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.