Hyperbaric oxygen enhanced the chemotherapy of mitochondrial targeting molecule IR-780 in bladder cancer

Abstract

Background

Bladder cancer has a high rate of recurrence and drug resistance due to the lack of effective therapies. IR-780 iodide, a near-infrared (NIR) mitochondria-targeting fluorescent agent, has been demonstrated to achieve higher selectivity than other drugs in different tumor types and exhibited tumor-killing effects in some cancers. However, this therapeutic strategy is rarely studied in bladder cancer.

Material and methods

The accumulation of IR-780 in bladder cancer was measured by NIR imaging. Human bladder cell lines (T24, 5637, and TCCSUP) were treated with IR-780 or combined IR-780 and hyperbaric oxygen (HBO). Cell viability, cell apoptosis, cellular ATP production, mitochondrial reactive oxygen species (ROS), and plasma membrane potential were detected. Mitochondrial complex I protein NDUFS1 was measured by western blot. To confirm the anti-tumor efficacy of IR-780 + HBO, mouse bladder cell line (MB49) tumor-bearing mice were established and tumor size and weight were recorded. Besides, cell apoptosis and tumor size were assessed in drug-resistant bladder cancer cells (T24/DDP) and xenografts to evaluate the effect of IR-780 + HBO on drug-resistant bladder cancer.

Results

IR-780 selectively accumulated in bladder cancer (bladder cancer cells, transplanted tumors, and bladder cancer tissue from patients) and could induce cancer cell apoptosis by targeting the mitochondrial complex I protein NDUFS1. The combination with HBO could significantly enhance the anti-tumor effect of IR-780 in vitro by promoting cancer cell uptake and inducing excessive mitochondrial ROS production, while suppressing tumor growth and recurrence in animal models without causing apparent toxicity. Moreover, this combination antitumor strategy was also demonstrated in drug-resistant bladder cancer cells (T24/DDP) and xenografts.

Conclusion

We identified for the first time a combination of IR-780 and HBO (IR-780 + HBO), which exhibits mitochondria-targeting and therapeutic capabilities, as a novel treatment paradigm for bladder cancer.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00432-022-04385-4.

Introduction

Bladder cancer is one of the most common cancer in the world and one of the costliest cancers to manage due to the necessity of multiple therapeutic interventions (Richters et al. 2020). Approximately 75% of newly diagnosed patients present with non-muscle-invasive bladder cancer (NMIBC). Although the 5-year survival rate of NMIBC is over 80%, the 5-year recurrence rate can reach up to 78% (Ploeg et al. 2009; Teoh et al. 2022). Cisplatin (DPP)-based chemotherapy is currently a standard treatment for muscle-invasive and metastatic bladder cancer (Alfred Witjes et al. 2017). However, the subsequent survival benefits can only be described as modest due to chemoresistance, underscoring the need for new agents to reduce the recurrence of bladder cancer and drug resistance.

Mitochondria, which play a pivotal role in the regulation of tumorigenesis and progression, have recently attracted increasing attention as the subcellular target suitable for cancer therapy (WX et al. 2016). Targeting mitochondria in cancer cells may provide a new strategy to tumor-targeting therapy, especially for drug-resistant cancer cells, which may be more dependent on mitochondria (JR et al. 2018; Y et al. 2019). Metabolic change occurs in bladder cancer have been identified, and mitochondrial dysfunction is considered to be closely related to bladder cancer occurrence, progression and drug resistance (Oresta et al. 2021; Sahu et al. 2017; Woolbright et al. 2018). Moreover, a number of chemo-therapeutics in bladder cancer induces apoptosis associated with mitochondrial pathway (Xiao et al. 2022; Xu et al. 2022). However, this mitochondria-targeted treatment strategy is rarely studied in bladder cancer. The practical application of current mitochondrial-targeted therapy is limited due to the poor tumor-specific distribution and reduced drug accumulation in mitochondria. Recently, a group of near-infrared heptamethine dyes with both tumor and mitochondrial targeting have attracted much attention. IR-780 iodide, a near-infrared (NIR) fluorescent agent, has been reported to achieve higher selectivity than other drugs for the mitochondria of different types of tumor cells and xenografts (Jiang et al. 2019; Luo et al. 2011; Wang et al. 2018a; Zhang et al. 2010). In addition, IR-780 has an absorption peak at 780 nm and can emit fluorescence with a high intensity in the 807–823 nm wavelength range, which facilitates its use for imaging applications (Kuang et al. 2017). However, the research on the anti-tumor effect and mechanism of IR-780 is limited. In this study, we revealed for the first time that IR-780 selectively accumulated in bladder cancer cells and disrupted the mitochondrial electron transfer chain (ETC), which promotes ROS production and leads to cell apoptosis. However, the hypoxic environment of solid tumors could limit or delay the ROS production, which may reduce antitumor effect of IR-780. Therefore, we speculated that providing adequate oxygen in tumor tissue while the mitochondrial respiratory chain is disrupted could promote ROS production and enhance the antitumor effect. Hyperbaric oxygen (HBO), an adjuvant treatment with chemotherapy has been demonstrated to alter the hypoxic environment of tumors (Stępień et al. 2016). However, the research on the combination of mitochondria targeting agent and hyperbaric oxygen is limited. Our further research found that combined treatment with IR-780 and HBO exerted an incredible anti-tumor effect, representing a novel candidate strategy for bladder cancer-targeted therapy.

Results

IR-780 selectively accumulates in mitochondria of bladder cancer cells

The ability of IR-780 to selectively accumulate in bladder tumors was studied with T24 and MB49 transplantation tumor models. The organs of nude mice with pre-established T24 tumor xenografts and C57BL/6 mice with pre-established MB49 transplanted tumor were imaged after intraperitoneal injection of 1 mg/kg IR-780. The fluorescence distribution of the dissected organs in Fig. 1a shows the tumors had stronger NIR fluorescence signal. In addition, the histopathological analysis of the tumors by confocal microscopy (Fig. 1b) confirmed the accumulation of IR-780 in the tumor tissues. To further clarify the time course of IR-780 distribution in tumor-bearing mice, the dissected organs were imaged at different times after IR-780 injection. Figure S1 shows that the accumulation of IR-780 in tumors gradually increased, reaching its peak at 4 h, while less uptake occurred in other (normal) tissues. Moreover, IR-780 preferentially accumulated in bladder tumor tissues from clinical samples (Fig. 1c), indicating that the NIR small-molecule dye IR-780 could target human bladder tumors. Subcellular uptake and localization analysis of IR-780 through assessment of IR-780 and MitoTracker Green colocalization in T24 and MB49 cells revealed that IR-780 exclusively accumulated in the mitochondria of bladder cancer cells (Fig. 1d); HK2 and SV-HUC-1 cells were used as controls.

Fig. 1.

IR-780 specifically targets tumor tissue and locates to cancer cell mitochondria. a Nude mice with T24 subcutaneous tumor xenografts and C57BL/6 mice with MB49 subcutaneous transplanted tumors were subjected to a single-dose intraperitoneal administration of IR-780 at 1 mg/kg. The dissected organs at 24 h after injection were subjected to near-infrared fluorescence (NIRF) imaging. b The preferential accumulation of IR-780 in tumor tissue was detected by confocal microscopy. Nuclei were stained with DAPI. Scale bar = 100 μm. c Tumor and the normal tissues from patients with MIBC were subjected to NIRF imaging after incubation with IR-780 at 10 μM for 2 h. 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 33,258. Scale bar = 25 μm. (*p < 0.05; ****p < 0.0001)

IR-780 induces cancer cell apoptosis by targeting the mitochondrial electron transport chain

Since IR-780 can target bladder cancer cell, its anti-tumor effect should be investigated. CCK8 shows that IR-780 could significantly inhibit bladder cancer cells (T24, 5637, TCCSUP, MB49) proliferation in a dose-dependent manner (Fig. 2a). The 50% inhibitive concentration (IC50) of IR-780 on T24, MB49, TCCSUP and 5637 cell was 11.26 μM, 10.38 μM, 9.34 μM and 8.17 μM, respectively. Apoptosis of T24 cells was analyzed by flow cytometry. The apoptosis rate after incubation with 15 μM IR-780 was more than 30% compared with 3% in the control group (Fig. 2b; p < 0.0001), suggesting that IR-780 could induce T24 cells apoptosis in a dose-dependent manner. As we confirmed that IR-780 specifically accumulated in mitochondria, its effect on mitochondrial function should be studied. Figure 2c demonstrates that 2.5 μM-15 μM IR-780 markedly reduced cellular ATP production (p < 0.0001). To evaluate the effect of IR-780 on mitochondrial ROS level and mitochondrial membrane potential, flow cytometry was performed with MitoSOX and TMRM, respectively. The result demonstrated that 2.5 μM-15 μM IR-780 increased mitochondrial ROS (Fig. 2d), and 10 μM-15 μM IR-780 decreased mitochondrial membrane potential (Fig. 2e) in a dose-dependent manner, which could activate apoptosis signaling pathways and induce cancer cell apoptosis. In addition, IR-780 significantly inhibited mitochondrial complex I activity (Fig. 2f) and reduced the expression of complex I subunit protein NDUFS1 (Fig. 2g) in T24 cells in a dose-dependent manner, consistent with the findings of our previous study confirming IR-34 (a NIR fluorescent dye) targets and cleaves the mitochondrial complex I protein NDUFS1 in cancer cells (Wang et al. 2018b). Clearance of the NDUFS1 protein disturbed electron transport in the ETC, which led to considerable electronic leakage, promoting mitochondrial ROS production.

Fig. 2.

The anti-tumor effect of IR-780 and influence on the mitochondrial activities of T24 cells. T24, 5637, TCCSUP, and MB49 bladder cancer cell viabilities 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 ATP production, d Mitochondrial ROS levels, 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 < 0.01; ***p < 0.001; ****p < 0.0001)

HBO promotes IR-780 anti-tumor efficacy in vitro

To investigate the anti-tumor activity of IR-780 + HBO in vitro, bladder cancer cell lines of different grades (T24, 5637, TCCSUP, and MB49) were incubated with the indicated doses of IR-780 combined or not with HBO. Cell viability was measured with a CCK-8 kit. Figure 3a shows that 2.5 μM -10 μM IR-780 + HBO inhibited cancer cell proliferation in a dose-dependent manner and displayed better anticancer activity than IR-780 alone (P < 0.0001). The 7.5 μM IR-780 + HBO treatment almost achieved the efficacy of 15 μM IR-780 treatment in T24 cells and had little effect on normal cells (Fig. S2a). T24 cells were treated with doxorubicin (DOX) or DDP with or without HBO to further investigate whether this combination strategy also applied to other chemotherapeutic drugs. As shown in Fig. S2b and Fig. S2c, there was no significant difference between drugs (DOX and DDP) combined or not with HBO. Since bladder cancer cells could self-renew, we first investigated the effect of IR-780 + HBO on this characteristic. Compared with the other groups, the group treated with 7.5 μM IR-780 combined with HBO inhibited the colony formation ability of T24 cancer cells (Fig. 3b). The cells were obviously wrinkled and rounded upon microscopic examination 16 h after treatment with 7.5 μM IR-780 + HBO (Fig. 3c), which suggested that cell death may be the main reason for cell number decrease. As shown in Fig. 3d, the apoptosis rate was significantly higher in the 7.5 μM IR-780 + HBO group than in the other groups, and the Calcein-AM/PI double-labeling assay further confirmed the occurrence of apoptosis (Fig. 3e). Moreover, we found that the protein levels of Cytochrome C and the apoptosis markers poly ADP-ribose polymerase (PARP), caspase3, and cleaved caspase9 (c-caspase9) were all increased by IR-780 + HBO (Fig. 3f). The apoptosis inhibitor z-VAD-FMK partly rescued cell death, decreasing c-caspase3 protein levels (Fig. 3g) and increasing cell viability (Fig. 3h). All these results suggested that HBO could enhance IR-780 anti-tumor efficacy by inducing classic apoptosis in T24 cells.

Fig. 3.

HBO promotes IR-780 anti-tumor 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 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. 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 Calcein Green was used to detected apoptosis of T24 cells being treated with IR-780 + HBO. Scale bar = 50 μm. f Expression of apoptosis-related proteins in T24 cells. Western-blot detection g, and viability of T24 cells (H) treated with IR-780 + HBO in the presence of Z-VAD (an apoptosis inhibitor). (**p < 0.01; ***p < 0.001; ****p < 0.0001)

The anti-tumor efficacy of IR-780 + HBO in vivo

Based on the excellent anti-tumor effect of IR-780 + HBO in vitro, we further studied its anti-tumor effect in vivo. MB49 tumor-bearing mice were injected intraperitoneally with IR-780 combined or not with HBO every 2 days for a total of 5 injections. Analysis of volumes of the tumors in different intervention groups showed that the IR-780 + HBO group exhibited greater tumor inhibition than other groups (Fig. 4a) P < 0.0001. The HBO-only treatment had no effect on tumor growth, and compared with the control, IR-780 alone exerted a modest tumor-inhibiting effect. The sizes and shapes of the tumors were photographed (Fig. 4b), and tumor weight was detected (Fig. 4c). These results also suggested that IR-780 + HBO had a stronger ability to inhibit tumor growth than the other treatments. The body weights among the groups did not significantly differ (Fig. 4d), and abnormal histopathological changes were not observed in normal tissues (Fig. 4e), which indicated that IR-780 + HBO treatment did not cause obvious side effects. Since bladder cancer has a high rate of recurrence, we further investigated whether IR-780 + HBO could delay tumor recurrence. We observed the growth of tumors for a longer time period and compared the effects of IR-780 + HBO with those of classic anti-tumor drug DPP. Interestingly, tumor volumes in the mice subjected to IR-780 + HBO therapy did not change significantly, while those in mice subjected to DDP therapy continued to increase after intervention (Fig. 4f). On day 28, the tumors from mice treated with IR-780 + HBO were much smaller than those from mice treated with DDP (Fig. 4g, p < 0.0001). These studies indicated that IR-780 + HBO could effectively inhibit the growth of tumors and delay tumor recurrence in vivo without causing obvious side effects.

Fig. 4.

The anti-tumor 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 tumors were photographed b and weighed (c). d The body weights of mice were measured every 3 days before excision. e H&E staining in major organs of mice. Scale bar = 5.0 μm. f 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 g. (*p < 0.05; ***p < 0.001; ****p < 0.0001)

HBO promotes uptake of IR-780 in cancer cells by increasing plasma 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 in T24 cells with different interventions was investigated. 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 et al. 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) is a lipophilic anionic fluorescent dye, which could enter cells through depolarized plasma membrane and enhance fluorescence intensity. Figure 5e shows that the fluorescence intensity of DiBAC4(3) in T24 cells was significantly decreased in the pre-IR-780 + HBO group (P < 0.0001) than in pre-IR-780 group, indicating hyperpolarization of plasma membrane and elevation of the plasma membrane potential. 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 potential (Fig. 5f) and then incubated with 2.5 μM IR-780 for 30 min. As shown in Fig. 5g and h, the fluorescence intensity of IR-780 decreased gradually with the increase in K⁺ concentration. In contrast, we hyperpolarized the plasma membrane potential with H₂O₂ (Fig. 5i), which resulted in enhanced cellular uptake of IR-780 (Fig. 5j). Furthermore, after 2.5 μM IR-780 treatment 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. 5k). 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 intensities between the two groups at corresponding times. Finally, T24 cells were incubated with 7.5 μM IR-780 or pre-IR-780 and combined with or without HBO treatment. As shown in Fig. S3c, 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 anti-tumor effects of IR-780 + HBO.

Fig. 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 min) + HBO and stained with DiBAC4 to detect the plasma membrane potential by flow cytometry. f Plasma membrane potential was tested after cell incubation with different doses of K⁺ medium for 1 h. Cells were pre-incubated for 1 h with the indicated doses of K⁺ medium followed by 2.5 μM IR-780 for 30 min, and the fluorescence intensity of IR-780 was examined (g and h). Scale bar = 50 μm. Cells were treated with various concentration of H₂O₂ for 12 h, and the fluorescence intensity of DiBAC4(3) I and IR-780 (J) was detected by flow cytometry. After 2.5 μM IR-780 treatment for 30 min, the cells were incubated with the indicated doses of K⁺ medium and treated with HBO, and the fluorescence intensity of IR-780 was examined (K). (**p < 0.01; ****p < 0.0001)

HBO enhances IR-780 anti-tumor efficacy by inducing excessive mitochondrial 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. Figure 6a shows 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. Figure 6b and Fig. 6c show that the MitoSOX fluorescence levels in T24 cells increased in the IR-780 + HBO group immediately after HBO treatment. The elevations in MitoSOX fluorescence were positively associated with cell death (Fig. 6d). Treatment with NAC and MitoQ (a mitochondrial ROS inhibitor) before treatment with IR-780 + HBO could obviously restrain the increases in MitoSOX levels and improve the survival rate of T24 cells by decreasing the expression of the apoptosis marker c-caspase3, however, this effect was limited by diphenyleneiodonium (DPI: an NADPH oxidase inhibitor) (Fig. 6e–g), 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 H₂O₂ for 48 h, and the apoptosis rate of the IR-780 + H₂O₂ group was significantly higher than that of the control group (Fig. 6h). These studies indicated that mitochondrial ROS played a critical role in IR-780 + HBO-induced cell death. The anti-tumor 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 anti-tumor 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 increased with increasing oxygen concentrations (Fig. 6i), and TTFA could partially reduce MitoSOX fluorescence (Fig. 6j), 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, then increased with time, and returned to normal by 24 h (Fig. S5b). However, 7.5 μM IR-780 combined with HBO further reduced the mitochondrial membrane potential (Fig. 6k), which could aggravate the damage to mitochondria. As shown by TEM imaging (Fig. 6l), mitochondrial vacuolation was induced by IR-780 + HBO treatment, which was closely related to cell death.

Fig. 6.

IR-780 + HBO induces excessive mitochondrial ROS production. a DCFH fluorescence intensity was measured using flow cytometry. b Fluorescence images of T24 cells labeled with DCFH and MitoSOX after treatment with IR-780 + HBO. Scale bar = 25 μm. c Mitochondrial ROS production detected using MitoSOX. d IR-780 + HBO-induced cellular mitochondrial ROS levels were positively correlated with cell death. e 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 (f). g Western blot detection of apoptosis induction by IR-780 + HBO in the presence or absence of MitoQ or NAC. h 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. Mitochondrial ROS levels were measured after treatment with IR-780 + HBO in the present of 21% O₂, 50% O₂, 100% O₂ (i) or TTFA (j). k Detection of mitochondrial membrane potential induced by IR-780 + HBO. L 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. Scale bar = 1 μm. (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001)

The effect of IR-780 + HBO on DDP-resistant T24 cells

DPP resistance is an important barrier in bladder cancer treatment, which has a close relationship with prognosis. Previous studies have demonstrated higher selective accumulation of IR-780 in drug-resistant lung cancer cells rather than in parental cells (Wang et al. 2014), therefore, we explored the accumulation of IR-780 in DPP-resistant T24 cancer cells (T24/DDP). In vivo, IR-780 was intraperitoneally injected into athymic nude mice bearing subcutaneous T24/DDP cell xenografts. The tumor xenografts were clearly demarcated in these mice 24 h after IR-780 injection (Fig. 7a). In vitro, as shown in Fig. 7b and c, the fluorescence intensity in T24/DDP cells was stronger than in T24 cells. Next, we validated the anti-tumor effect of IR-780 + HBO on T24/DDP cells. As shown in Fig. 7d, IR-780 + HBO exhibited better anti-tumor activity than IR-780 alone. The apoptosis rate in 7.5 μM IR-780 + HBO group was significantly higher than that in 7.5 μM DDP group (Fig. 7e). 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 (2/5) in the IR-780 + HBO group appeared to be ruptured (Fig. 7h), which may be due to the rapid excessive production of ROS.

Fig. 7.

The effects of IR-780 + HBO on DDP-resistant T24 cells. a NIRF imaging of T24/DDP subcutaneous tumor xenografts using IR-780. b Staining of IR-780 in T24 and T24/DDP cancer cells was imaged using a confocal microscope, and the fluorescence intensity was determined by flow cytometry (c). Scale bar = 50 μm. d Viability of T24/DDP cells treated with IR-780 or IR-780 + HBO. e Cells were treated with 7.5 μM IR-780 + HBO or 7.5 μM DDP for 24 h and then harvested for detection of apoptosis using flow cytometry. 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 < 0.0001)

Discussion

Due to the lack of effective methods to reduce the recurrence and drug resistance, bladder cancer treatments are very limited, with poor efficacy and quality of life for patients. 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.

Mitochondria in cancer cells are essentially intact and play key roles in energy production and apoptotic pathways (Jose et al. 2011; Xiao et al. 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 and Chandel 2015; Wen et al. 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 et al. 2017; Sabharwal and Schumacker 2014). Evidence shows that most cancer cells have normal mitochondrial respiratory function, and some cancer cells even show a more active ETC than normal cells because of the rapid turnover (Deribe et al. 2018). Inhibition of the ETC has been shown to exert anti-tumor effects in different types of cancers (Hao et al. 2010; Roesch et al. 2013). However, the poor tumor targeting of these drugs limits their further clinical application. Recently, IR-780, a near-infrared fluorescent dye of both mitochondrial and tumor targeting, has been widely observed and researched. The mechanism of IR-780 selectively accumulates in the mitochondria of tumor has not been clarified. Erlong et al. have found that the selective uptake of IR-780 is associated with high glycolytic levels and plasma membrane potential in tumor cells (Erlong et al. 2014). Wang et al. demonstrated that IR-780 uptake is mediated by mitochondrial transporter ABCB10, which may be the reason why IR-780 accumulates in mitochondria (Wang et al. 2018a). Current strategies for IR-780 mainly involve nanotechnology or multistep chemical coupling of numerous functional agents, including tumor-specific ligands and anti-tumor drugs (Cao et al. 2017; Uthaman et al. 2018; Yan et al. 2016). Although these strategies have revealed some anti-tumor effects, there are still great challenges hindering their further application, such as adverse immunogenic reactions, inefficient drug delivery systems, the complexity of nanomedicine, and difficulties in large-scale manufacturing. (Desai 2012; Zhu et al. 2022). 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 et al. 2015; Lucky et al. 2015), it is still difficult to apply it to internal organs in the human body because the NIR excitation cannot reach these organs. Since oxygen can be transported to various tissues in the body during HBO treatment, in this study, we chose oxygen as a sensitizer of IR-780. 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. NDUFS1 is the largest subunit in complex I (thus considered as a marker of complex I), has nicotinamide adenine diphosphate hydride (NADH) dehydrogenase and oxidoreductase activities, can transfer electrons from NADH to the respiratory chain. Under normal physiological conditions, the premature leak of electrons from complex I to mediate the one electron reduction of oxygen to superoxide, which is considered to be the most significant source of ROS in mitochondria((Nolfi-Donegan et al. 2020). Disruption of mitochondrial complex I has been reported to increase the electron leakage from ETC and enhance the ROS generation (Fang and Maldonado 2018; Zhao et al. 2022). In this study, we found that 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. 6C), 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 et al. 2019). HBO, which is used as an adjuvant treatment with chemotherapy, has been demonstrated to significantly improve the efficacy of various chemotherapeutic drugs (Stępień et al. 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 anti-tumor 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 et al. 2003), which will largely 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 be related to the 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, and the results indicated that plasma membrane potential plays a critical role in IR-780 iodide uptake in tumor cells (Zhang et al. 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 et al. 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 et al. 2000). In the current study, our data showed that HBO increased the ROS production in bladder cancer cells pretreated with IR-780. 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. Checkpoint inhibitors represent a recent treatment for cisplatin resistance bladder cancer. Unfortunately, only 20% to 30% of patients show a clinical response, and long-term data indicate no improvement in disease-specific survival, new therapeutics are sorely needed (Teo and Rosenberg 2018). 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 ETC and causing death of DPP-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, IR-780 could obviously promote apoptosis of T24/DDP cells, suggesting a new therapeutic strategy for DDP-resistant bladder cancer.

Conclusion

In this study, we identified a mitochondria-targeted fluorescent small molecule, IR-780. It can selectively target the mitochondria of bladder cancer cells, including DPP-resistant cells, and induce cancer cell apoptosis by targeting the electron transport chain. Moreover, when combined with HBO, IR-780 exhibited effective anti-tumor activity by promoting cancer cell uptake of IR-780 and inducing excessive mitochondrial ROS production. This discovery potentially offers a novel treatment paradigm for human bladder cancer.

Materials and methods

Materials

IR-780 iodide, 2-thenoyltrifluoroacetone (TTFA), diphenyleneiodonium chloride (DPI), H₂O₂, and N-acetylcysteine (NAC) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Cis-diamminedichloroplatinum (DDP) and doxorubicin hydrochloride (DOX) were obtained from Aladdin (Shanghai, China). MitoTracker Green, MitoSOX, and the mitochondrial membrane potential indicator TMRM were purchased from Invitrogen (Carlsbad, CA, USA). A Complex I Enzyme Activity Microplate Assay was purchased from Abcam (Cambridge, MA, USA). DiBAC4(3) was purchased from MedChem Express (Monmouth Junction, NJ, USA). A Reactive Oxygen Species (ROS) Assay Kit, Hoechst 33,258, Crystal Violet Staining Solution, and an ATP Assay Kit were obtained from Beyotime (Shanghai, China). Primary antibodies were rabbit (Rb) anti-Cytochrome C (1:1000, CST, 11940 T), Rb anti-Ndufs1 (1:1000, abcam, ab169540), Rb anti-Caspase-9 (1:1000, abcam, ab202068), Rb anti-Caspase 3 Antibody (1:1000, proteintech, 19,677–1-AP), Rb anti-cleaved Caspase 3 Antibody (1:1000, abcam, ab13847), Rb anti-PARP (1:1000, proteintech, 13,371–1-AP) and mouse (Ms) anti-β-action (1:5000, proteintech, 6008–1-Ig).

Cell lines

Human bladder cell lines (T24, 5637, and TCCSUP) were purchased from the ATCC (Manassas, VA,

USA), and human normal cell lines (HK2 and SV-HUC-1) were obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China). A mouse bladder cell line (MB49) was donated by Dr. Chunmeng Shi from the Institute of Rocket Force Medicine, Third Military Medical University (Chongqing, China). All cells were cultured in their recommended medium supplemented with 10% FBS (Gibco, USA) and 1% penicillin/streptomycin (Beyotime). All cells were passaged at 1:3 ratios and cultured in an incubator at 37 °C with 5% CO₂. T24/DDP, a DPP-resistant bladder cancer cell line, was established by a concentration gradient method.

HBO exposure

An HBO exposure experiment was performed in a temperature-adjustable hyperbaric chamber provided by the neurosurgery department of Southwest Hospital (Chongqing, China). The HBO administration was performed by giving 2.5 atmospheres absolute (ATA) of pure oxygen for 120 min, and the program included 15 min of compression and decompression at a constant velocity. The temperature was set to 37 °C for cells, and 25 °C for animals. Cells in different plates were removed from the chamber after HBO exposure and cultured routinely in an incubator.

Tumor models

MB49 cancer cells grown to 70% confluence were suspended in phosphate-buffered saline (PBS). MB49 transplanting tumor model were established in female C57BL/6 mice (6–8 weeks old and weighing 17-20 g) by subcutaneously implanting each mouse with 1*10⁷ cells. T24, T24/DDP tumor xenografts were established in female nude mice in the same way.

Optical imaging

In vivo fluorescence imaging: mice bearing T24, MB49 transplanted tumors were sacrificed at 24 h after intraperitoneal injection with IR-780 at a dose of 1 mg/kg. The dissected organs were subjected to NIR imaging with a Kodak In-Vivo FX Professional Imaging System (New Haven, CT). Cellular dye uptake and the subcellular localization: cells of different cell lines (T24, MB49, HK2, and SV-HUC-1) were seeded at the same densities in culture dishes and cultured for 24 h. Then, the cells were incubated with 2.5 μM IR-780 in 1640 culture medium for 15 min in the incubator before being incubated for 20 min with 1 μM MitoTracker Green. Nuclei was stained with Hoechst 33,258. Finally, these cells were imaged under a confocal microscope.

Cell viability evaluation

Bladder cancer cell lines of different grades (T24, 5637, TCCSUP, T24/DDP and MB49) were treated with the indicated doses of IR-780 with or without HBO exposure, and cell proliferation after 48 h was measured by using a CCK-8 kit (Dojindo Kumamoto, Japan). T24 cells after treatment with or without different inhibitors were placed in 7.5 μM IR-780 + HBO, and cell viability was detected after 48 h.

In vivo IR-780 + HBO evaluation

In vivo, treatment was administered on the fifth day of transplanted tumor establishment through intraperitoneal injection of 3 mg/kg IR-780. The IR-780 + HBO group and the HBO-only group were then placed in a hyperbaric chamber 4 h after IR-780 injection. The change in tumor volume was monitored every other day during the experiment. The mice, administered every two days for a total of 5 interventions, were weighed and sacrificed, and the transplanted tumors from the animals were photographed and weighed. Subsequently, the tumor tissues and organs were dissected and fixed in 4% paraformaldehyde. Hematoxylin and eosin (H&E) staining was performed to observe the toxicity in normal tissues. To study the long-term development of tumors after IR-780 + HBO treatment, tumor volume was monitored in the IR-780 + HBO group and DDP group. After 5 injections of 3 mg/kg IR-780 and 3 g/kg DDP, the mice were continuously fed for 28 days. To compare the anti-tumor effect of IR-780 + HBO and DDP on drug-resistant bladder cancer, 3 mg/kg IR-780 and 3 mg/kg DDP were injected into transplanted tumor mice in the IR-780 group and DDP group respectively. After the treatment for a total of 5 interventions, the mice were photographed and sacrificed, and the transplanted tumor from the animals were photographed and weighed.

Flow cytometry

Apoptosis was detected by flow cytometry using an Annexin V/PI detection kit (BD Biosciences) according to the protocol description of the assay. For the ROS assay, cells were incubated with the probe DCFH-DA at a concentration of 10 μM after the different treatments for 30 min at 37 °C, and then flow cytometry was used for fluorescence intensity detection. For the mitochondrial ROS assay, cells subjected to the different treatments were incubated with the probe MitoSOX at a concentration of 5 μM for 10 min at 37 °C, and then flow cytometry was used for fluorescence intensity detection. For the plasma membrane potential assay, cells were incubated in KRH (Krebs–Ringer-HEPES) buffer containing 100 nM DiBAC4(3) for 20 min at room temperature before flow cytometry detection. For the mitochondrial membrane potential (MMP) assay, TMRM was also detected by flow cytometry.

Colony formation assay

After intervention for 6 h, cells were seeded in six-well plates at a density of 1000 cells per well and cultured overnight to attach. The medium was replaced with fresh medium after 24 h, and the cells were cultured for 12 days. Subsequently, the cells were fixed with paraformaldehyde for 10 min and subjected to crystal violet (Beyotime, China) staining for 5 min. The samples were photographed, and the colonies > 0.2 mm in diameter were counted under a microscope.

Transmission electron microscopy (TEM)

After 24 h of treatment, cells were collected and fixed overnight at 4 °C in 2.5% glutaraldehyde. Then, the cells were subjected to secondary fixation in 2% osmium tetroxide followed by embedding in resin. Images of thin sections were obtained by TEM.

Western blot analysis

Cells were harvested after treatment for 24 h, and total proteins were extracted with RIPA lysis buffer (Beyotime). After separation using 12% SDS–polyacrylamide gels, the proteins were transferred onto PVDF membranes (Millipore, USA). The PVDF membranes were blocked with Quick Blocking Buffer (Beyotime) for 15 min and then incubated overnight with primary antibodies at 4 °C. The antibodies described above were used. After incubation for 1 h with secondary antibodies in an incubator at 37 °C, the signals were identified by chemiluminescence detection.

Statistical analysis

All data were expressed as the means ± standard derivation at least three independent experiments. Statistical analysis of experimental data was performed using GraphPad Prism 8.0. Comparisons between two groups were performed using the student’s  t-test, one-way analysis of variance (ANOVA) or two-way ANOVA were used for multiple groups. P-values < 0.05 were considered to indicate statistical significance.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

LWB & SCM conceived and designed the experiments, supervised the experiments and revised the manuscript. SCX performed the experiments, analyzed the data and drafted the manuscript. YXF and WJW took part in the animal experiments, drafted the manuscript and analysis of optical properties. DLY and LJJ participated in Flow Cytometer and western blot analysis. FQ and ZY contributed to collecting human tissues. All authors have read and approved the final manuscript.

Funding

This work was supported by National Natural Science Foundation of Chongqing Province (No.cstc 2018jcyjAX0034), National Key Research and Development Program (2016YFC1000805, to C. Shi), National Natural Science Foundation of Chongqing Province (No.cstc 2021 jcyj-bsh0238 to X. Yue), Key research incubation in the Third Affiliated Hospital of Chongqing Medical University (KY08026, to Y. Zhi), Science and technology project of Yubei District, Chongqing (2021-NS-38, to Y. Zhi).

Availability of data and materials

The datasets used during the current study are available from the corresponding author on reasonable request.

Declarations

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval and consent to participate

The collection of human tumor samples and related procedures were approved by the Ethical Committee of the Third Affiliated Hospital, Chongqing Medical University. The animal experiments in our study were conducted in accordance with the Helsinki Declaration and approved by the Laboratory Animal Welfare and Ethics Committee of Third Military Medical University.

Consent for publication

Not applicable.