IR-780 selectively accumulates in the mitochondria of bladder cancer cells
The ability of IR-780 to selectively accumulate in bladder tumors was studied with T24 and MB49 transplanted tumors. The organs of nude mice with pre-established T24 tumor xenografts and C57 BL/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 shown in Fig. 1A and B confirmed the preferential aggregation of IR-780 in tumors at 24 h after injection. 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. Fig. S1 revealed that the accumulation of IR-780 in tumors gradually increased with time, 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 and D), indicating that the NIR small-molecule dye IR-780 had the ability to target 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. 1E); HK2 and SV-HUC-1 cells were used as controls.
IR-780 induces cancer cell apoptosis bytargeting the mitochondrial electron transport chain
Since IR-780 has the ability to target bladder cancer cell, its antitumor effect should be investigated. Fig.2A shows that IR-780 could inhibit bladder cancer cells (T24, 5637, TCCSUP, MB49) proliferation in a dose-dependent manner (p<0.05). The apoptosis rate of T24 cells after incubation with 15 μM IR-780 was more than 30% compared with 3% in the control group (Fig. 2B; p<0.05), suggesting that IR-780 could induce T24 cells apoptosis in a does-dependent manner. As we confirmed that IR-780 specifically accumulated in mitochondria, the influence on mitochondrial function should be studied. Fig. 2C demonstrates that IR-780 markedly increased mitochondrial ROS, reduced cellular ATP production (Fig. 2D) and 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., 2018). Clearance of the NDUFS1 protein disturbed electron transport in the ETC, which led to considerable electronic leakage, promoting mitochondrial ROS production.
HBO promotes IR-780 antitumorefficacy in vitro
To investigate the antitumor 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. Fig. 3A shows that IR-780+HBO inhibited cancer cell proliferation in a dose-dependent manner and displayed better anticancer activity than IR-780 alone. The 7.5 μM IR-780 +HBO treatment almost achieved the efficacy of the 15 μM IR-780 treatment in T24 cells and had little effect on normal cells (Fig. S2A). T24 cells were treated with 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, there was no significant difference between drugs (DOX and DDP) combined or not with HBO. Since bladder cancer cells have the ability to 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 significantly inhibited the colony formation ability of T24 cancer cells (Fig. 3B and C). The cells were obviously wrinkled and rounded upon microscopic examination 16 h after treatment with 7.5 μM IR-780 +HBO (Fig. 3F), which suggested that cell death may have been the main cause of the decrease in cell numbers. As shown in Fig. 3D and E, the apoptosis rate in the 7.5 μM IR-780+HBO group was significantly higher than in the other groups. Moreover, we found that the levels of Cytochrome C and the apoptosis markers PARP, caspase3, and cleaved caspase9 (c-caspase9) were all increased by IR-780+HBO (Fig. 3G). The apoptosis inhibitor z-VAD-FMK partly rescued cell death, increasing cell viability (Fig. 3H). All these results suggested that HBO could enhance IR-780 antitumor efficacy by inducing classic apoptosis in T24 cells.
The antitumor efficacy of IR-780+HBO in vivo
Based on the excellent antitumor 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 two days for a total of 5 injections. Analysis of the volumes of the tumors in the different intervention groups showed that the IR-780+HBO group exhibited the best tumor inhibition among the groups (Fig. 4A). 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. 4G), 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 the classic antitumor drug cisplatin. Interestingly, the volumes of the tumors in the mice subjected to IR-780+HBO therapy did not change significantly, while those of the tumors in mice subjected to DDP therapy continued to increase after intervention (Fig. 4E). On day 28, the tumors from mice treated with IR-780+HBO were much smaller than those from mice treated with DDP (Fig. 4F). 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.
HBO promotesuptake of IR-780 in cancer cells byincreasingplasma