membrane potential
We have confirmed that IR-780 alone can induce apoptosis in cancer cells
in a dose-dependent manner. To test whether HBO promotes the absorption
of IR-780, the uptake of IR-780 was compared in T24 cells with different
interventions.
As
shown in Fig. 5A and B,
the
fluorescence intensity was obviously enhanced in the group treated with
combined IR-780 and HBO compared with the IR-780-only group. In
addition, HBO did not change the fluorescence properties of IR-780 (Fig.
5C), and the levels of NDUFS1, the complex I subunit protein, were
decreased in the
7.5
μM
IR-780 +HBO group compared with the IR-780-only group (Fig. 5D), further
confirming that HBO enhanced the accumulation of IR-780. Our previous
study has shown that the
plasma
membrane potential plays a critical role in the uptake of IR-780 in
cancer cells(Zhang, Luo, Tan & Shi,
2014).
Therefore,
plasma membrane potential was measured using the voltage-dependent
fluorescent oxonol dye DiBAC4(3) in T24 cells after pre-IR-780 (IR-780
was removed after incubation for 30 min) treatment combined or not with
HBO treatment.
DiBAC4(3)
was a lipophilic anionic fluorescent dye, which could enter cells
through
depolarized
plasma membrane and enhance fluorescence intensity. Fig. 5E revealed
that the
fluorescence
intensity of
DiBAC4(3)
in T24 cells was significantly decreased in the pre-IR-780+ HBO group,
indicating hyperpolarization of plasma membrane, and
the
plasma
membrane potential was elevated. To further clarify the principal role
of plasma membrane potential in the accumulation of IR-780 in T24
cells,
cells were pre-incubated for 1 h with different doses of
K+medium to depolarize
the
plasma membrane (Fig. 5F) and then incubated with
2
μM IR-780 for 10 min. As shown in Fig. 5G and H, the fluorescence
intensity of IR-780
decreased
gradually with the increase in K+ concentration.
Moreover,
after
2 μM IR-780 treated for 30 min, T24 cells were incubated with
different
does of
K+medium and then treated with
HBO.
The fluorescence intensity revealed that the absorption enhancement of
IR-780 caused by HBO was gradually weakened with the increase in
K+ concentration (Fig. 5I). Taken together, these
studies strongly suggested that HBO promoted the accumulation of IR-780
in T24 cells by changing the plasma membrane potential.
We
ascertained that high doses of IR-780 could lead to cell death in our
previous study, but we wondered whether the enhanced uptake of IR-780
was the primary cause of cell death during IR-780+HBO treatment.
Therefore, we first validated that the fluorescence intensity of IR-780
reached its peak at 30 min when cells were incubated with 7.5 μM IR-780
(Fig. S3A). Second, we compared the fluorescence intensity between cells
incubated with IR-780 and cells pre-incubated with IR-780 at different
times. As shown in Fig.S3B, there were no significant differences in
fluorescence intensity between the two groups at corresponding times.
Finally, T24 cells were incubated with 7.5 μM IR-780 or pre-IR-780 and
combined or not with HBO treatment. As shown in Fig. 5J, pre-IR-780+HBO
displayed better tumor inhibition than IR-780 alone; however,
pre-IR-780+HBO was not as effective as IR-780+HBO. These results
indicated that the enhanced uptake of IR-780 was one of the causes of
the antitumor effects of IR-780+HBO.
HBOenhances
IR-780 antitumor efficacy by inducing excessivemitochondrial
ROS
To further explore the main mechanism of anti-tumor effect of
IR-780+HBO, ROS production was measured with the probe DCFH-DA in T24
cells. Fig. 6A and B revealed that ROS production in T24 cells was
significantly enhanced in the IR-780+ HBO group. Given that IR-780
localizes to
mitochondria,
we tested mitochondrial ROS production with the probe mitoSOX. Fig. 6C
and D show that the mitoSOX fluorescence levels in T24 cells rapidly
increased in the IR-780+HBO group immediately after HBO treatment.
The
elevations in mitoSOX fluorescence were positively associated with cell
death
(Fig.
6E). Treatment with NAC and MitoQ (a mitochondrial ROS inhibitor) before
treatment with IR-780+HBO could obviously restrain the increases in
mitoSOX levels, and increasing the survival rates of T24 cells also
decreased the expression of the apoptosis marker c-caspase3, but this
effect was limited by diphenyleneiodonium (DPI; an NADPH oxidase
inhibitor) (Fig. 6F-H), further confirming that IR-780+HBO-induced ROS
was mainly from mitochondria. Moreover, T24 cells incubated with or
without
7.5
μM IR-780 for 30 min were treated with different doses of
H2O2for 48 h, and the apoptosis rate of the IR-780+
H2O2 group was significantly higher than
the control group (Fig. 6I). These studies indicated that mitochondrial
ROS played a critical role in IR-780+HBO-induced cell
death.
The antitumor effects of IR-780+HBO could be eliminated when the oxygen
concentration was reduced to 21% (Fig. S4A). However, with the
increased oxygen concentration, the cell survival rate gradually
decreased (Fig. S4B), which suggested that oxygen, rather than pressure,
played an important role in the antitumor effect. To further explore the
mechanism by which IR-780+HBO increased mitochondrial ROS, the
concentration of
oxygen
was changed during HBO treatment, and TTFA (a mitochondrial complex II
inhibitor), which can inhibit back-propagation of
electrons,
was used before IR-780+HBO treatment. mitoSOX fluorescence decreased
with decreasing
oxygen
concentrations (Fig. 6J), and TTFA could partially reduce
mitoSOX
fluorescence (Fig. 6K), suggesting that oxygen and electrons are the
crucial factors mediating the increased mitochondrial ROS
induced by IR-780+HBO. Furthermore,
mitochondrial
membrane potential, which plays an important role in regulating
apoptosis, decreased rapidly when T24 cells were treated with IR-780 for
30 min (Fig. S5A). Further research revealed that when
the
concentration of IR-780 was 7.5 μM, the
mitochondrial
membrane potential was decreased at 30 min but increased with time and
returned to normal by 24 h (Fig. S4B). However, 7.5 μM IR-780 combined
with HBO further reduced the mitochondrial membrane potential (Fig. 6L),
which could aggravate the damage to mitochondria.
As
shown by TEM imaging (Fig. 6M), mitochondrial vacuolation was induced by
IR-780+HBO treatment, which was closely related to cell death.
The
effects of IR-780+HBO onDDP-resistant
T24 cells
Cisplatin resistance is an important barrier in bladder cancer treatment
that has a close relationship with prognosis. Previous studies have
demonstrated higher selective accumulation of IR-780 than other drugs in
drug-resistant lung cancer cells. To verify whether this accumulation
advantage occurs in
cisplatin-resistant
bladder cancer cells, we explored the accumulation of IR-780
in
cisplatin-resistant T24 cancer cells (T24/DDP). As shown in Fig. 7A and
B,
the
fluorescence intensity in T24/DDP cells was stronger than in T24 cells.
IR-780 was intraperitoneally injected into athymic nude mice bearing
subcutaneous
T24/DDP
cell
xenografts to further validate the accumulation ability in vivo. The
tumor xenografts were clearly
demarcated
in these mice 24 h after IR-780 injection (Fig.
7C).
Next, we validated the anticancer effect of
IR-780+HBO
on T24/DDP cells. As shown in Fig. 7E, IR-780+HBO inhibited T24/DDP cell
proliferation in a dose-dependent manner and exhibited better anticancer
activity than DDP and IR-780
alone.
In vivo, T24/DDP tumor-bearing nude mice were injected intraperitoneally
with IR-780 (combined with HBO) or DDP; the treatment was administered
every two days for a total of 5 treatments. IR-780+HBO showed a more
pronounced tumor-inhibiting effect than the other treatments (Fig. 7F
and G). Interestingly, we found that some tumor xenografts (4/7) in the
IR-780+HBO group appeared to be ruptured (Fig. 7H), which may have been
due to the rapid excessive production of ROS.
Discussion
The present treatments for
bladder
cancer are unsatisfactory in terms of the treatment methods, therapeutic
effects and quality of life of the
patients,
due to the lack of effective methods to reduce the recurrence of bladder
cancer and drug resistance. Even with the advent of neoadjuvant therapy
and immunotherapy, there was no significant change in prognosis.
Therefore,
there are still obvious unmet medical needs, and more treatments are
needed.
IR-780,
a
mitochondria-targeted fluorescent small molecule, has been confirmed to
delay tumor recurrence in a lung tumor model(Wang, Liu, Zhang, Luo, Tan
& Shi, 2014). In the current study, we investigated the effect of
IR-780 in bladder cancer. It was
confirmed
that IR-780 could preferentially accumulate in the mitochondria of
bladder cancer
cells.
Further studies have shown that IR-780 rapidly targets the mitochondrial
complex I protein NDUFS1 of cancer cells, resulting in a large number of
electron leakage for ROS production and can induce cancer cell
apoptosis. Moreover, IR-780 combined with HBO could significantly
enhanced the antitumor effect of IR-780 both in vitro and vivo,
indicating that the combination of IR-780 and HBO could be a therapeutic
strategy for bladder cancer.
Mitochondria
in cancer cells are essentially intact
and
play key roles in energy production and apoptotic pathways(Jose,
Bellance & Rossignol, 2011; Xiao, Fan, Huang, Gu, Li & Liu, 2010).
Increasing
evidence indicates that mitochondrial biosynthesis, bioenergetics and
signaling are essential for tumorigenesis. Hence, mitochondria have been
considered as subcellular targets for tumor targeting and therapy
(Weinberg & Chandel, 2015; Wen, Zhu & Huang, 2013). In particular,
as
increased levels of ROS and altered redox statuses have been observed in
cancer cells(Berkenblit et al., 2007), mitochondria in cancer cells
are
believed
to be more susceptible to increased ROS than those in normal cells.
Dysfunctional
mitochondria are essential sources of ROS, which are produced by leakage
of the
ETC,
and can activate intrinsic apoptosis(Porporato, Filigheddu, Pedro,
Kroemer & Galluzzi, 2017; Sabharwal & Schumacker, 2014).
Evidence
shows that most cancer cells have normal mitochondrial respiratory
function, and some cancers even show a more active ETC than normal cells
because of the rapid turnover of cancer cells(Deribe et al., 2018).
Inhibition
of the ETC has been shown to exert antitumor effects in
different
types of cancers (Hao, Chang, Tsao & Xu, 2010; Roesch et al., 2013).
However,
the
poor tumor targeting of these drugs limits their further clinical
application.
Recently,
a
near-infrared fluorescent dye, IR-780, which has both mitochondrial and
tumor targeting, has been widely observed and researched.
Current
strategies for IR-780 mainly involve nanotechnology or multistep
chemical coupling of
numerous
functional agents, including tumor-specific ligands and antitumor
drugs(Cao et al., 2017; Uthaman et al., 2018; Yan et al., 2016).
Although these strategies have revealed some antitumor effects, there
are still great challenges hindering their further application, such as
adverse immunogenic reactions, inefficient drug delivery systems and
difficulties in mass production (Desai, 2012). Very recently, a number
of photosensitizers for mitochondrial targets have been designed and
administered
to absorb light energy and used to induce excessive ROS production,
leading to cancer cell death(Luo et al., 2016; Tan et al., 2017).
However,
though
NIR excitation has better tissue penetration than ultraviolet excitation
(Idris, Jayakumar, Bansal & Zhang, 2015; Lucky, Muhammad Idris, Li,
Huang, Soo & Zhang, 2015), it is still difficult to apply it to
internal organs in the human body because the NIR excitation cannot
reach these organs. In this
study,
we chose oxygen as a sensitizer of IR-780, as oxygen can be transported
to various tissues in the body during HBO treatment.
We
confirmed that IR-780 could preferentially accumulate in the
mitochondria of bladder cancer cells
and
disturb electron transport in the ETC by targeting the
complex
I subunit protein NDUFS1, which
caused
many electrons to leak out and promote ROS
production.
The
mitoSOX fluorescence in T24 cells increased gradually and reached its
peak
at
3 h
upon
treatment with 7.5 μM IR-780 alone
(Fig.
S5C).
However,
when IR-780 was combined with HBO, mitoSOX fluorescence at 3 h was more
than twice as high as that after IR-780 treatment alone (Fig. 6D), which
could have been the result of a combination of enough oxygen and leaked
electrons.
Moreover,
we found that IR-780 rapidly reduced the mitochondrial membrane
potential, which may have been related to its rapid entry into
mitochondria.
The
mitochondrial membrane potential gradually recovered with time after
treatment with
7.5
μM IR-780 alone. However, the mitochondrial membrane potential remained
at low levels during the 2 h of combined treatment with HBO, which made
the mitochondria more vulnerable to increased ROS levels and led to
apoptosis through the mitochondrial pathway.
Hypoxia,
a common characteristic of most solid tumors, remains a significant
barrier to therapeutic efficacy. Therefore, various strategies for
increasing oxygen tension in hypoxic solid tumors have been urgently
pursued(Dewhirst, Mowery, Mitchell, Cherukuri & Secomb, 2019). HBO,
which is used as an adjuvant treatment with chemotherapy, has been
demonstrated to significantly improve the efficacy of various
chemotherapeutic drugs(Stępień, Ostrowski & Matyja, 2016). Alteration
of the hypoxic environment of tumors, promotion of the absorption of
drugs in tumors, and enhancement of the sensitivity of tumors to drugs
are considered to be the main mechanisms by which HBO sensitizes cells
to chemotherapy. However, HBO may aggravate the toxicity of some drugs
(DDP and DOX) toward normal tissues(Selvendiran et al., 2010), which
limits its further application. In this study,
we
found that HBO, when combined with IR-780, affected the antitumor
process not only by increasing the killing capacity of the drug itself
but also by providing the necessary oxygen support for the explosive
production of ROS (as indicated by mitoSOX fluorescence), which could
have been the main cause of cell death.
The
existence of hypoxia in bladder tumor has been confirmed(Hoskin,
Sibtain, Daley & Wilson, 2003), which will seriously affect the ROS
production after IR-780 enters the tumor. However, the combination with
HBO could compensate for this defect and maximize the anti-tumor effect
of IR-780. In addition, our study also indicated that IR-780+HBO did not
produce significant toxicity in normal tissues, which may have been
related to the strong tumor-targeting ability of IR-780.
The
mechanism
by which IR-780 iodide selectively accumulates in tumor cell has been
investigated in our previous research, which indicated that plasma
membrane potential plays a
critical
role
in
IR-780 iodide uptake in tumor cells(Zhang, Luo, Tan & Shi, 2014).
IR-
780 iodide is a lipophilic cation, and some researchers have confirmed
that
in normal and carcinoma cells, the difference in accumulation of
lipophilic cation can be directly attributed to the difference in
membrane potential(Davis, Weiss, Wong, Lampidis & Chen, 1985).
It
is also have been reported that the accumulation of any charged species
across the membrane is determined by the membrane potential(Zielonka et
al.,
2017).
Our data indicated that the absorption of IR-780 in bladder cancer cells
was significantly reduced after the plasma membrane was depolarized by
increasing the concentration of K+ in cell culture
medium, which further confirmed the critical role of
plasma
membrane potential for IR-780 iodide uptake in bladder cancer.
Interestingly, we found that HBO enhanced the uptake of IR-780 in
bladder cancer cells in a plasma membrane potential-mediated manner.
Inhibiting
the
plasma membrane potential with a certain concentration of
K+ could reduce the absorption of IR-780 caused by
HBO.
However,
the mechanism by which HBO altered the plasma membrane potential of
cells
pretreated
with IR-780 is not well
understood.
Increased ROS has been
reported
to cause the changes in plasma membrane potential by hyperpolarizing the
plasma membrane in macrophages(Klyubin, Kirpichnikova, Ischenko, Zhakhov
& Gamaley, 2000). In the current study, our data showed that HBO
increased the ROS production in bladder cancer cells pretreated with
IR-780,
and ROS production induced by H2O2 could
change the plasma membrane potential of bladder cancer cell, resulting
in enhanced absorption of IR-780 (Fig. S6). The plasma membrane
potential of epithelial cells is considered to be primarily a potassium
diffusion potential. Therefore, we speculate that the elevated ROS
during HBO may cause intracellular K + to flow out, leading to changes
in plasma membrane potential. However, the specific mechanism requires
further exploration.
Drug
resistance is one of the main causes of recurrence after bladder cancer
radiotherapy and chemotherapy. The mechanisms related to drug resistance
include reduced drug accumulation in cancer cells, increased
antiapoptotic ability, and overexpression of certain
multiple
drug resistance (MDR) proteins, such as the
ATP
binding cassette (ABC) transporter(Housman et al., 2014). However, most
antidrug behavior is closely related to mitochondria. Targeting of
mitochondria and modulation of ROS have been shown to be an effective
strategy against different types of drug-resistant cancer cells(Cui et
al., 2018). Alexander Roesch (A et al., 2013)confirmed that restraint of
the ETC overcomes multidrug resistance in melanoma and revealed
long-term effects. Elesclomol, an ETC-targeting compound, can cause
mitochondrial ROS by disrupting the ETC and causing the death of
cisplatin-resistant melanoma cells (Cierlitza et al., 2015; Santos et
al., 2012). Rotenone, an ETC complex I inhibitor, can increase ROS
production and induce apoptosis in DOX-resistant cancer cells(Wu et al.,
2018). However, to the best of our knowledge, studies investigating
mitochondrial targeting in drug-resistant bladder cancer are limited. In
the current study, we demonstrated that IR-780 could target the
oxidative respiratory chain of bladder cancer cells and accumulate to
greater levels in T24/DDP than T24 cells. When combined with HBO, it
could obviously promote apoptosis of T24/DDP cells, suggesting a new
therapeutic strategy for DDP-resistant bladder cancer.
Conclusions
In this study,
we
identified a mitochondria-targeted fluorescent small molecule, IR-780,
which can selectively target the mitochondria of bladder cancer cells,
including cisplatin-resistant cells, and
induce
cancer cell apoptosis by targeting the electron transport chain.
Moreover, when IR-780 combined with HBO, it exhibited effective
antitumor activity by promoting cancer cell uptake of IR-780 and
inducing excessive mitochondrial ROS production (as illustrated in
Scheme 1). This discovery potentially offers
a
novel treatment paradigm for human bladder cancer.