Dual-target cancer theranostic for glutathione S-transferase and hypoxia-inducible factor-1α inhibition

Zan Li a, Jie Ding b, Chunxia Chen a, Jiayin Chang a, Binghuan Huang a, Zhirong Geng *a and Zhilin Wang *a
aState key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Collaborative Innovation Center of Advanced Microstructures, Nanjing University, Nanjing 210093, P. R. China. E-mail: gengzr@nju.edu.cn; wangzl@nju.edu.cn
bDepartment of General Surgery, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing 210008, P. R. China

Received 23rd October 2017 , Accepted 30th October 2017

First published on 30th October 2017

We developed a dual-target theranostic F671, which could exhibit synergetic anticancer effects for inhibiting the activities of glutathione S-transferase and the accumulation of hypoxia inducible factor-1α. F671 undergoes self-immolative cleavage when exposed to GSTP1-1 in live cancer cells, facilitating the visualization of molecule release and distribution, as well as confirming the autophagy-induced apoptosis.

Cancer is one of the most intricate and refractory diseases with increasing morbidity in the world, and chemotherapy remains the primary option for cancer therapy. Exposure to anticancer agents may lead to drug resistance, a key element in the failure of chemotherapy.1 Therefore, it is critical to develop effective strategies to overcome drug resistance for cancer treatment.2

Previous studies revealed that glutathione S-transferases (GSTs) have been implicated in the development of carcinogenesis and drug resistance, concluding that GSTs can be destined as protein markers for cancer.3 Among the different GST isoenzymes, GSTP1-1 has received the most attention because of its high levels in tumors.4 Hypoxia inducible factor-1α (HIF-1α), which upregulates the expression of genes associated with tumor initiation, malignant progression, metastasis, and resistance to radiotherapy and chemotherapy, is overexpressed in several human cancers.5,6 Targeting HIF-1α has been proven to significantly slow tumor growth in xenograft models and render hypoxic cells more susceptible to conventional therapies.7 Given the significant roles of GST and HIF-1α in the drug resistance of human cancers, a growing number of therapeutic agents that target GST and HIF-1α have gained considerable traction and attention in the medical and scientific communities.8

Theranostics, combining diagnosis with targeted therapy in one molecular system, have received considerable attention in recent years.9a–c However, the problem of drug resistance when using a mono-target molecular system is still unresolved.9d Synchronous administration of multiple drugs is an attractive way to maximize therapeutic efficacy and minimize the occurrence of multidrug resistance compared to mono-drug treatment. Right now the design and synthesis of dual-target cancer theranostics for enhanced synergetic anticancer efficacy is still challenging.

The synthesized compounds, F671 and F335, were characterized by 1H NMR, 13C NMR and ESI-MS. 7-Nitro-2,1,3-benzoxadiazol (NBD) was introduced into F671 due to its GSTP1-1 targeting and inhibiting abilities. Furthermore, the high fluorescence of F508 (Φ = 0.192) could be quenched by NBD (Fig. S1, ESI).10,11 It is anticipated that upon initiation of F671 (Φ = 0.025) by GSTP1-1, subsequent cleavage of the linker occurs to afford F508, and the HIF-1α level could be decreased simultaneously. In the meantime, the enhanced fluorescence allows for GSTP1-1-overexpressed cancer cells to be imaged and identified (Scheme 1).

image file: c7cc08162f-s1.tif
Scheme 1 Proposed mechanism of the reaction of GSTP1-1 in the presence of GSH.

The apparent dissociation constant of F671 and GSTP1-1 in the presence of GSH is 75.79 μM (Fig. S2, ESI).10a This value is close to the order of magnitude for the IC50 value found for cancer cell lines (Table S1, ESI). The affinity of F671 toward GSTP1-1 distinctly decreased (Kd 877.26 μM) in the absence of GSH, which demonstrated that GSH is vital in the GST inhibition of F671. The relative fluorescence intensity of F671 is stable in the pH range 7.0–10.0 (Fig. S3, ESI), which clearly demonstrated that F671 is stable under physiological conditions. After incubating F671 with GSTP1-1 and GSH, the fluorescence exhibited a steady enhancement (Fig. 1a). However, upon adding the potent GSTP1-1 inhibitor TER 199 into the former system, the fluorescence did not change significantly.4 Similarly, the interaction between F671 and GSH (without GSTP1-1) yielded negligible fluorescence changes (Fig. 1b). The results indicated that GSTP1-1 initiated the decloaking of F671 and revealed free F508 in the presence of GSH. The proposed mechanism is shown in Scheme S2 (ESI).10b As NBD-GSH (Φ = 0.002) had low quantum yields, the fluorescence interference of NBD-GSH could be neglected in the fluorescence spectra and confocal imaging spectra (Fig. S4, ESI).

image file: c7cc08162f-f1.tif
Fig. 1 Spectroscopic properties of F671 (10 μM) in Tris–HCl (0.02 M) solution (DMSO/Tris–HCl = 1[thin space (1/6-em)]:[thin space (1/6-em)]9 v/v, pH 7.4) at 37 °C. (a) Time-dependent fluorescence spectra of F671 to GSTP1-1 (5 U) in the presence of GSH (1 mM). (b) Time-dependent fluorescence intensity of F671. (■), in the presence of GSTP1-1 (5 U) and GSH (1 mM). (image file: c7cc08162f-u1.tif), in the presence of GSTP1-1 (5 U), GSH (1 mM) and TER 199 (200 μM). (image file: c7cc08162f-u2.tif), in the presence of GSH (1 mM). The excitation wavelength was 480 nm. The error bars represent ±S.D. (n = 3).

Moreover, the ESI-MS spectra of the enzymatic reaction solution were examined. The m/z signal at 509.34 ([M + H]+) represents the existence of compound F508 (calculated value is 509.34) and the m/z signal at 469.06 ([M − H]) represents NBD-GSH (calculated value is 469.08) (Fig. S5 and S6, Scheme S2, ESI), further confirming the formation of F508 and NBD-GSH. The in vitro anticancer activity of F671 and other compounds was tested against several human cancer cell lines (HepG-2, HeLa, MCF-7, A549, and A549cisR) and the human hepatic cell line LO2 (Table S1, ESI). F671 exhibited approximately 1.5-fold higher cytotoxicity against HepG-2 cells than against LO2 cells. Intriguingly, the cytotoxicity toward A549cisR was slightly higher (1.3-fold) than toward A549, which indicated that F671 is more efficient when treating a drug resistant cancer cell line.

GSTP1-1 is overexpressed in many cancer cell lines, where the estimated concentrations of GSTP1-1 may reach up to 50 μM.10a So we employed F671 for imaging GSTP1-1 in HepG-2 cells (Fig. 2a). The green fluorescence from the cytoplasm showed an obvious 8-fold enhancement after 6 h (Fig. 2a and c1), indicating that F671 was consumed by GSTP1-1 in the cytoplasm. This is consistent with evidence that the cytosolic GSTP1-1 accounts for the predominant part of the total GST protein.12 Conversely, the fluorescence enhancement was remarkably reduced in the presence of TER 199 (Fig. 2c1). In the LO2 cells, the green fluorescence changes of F671 were barely recognizable from 0.5 h to 6 h (Fig. 2b and c2). The fluorescence images of F508 and DND-99 can be merged well in the colocalization experiments (Fig. S7, ESI). The Pearson Coefficient is 0.85, which confirmed that F508 can specifically target the lysosomes. Above all, the experimental results demonstrated that F671 can be applied for the fluorescence imaging of GSTP1-1 in cancer cells and for fluorescently distinguishing cancer cells from normal cells.

image file: c7cc08162f-f2.tif
Fig. 2 Time-dependent GSTP1-1 imaging in HepG-2 cells and LO2 cells. Cells were cultured with F671 (1 μM) for 20 min. Confocal fluorescence images were obtained at different time points. (a) Confocal imaging after incubation of the HepG-2 cells with F671 (top) and TER 199 (200 μM) + F671 (bottom). (b) Confocal imaging after incubation of the LO2 cells with F671 (top) and TER 199 (200 μM) + F671 (bottom). (c) Quantitative analysis of the fluorescence changes of F671 in the absence or presence of TER 199 in panels (c1) and (c2).

The intracellular fluorescence distribution altered dramatically while incubating with F671 (Fig. 3). At 12 h, the green fluorescence was distributed in the cytoplasm, while at 18 h, the fluorescence was concentrated mainly in the lysosomes. The fluorescence was transformed into the green punctate dots after 24 h. Moreover, cell apoptosis was evaluated by quantitatively measuring cellular propidium iodide (PI) uptake using flow cytometry analysis. Only 2.1% of the HepG-2 cells exhibited positive PI uptake after 0.5 h (Fig. S8, ESI), which indicated that a short time of incubation with F671 did not induce cell apoptosis. The percentage of PI positive cells was 99.4% at 24 h, which indicated that F671 caused apoptosis after a long incubation time. The co-staining and PI staining images in Fig. S9 (ESI) indicate that F508 could also induce lysosome morphology variation and cell apoptosis. The integrity of the lysosome membrane in HepG-2 cells was evaluated by the activity of released β-N-acetylglucosaminidase (NAG). Compared with the control group, the activity of the released NAG was remarkably enhanced after treating with F671 and F508 (Fig. S10, ESI). It is concluded that the lysosome membrane was disrupted by F671 and F508.

image file: c7cc08162f-f3.tif
Fig. 3 Lysosome morphology variation in HepG-2 cells after different incubation times (12 h, 18 h and 24 h) with F671 (1 μM) treatment. (a1, b1 and c1) The green fluorescence distribution. (a2, b2 and c2) Lyso-Tracker DND-99 staining reveals the morphological alterations of the lysosomes. (a3, b3 and c3) The merged images of green fluorescence and red fluorescence. (a4, b4 and c4) Flow cytometry analyses of the propidium iodide positive cells.

To further verify the formation of F508 in living cancer cells, the lysosomes of the HepG-2 cells after incubation with F671 were isolated. As shown in Fig. S11 (ESI), the m/z signal at 509.32 ([M + H]+) represents the existence of F508, demonstrating the generation of F508 in the lysosomes of HepG-2 cells. The results supported the proposed reaction raised formerly and further confirmed the exact formation of F508 in living cells, as well as the distinct fluorescence response of GSTP1-1.

Previous studies revealed that nitrogen-containing molecules and metal complexes can induce autophagy in cancer cells.13 We further investigated whether F671 could induce autophagy in HepG-2 cells by transmission electron microscopy (TEM).14 Most of the cisplatin- and F671-treated cells were found to display morphological characteristics of autophagy compared with the control group (Fig. S12, ESI). Western blot analysis showed that the ratio of LC3-II/LC3-I is markedly enhanced after F671 treatment (Fig. S13a, ESI).15 Collectively, F671 could induce autophagic apoptosis of cancer cells and could be utilized to trace the autophagic apoptosis process with bright green fluorescence.

Our laboratory has exploited manganese complexes as neuroprotection agents based on the regulation of HIF-1α levels.16 The effect of F671 on HIF-1α accumulation in HepG-2 cells was studied by western blot analysis.17 As illustrated in Fig. 4, F671 suppressed HIF-1α accumulation in a dose-dependent manner. Reverse transcription-PCR analysis showed that F671 exerted no effect on the mRNA level of HIF-1α, indicating that the decreased concentration of HIF-1α was regulated at the protein level. As indicated in Fig. S14 (ESI), accumulation of HIF-1α was also suppressed by F508 in an analogous manner to that of F671, which confirmed that the inhibition of HIF-1α by F671 is actually executed through F508. Therefore, F508 is the key element in targeting HIF-1α.

image file: c7cc08162f-f4.tif
Fig. 4 Effects of F671 treatment on protein and mRNA levels of HIF-1α in HepG-2 cells under hypoxia (1% O2). HepG-2 cells were incubated with various concentrations of F671 for 24 h. (a) Western blot analysis of HIF-1 α accumulation in whole cell extracts. β-Actin was used as a loading control. (b) RT-PCR analysis of HIF-1α gene expression. β-Actin was used as a loading control. (c) Quantification of HIF-1α protein expression levels by densitometry relative to β-actin. (d) Quantification of HIF-1α gene expression levels by densitometry relative to β-actin. The error bars represent ±S.D. (n = 3).

The fluorescence intensity of dihydroethidium (DHE) in HepG-2 cells is independent of concentration and time (Fig. S15 and S16, ESI), indicating that the generation of excess ROS does not occur in the presence of F508 and F671. This in turn prevents the ROS-mediated inhibition of the prolyl hydroxylase domain (PHD), and consequently reduces HIF-1α protein levels under hypoxic conditions.18 The mode of action of F671 can also be regarded as a restoration of PHD activity.19 As shown in Fig. S13b (ESI), a down-regulation of Bcl-220 expression was observed after treating HepG-2 cells with various concentrations of F671. The active-caspase-3 (17 kDa)21 level was enhanced with the increasing concentrations of F671 (Fig. S13c, ESI). Thus, F671 acts as a potent inhibitor to suppress the Bcl-2 levels, while promoting the induction of apoptosis in HepG-2 cells.

The in vivo therapeutic efficacy of F671 was evaluated using tumor-bearing BALB/c mice. There was no statistically significant difference in the weight changes among the groups over 16 days (Fig. 5b). However, the tumor volume of the control group increased rapidly compared with other groups (Fig. 5a). The mice administrated with a high-dose of F671 showed an obvious suppression of the tumor compared to other doses (Fig. 5c), indicating effective antitumor therapy in vivo. In addition, there was no fluorescence signal from other organs of the F671-treated group, while only the tumor was fluorescent in ex vivo fluorescence imaging (Fig. 5d and Fig. S17, ESI). This enhanced fluorescence signal in the solid tumor sample indicated the tumor-specific accumulation of F671, which is consistent with the in vitro fluorescence results in Fig. 2.

image file: c7cc08162f-f5.tif
Fig. 5 Antitumor efficacy of F671. (a) The HepG-2 tumor growth curves after different treatments. (b) The body weight variation of HepG-2 tumor-bearing mice during treatment. (c) Representative images of the HepG-2 tumors after dealing with different treatments on day 16 (1: control, 2: low-dose, 3: medium-dose, 4: high-dose). (d) Fluorescence images of the main internal organs after anatomy dissection (1: control group, 4: high-dose group). The error bars represent ±S.D. (n = 3). *P < 0.05, **P < 0.01.

In summary, we report a novel synergetic anticancer theranostic, F671, which undergoes self-immolative cleavage when exposed to GSTP1-1 in live cancer cells. Thus the imaging and distinguishing of GSTP1-1-overexpressed cancerous cells is realized with the aid of F671. The released F508 in live cells could act as a potent inhibitor to suppress the levels of HIF-1α, and facilitate the visualization of molecule release and distribution, as well as the verification of autophagic apoptosis induction. More importantly, F671 displays anticancer activity in various cancer cell lines and the HepG-2 tumor-bearing murine model. The specific cytotoxicity toward cancer cells and drug resistant cancer cells renders F671 a promising antitumor agent. To the best of our knowledge, this is the first time a dual-target BODIPY-based theranostic agent tested in vitro and in vivo has been reported.

This work is supported by the National Natural Science Foundation of China (21475059, 21527809 and 21775069).

Conflicts of interest

There are no conflicts to declare.

Notes and references

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Electronic supplementary information (ESI) available. See DOI: 10.1039/c7cc08162f

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