H2O2 for 24 h and then harvested for
detection of apoptosis using flow cytometry (I). Mitochondrial ROS
levels of T24 cells were measured after treatment with IR-780+HBO in the
present of 21% O2, 50% O2, 100%
O2 (J) or TTFA (K). (L) Detection of mitochondrial
membrane potential induced by IR-780+HBO. Mitochondrial morphology in
T24 cells after treatment with 0 or 7.5 μM IR-780 combined or not with
HBO was observed by transmission electron microscopy (M). Scale bar =1
μm. (*, p<0.05).
Figure 7 . The effects of IR-780+HBO on DDP-resistant T24 cells.
(A) Staining of IR-780 in T24 and T24/DDP cancer cells was imaged using
a fluorescence microscope, and the fluorescence intensity was determined
by flow cytometry (B). Scale bar =50 μm. (C) NIRF imaging of T24/DDP
subcutaneous tumor xenografts using IR-780. (D) Viability of T24 cells
and T24/DDP cells treated with DDP. (E) Cell viability of T24/DDP cells
treated with IR-780, IR-780+HBO or DDP. (F) The volumes of T24/DDP cell
tumors were observed after nude mice were treated with IR-780+HBO or
DDP. Images of the xenografts after excision from the mice at day 19 are
shown in (G). Scale bar = 1 cm. (H) In the IR-780+HBO treatment group,
the T24/DDP tumor xenografts appeared to be ruptured. (*,
p<0.05).
Figure 8 . Proposed model of mitochondrial apoptosis-inducing
mechanism by IR-780+HBO. IR-780 preferentially accumulates in the
mitochondria of tumor cells by targeting mitochondrial protein NDUSF1,
causing many electrons to leak out; combination with HBO could induce
cancer cell apoptosis by promoting cancer cell uptake of IR-780 and
inducing excessive mitochondrial ROS production.