Dynamin‐related protein 1‐mediated mitochondrial fission contributes to IR‐783‐induced apoptosis in human breast cancer cells

Abstract IR‐783 is a kind of heptamethine cyanine dye that exhibits imaging, cancer targeting and anticancer properties. A previous study reported that its imaging and targeting properties were related to mitochondria. However, the molecular mechanism behind the anticancer activity of IR‐783 has not been well demonstrated. In this study, we showed that IR‐783 inhibits cell viability and induces mitochondrial apoptosis in human breast cancer cells. Exposure of MDA‐MB‐231 cells to IR‐783 resulted in the loss of mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) depletion, mitochondrial permeability transition pore (mPTP) opening and cytochrome c (Cyto C) release. Furthermore, we found that IR‐783 induced dynamin‐related protein 1 (Drp1) translocation from the cytosol to the mitochondria, increased the expression of mitochondrial fission proteins mitochondrial fission factor (MFF) and fission‐1 (Fis1), and decreased the expression of mitochondrial fusion proteins mitofusin1 (Mfn1) and optic atrophy 1 (OPA1). Moreover, knockdown of Drp1 markedly blocked IR‐783‐mediated mitochondrial fission, loss of MMP, ATP depletion, mPTP opening and apoptosis. Our in vivo study confirmed that IR‐783 markedly inhibited tumour growth and induced apoptosis in an MDA‐MB‐231 xenograft model in association with the mitochondrial translocation of Drp1. Taken together, these findings suggest that IR‐783 induces apoptosis in human breast cancer cells by increasing Drp1‐mediated mitochondrial fission. Our study uncovered the molecular mechanism of the anti‐breast cancer effects of IR‐783 and provided novel perspectives for the application of IR‐783 in the treatment of breast cancer.

optical imaging takes advantage of the wavelength range of approximately 700-1000 nm, wherein the interference from tissue autofluorescence is minimized. 1 There are several kinds of NIR in use, including the organic NIR dyes such as BODIPY derivatives, cyanine dye, the inorganic dye-like gold nanoparticle, and carbon-based and copper sulphide-based nanomaterials. 2 Among various NIR agents, indocyanine green (absorption at~780 nm and emission at 820 nm) is the only FDA-approved NIR dye for clinical applications in angiography, blood flow evaluation and liver function assessment.
Moreover, recent studies have shown that indocyanine green is well-tolerated and can be injected into patients for NIR cancer imaging during cancer surgery. 3,4 However, indocyanine green is not suitable for diagnostic and therapeutic applications due to the lack of penetration of the emitted light through tissues and the skin and it is without selective antitumour activity. 5 Thus, it is very important to find a new NIR dye with diagnostic and therapeutic activity that will provide a new possible therapeutic strategy for cancer treatment.
Recently, several reports showed that another kind of NIR, heptamethine cyanine dyes, such as IR-780 iodide, 6,7 IR-783 8 and MHI-148 9 with dual imaging and tumour targeting properties, can be further exploited to improve cancer detection, diagnosis and therapy. 10 In particular, IR-783 is a promising imaging reagent for human cancer treatment due to its highly selective cancer targeting activity that can target tumours of the prostate, bladder, pancreas and kidney in vitro and in vivo, and it can reduce the cell viability of cancer cells. 8,10,11 Moreover, IR-783 is a low toxicity water-soluble heptamethine cyanine dye with rapid clearance and is unlikely to be trapped in the reticular and endothelium of the liver or spleen. 12 Therefore, IR-783 is a prospective compound that could be further exploited for cancer treatment. However, the exact molecular mechanism by which IR-783 exerts anticancer effects remains unclear.
It has been reported that the selective antitumour activity of IR-783 may be mainly related to mitochondria. 10,13 Yuan et al reported that a strong IR-783 signal was detected in prostate cancer tissues but not in normal tissues, and their research showed that IR-783 staining was almost completely colocalized with MitoTracker (a mitochondrial-selective probe) in cancer cells. Moreover, they also identified that the organic anion transporter polypeptide (OATP), which is highly expressed in cancer cells, mediates the uptake of heptamethine cyanine dyes such as IR-780, 14,15 MHI-148 9 and IR58 16 into the mitochondria of tumour cells. These results all suggest that the anticancer property of IR-783 is related to mitochondria.
Mitochondria, crucial double membrane-bound organelles in cells, play an important role in a diverse range of physiological processes, including cell metabolism, proliferation and differentiation, survival and apoptosis. 17,18 Mitochondria exist as dynamic networks that maintain the normal shape, structure, quantity and function of mitochondria, and these dynamics are maintained by 2 opposing processes: fission and fusion. 19 Mitochondrial fission and fusion appear to be essential for cell function and tissue development, and imbalanced fission and fusion always lead to mitochondrial structural changes and dysfunction. Notably, dynamin-related protein 1 (Drp1) is essential for mitochondrial fission. Drp1, a member of the dynamin family of GTPases, can translocate from the cytosol to the mitochondria and mediates mitochondrial fission, resulting in a loss of mitochondrial membrane potential and apoptosis. 20 Inhibition of Drp1 blocks mitochondrial fission and cell death. 21 Recently, a study reported that Drp1 was up-regulated in certain types of cancers, such as lung and breast cancer. 22 Moreover, Drp1 is a newly discovered therapeutic target for tumour initiation, 23 migration 24 and proliferation. 25 In 2013, Zhao et al reported that cancer cell migration and invasion were regulated by mitochondrial dynamics. 17 Thus, targeting Drp1-dependent mitochondrial dynamics may provide a novel therapeutic strategy for cancer treatment.
In this study, we found that IR-783 induced breast cancer cell apoptosis by increasing mitochondrial fission. IR-783 treatment upregulated the mitochondrial fission proteins Drp1, mitochondrial fission factor (MFF) and fission-1 (Fis1), and down-regulated the fusion proteins mitofusin1 (Mfn1) and optic atrophy 1 (OPA1). Importantly, knockdown of Drp1 attenuated IR-783-mediated mitochondrial fission, mitochondrial injury and apoptosis, and inhibition of tumour growth by IR-783 in an MDA-MB-231 xenograft mouse model was also related to the mitochondrial translocation of Drp1 in vivo. Our findings provide a novel mechanistic basis for the application of IR-783 in the treatment of breast cancer.

| Cell culture
The human breast cancer cell line (MDA-MB-231) and normal hepatocyte cell line (LO 2 ) used in this study were purchased from ATCC (Manassas, VA), cultured in DMEM (Gibco) with 10% foetal bovine serum (TBD Science, TianJin), and incubated at 37°C with 5% CO 2 .  was added to the culture medium. After 4 h incubation, the medium was removed carefully and 150 lL of DMSO was added to each well. The plates were detected at a wavelength of 490 nm using a microplate reader (Multiskan Go Thermo Scientific).

| Measurement of cellular content of adenosine triphosphate (ATP)
To detect cellular ATP production, the ATP Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China, S0026) was used. After drug treatment, cells were collected and washed twice with PBS.
Then, we added ATP Cell Lysis solution to dissolve the cells, followed by centrifugation at 12 000 g for 10 minutes at 4°C, and the supernatant was removed and mixed with dilution buffer containing luciferase. The luminescence value was detected using a microplate reader (Thermo Varioskan TM LUX) according to the manufacturer's instructions. A fresh standard curve was prepared each time and the ATP content was calculated using this curve. The results are expressed as a percentage of the control, which was set at 100%.

| Measurement of mitochondrial permeability
transition pore (mPTP) opening mPTP opening analysis was performed as previously described. 26 Briefly, after drug treatment, the cells were washed twice with PBS and stained with calcein-acetoxymethyl ester (calcein-AM) and CoCl 2 in serum-free medium for 15 minutes at 37°C. After that, the medium was removed and fresh medium was added for detection. The extramitochondrial Ca 2+ concentration was measured by a fluorescence microplate reader (Thermo Varioskan TM LUX) at the excitation wavelength of 488 nm and the emission wavelength of 525 nm. The results are expressed as a percentage of the control, which was set at 100%.

| Transmission electron microscopy assay
For electron microscopy, cells were fixed in 2.5% glutaraldehyde at 4°C for 24 hours, fixed in 2% osmium tetroxide at 4°C for 2 hours, dehydrated with a series of ethanol and embedded in Epon Ultrathin. Subsequently, sections were prepared using a microtome (UC7, Leica, Germany) and stained with uranyl acetate and lead citrate.
Mitochondria were examined with a Tecnai 10 transmission electron microscope (Philips, Netherlands).

| Immunofluorescence
MDA-MB-231 cells were plated on coverslips and cultured in 24-well plates for 24 hours, and after drug treatment, the cells were stained with 100 nmol/L MitoTracker Red CMXRos for 30 minutes, then washed with culture medium 5 times. Then, the cells were fixed, permeabilized and blocked with 5% milk.
Immunostaining was performed using antibodies including anti-Drp1 (1:50), followed by incubation with a secondary antibody Alexa Fluor 488 goat antimouse (A1101, Molecular Probes). A laser-scanning confocal microscope (LSM780NLO, Zeiss, Germany) with a 639 oil objective was used for cell imaging. Mitochondrial length was measured by an investigator blinded to the samples performed with the polygon tool of Imaris 3D software.
The colocalization of Drp1 and mitochondria was analysed using ImageJ software.

| Animal experiments
All animal studies were approved by the Army Medical University Institutional Animal Care and Use Committee. Female nude mice

| Statistical analysis
All data are shown as the mean AE SD. Comparisons between individual groups were made with a 2-tailed Student's t-test. Comparisons between multiple groups were made with analysis of variance, and *P < .05, **P < .01 or ***P < .001 were considered significant. Adenosine triphosphate is an energy molecule that plays an important role in physiology and pathology. The synthesis of ATP occurs via the oxidative phosphorylation of glucose or via the canonical mitochondrial oxidation pathway from ATP synthetase, and thus cellular metabolism is maintained in a tight energy homeostasis through regulation of ATP levels. 29 Cancer cells have been shown to have higher ATP levels than normal cells in vitro, and decreasing ATP means the cancer cells are entering conditions of apoptosis or necrosis. 30 As shown in Figure 2C Figure 2D). Western blot analysis showed that treatment with IR-783 resulted in increased release of Cyto C from the mitochondria to the cytosol (Figure 2E). Altogether, these data demonstrate that IR-783 induces mPTP opening-dependent mitochondrial injury.

MB-231 cells
Increasing evidence indicates that mitochondrial fission promotes the initiation of mitochondrial apoptosis. 34

| Knockdown of Drp1 blocked IR-783-induced mitochondrial fission and mitochondrial apoptosis
To prove the important role of Drp1-regulated mitochondrial fission in the process of IR-783 induced MDA-MB-231 cell death, a lentiviral transduction approach was used to stably knockdown Drp1 expression ( Figure 4A). We first evaluated the effects of Drp1 knockdown on mitochondrial morphology. Immunofluorescence microscopic studies showed that knockdown of Drp1 significantly increased the average length of mitochondria and blocked the mitochondrial fission induced by IR-783 treatment ( Figure 4B,C). Mitochondrial fission is considered to be universally associated with the initiation of apoptosis through the mitochondrial apoptotic pathway. 38,39 We then inves- | 4479 statistically significant changes in bodyweight between the IR-783treated mice and control animals ( Figure 5B). Moreover, the representative liver and kidney tissues were sectioned and subjected to haematoxylin and eosin (H&E) staining. The results showed that there were no morphological differences between the IR-783-treated mice and control animals, indicating that IR-783 has a low liver and kidney toxicity ( Figure 5C). To further analyse the mechanism whereby IR-783 inhibited tumour growth, the representative tumour tissues were sectioned and subjected to H&E staining for tissue morphology, TUNEL analysis and immunohistochemistry staining of C-Caspase-3 for cell death. As shown in Figure 5D   that IR-783 is a prospective compound that could be further exploited for cancer treatment. 8,10,11 Therefore, it is important to reveal the exact molecular mechanism behind the anticancer effects of IR-783.
Mitochondria are important organelles in eukaryotic cells that exert vital and lethal effects on many biochemical functions, including cell metabolism, growth, differentiation, survival and programmed cell death. 18 Owing to its roles in the regulation of fundamental cellular functions, it is not surprising that mitochondria have been implicated in multiple aspects of tumour processes. 17,38 MMP, which reflects mitochondrial functional status, is thought to maintain the respiratory chain to generate adenosine triphosphate, and a decrease in MMP accelerates cellular depletion of ATP, followed by the mPTP opening with subsequent Cyto C release. mPTP opening has been implicated as the determinant cell death pathway in cancer. 32,40,41 It has been previously reported that the location of IR-783 was almost completely congruent with MitoTracker (a mitochondrial-selective probe) in prostate cancer cells, and the cancer-specific uptake of these organic dyes occurs primarily via OATP1B3. Our results are consistent with these prior findings that IR-783 induces mitochondrial-dependent apoptosis based on the following evidence. Mitochondrial fission and fusion appear to be essential for cell function and tissue development. 42 Changes in mitochondrial morphology are tightly regulated by the balance of fusion and fission processes. 19 Mitochondrial fission is considered to be universally associated with the initiation of the mitochondrial apoptotic pathway. Notably, Drp1, a highly conserved dynamin-related cytoplasm GTPase, is essential for mitochondrial fission. After stimulation by fission signals, Drp1 migrates to mitochondria and mediates mito-

CONFLI CTS OF INTEREST
No potential conflict of interests were disclosed. F I G U R E 5 IR-783 inhibited tumour growth in vivo by induction of the mitochondrial translocation of Drp1. A, Tumour volumes in xenograft mice were measured every week in the control and IR-783 group *P < .05. B, The bodyweight of mice after 4 weeks of IR-783 treatment. C, Representative liver and kidney tissues were sectioned and subjected to haematoxylin-eosin (H&E) staining. Scale bars: 100 lm. D, Representative tumour tissues were sectioned and subjected to H&E, TUNEL analysis and immunohistochemical staining for C-Caspase-3. Scale bars: 100 lm. E, The mitochondrial fractions of representative tumour tissues were prepared and subjected to western blot analysis using an anti-Drp1 antibody. Cox IV was used as the loading control for mitochondrial fractions. F, The representative tumour tissues were sectioned and subjected to immunofluorescence using the anti-Drp1 (green) and anti-TOM20 (red, a mitochondrial marker) antibody. Scale bars: 20 lm