Hydrogen sulfide triggered molecular agent for imaging and cancer therapy

Article abstract of DOI:10.1039/d0cc07982k

We developed an activatable molecular reagent, PNF, triggered by intracellular H₂S in the lysosome to release the therapeutic drug amonafide, which can escape from the lysosome into the nucleus to induce autophagy of cancer cells. PNF exhibits potent inhibitory activity against cisplatin-resistant tumor cell lines.

Full text of DOI:10.1039/d0cc07982k

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Hydrogen sulfide triggered molecular agent
for imaging and cancer therapy†
Cite this: Chem. Commun., 2021,
7, 1931
a
a
bc
a
a
a
5
Chao Ge,
Xiaojiao Di, Siqi Han, Mengmeng Wang, Xiaoting Qian, Zhi Su,
a
a
Received 8th December 2020,
Accepted 20th January 2021
Hong-Ke Liu * and Yong Qian
*
DOI: 10.1039/d0cc07982k
rsc.li/chemcomm
We developed an activatable molecular reagent, PNF, triggered by such as high glutathione levels, reduction species, and gaseous
9
intracellular H
amonafide, which can escape from the lysosome into the nucleus to
induce autophagy of cancer cells. PNF exhibits potent inhibitory diseases, such as diabetes, liver cirrhosis, Alzheimer’s disease,
2
S in the lysosome to release the therapeutic drug signal molecules, especially hydrogen sulfide (H S).
2
Dysregulation of H S has been found that linked to many
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1
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activity against cisplatin-resistant tumor cell lines.
hypertension, and Down’s syndrome. Recently, increasing
evidence suggested that cancer cells also produce large
Theranostic approach, an emerging technology in biomedicine, amounts of H
2
S, and their proliferation, migration, and inva-
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exhibits potential therapeutic utility and imaging capabilities, sion are dependent on the content of H
2
S.
Overexpressed
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leading to the extensive development of theranostic prodrugs.
H S has been identified as a biomarker for various cancers,
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Typically, these prodrugs are equipped with fluorophores, such and efficient tools for the visualization of endogenous H2S
as naphthalimide, coumarin, BODIPY, rhodol, and Cy7, which should be valuable for exploring H S-related biology and
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1
4
monitor the drug release process based on their fluorescence further for diagnostic and therapeutic purposes. Thus, many
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signals. Naphthalimide moiety has been widely used for the
preparation of responsive sensors because it exhibits good different strategies, in which some of them have been utilized
stability, strong fluorescence with large Stokes shifts, and to image H S either by exogenously supplemented or in situ
2
H S-specific fluorescence probes have been developed with
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5
2
3
,4
16,17
feasibility of structure modification. Moreover, the develop- synthesized by enzymes in live cells.
On the other hand, by
ment of functional 1,8-naphthalimide derivatives as anticancer utilizing highly expressed endogenous H S as a trigger, several
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or neuroprotective agents is a fast-growing field (Scheme S1, H S-activated nanoplatforms have also been reported with high
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5
,6
18,19
ESI†) and several derivatives have entered clinical trials. As therapeutic efficacy against cancer.
However, few H S-activated
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an early representative, amonafide (ANF) has been selected for fluorescent small-molecule systems have been designed for the
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the treatment of cancer in phase I and II clinical trials. It can integration of cancer diagnosis and treatment.
induce DNA strand breakage, protein–DNA crosslinking, and
Considering the above-mentioned information, herein, we
inhibition of nucleic acid synthesis. However, due to limited have developed a fluorogenic small molecule platform that can
intracellular accumulation and possible cross-resistance be activated by selective H S-mediated azide reduction for the
8
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mechanisms, ANF has shown limited therapeutic efficacy in synergic treatment of drug-resistant lung cancer. We termed this
drug-resistant cell lines. We envisioned that these defects small-molecule as PNF, in which the azide moiety acts as a
might be partially overcome by developing more hydrophobic triggered group that can be reduced to an amino group by H S,
2
forms of small-molecules to increase cell uptake, which can be thereby releasing active amonafide (ANF) and turning on its
activated and released in cancer-specific microenvironments, fluorescence (Scheme 1). The activated ANF was found to escape
from the lysosome into the nucleus, further inducing DNA
a
damage. Furthermore, mitochondrial autophagy (mitophagy)
and the down-regulated expression of multidrug-resistant protein
1 (P-glycoprotein) were observed in PNF treated cisplatin-resistant
lung cancer cells (A549R). Our observations indicated that PNF
can serve as a potential anticancer agent towards drug-resistant
cancers, which providing cytotoxic chemotherapy response
and promoting fluorescence imaging of cancer cells to monitor
cellular uptake, activation, and release processes of the drug.
School of Chemistry and Materials Science, Jiangsu Key Laboratory of
Biofunctional Materials, Nanjing Normal University, Nanjing, 210023,
P. R. China. E-mail: liuhongke@njnu.edu.cn, yongqian@njnu.edu.cn
Department of Medical Oncology, Jinling Hospital, The First School of Clinical
Medicine, Southern Medical University, Nanjing, 210002, P. R. China
Department of Medical Oncology, Jinling Hospital, School of Medicine,
Nanjing University, Nanjing, 210002, P. R. China
b
c
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0cc07982k
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Scheme 1 The activated mechanism of molecular agent PNF to form the
anticancer drug amonafide (ANF) triggered by H
resistance lung cancer cells (A549R).
2
S within cisplatin-
Fig. 1 (a) Fluorescence spectra of prodrug PNF (10 mM) in PBS buffer
(10 mM, 5% DMSO, 1 mM CTAB, pH 7.4) recorded in the presence and
absence of H
2
S (100 mM). (b) Fluorescence responses of PNF (10 mM)
towards increasing concentrations of NaHS (0, 5, 10, 15, 20, 25, 30, 40, 50,
ANF was synthesized according to the pathway as shown in 60, 70, 80, 90, 100 mM) for 1 h. (c) The linear relationship between the
emission at 598 nm and the concentrations of NaHS (0, 5, 10, 15, 20, 25,
30, 40, 50 mM) in PBS buffer. (d) The selectivity of PNF (10 mM) for various
relevant analytes, NaHS (100 mM), other reactive oxygen species (200 mM
Scheme S2 (ESI†). ANF was obtained following previously
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reported methods,
and then reacted with concentrated
sulfuric acid and sodium nitrite to obtain a reactive intermediate,
À
À
), sulphur-
ClO , H
2
O
2
, TBHP), reactive nitrogen species (200 mM NO
2
2À
2À
which was continuously reacted with sodium azide to yield the containing inorganic ions (200 mM S O3 , SO3 ), and inorganic salts
2
2 2 4
desired compound, PNF. The relevant products were well char- (1 mM NaCl, 1 mM KCl, 100 mM FeCl , 100 mM CuCl , 100 mM ZnSO , 1 mM
1
13
MgCl , 1 mM CaCl , 200 mM Na CO , 200 mM NaHCO , 200 mM Na SO ,
acterized by H, C NMR spectroscopy, mass spectrometry (MS),
and infrared spectroscopy (IR) (Fig. S1, ESI†).
2
2
2
3
3
2
4
2
00 mM NaHSO
4
, 200 mM NaH
2
PO
4
2
, 200 mM Na HPO
4
, 100 mM NaOAc).
The error bars were Æ SD (n = 3). Excitation: 415 nm, slit/slit: 10 nm/10 nm.
We first investigated the fluorescence response behavior of
PNF towards H S in vitro. As shown in Fig. 1a, PNF exhibited a
2
barely visible emission peak at around 598 nm in the absence respectively. Taken together, these observations demonstrate
of NaHS. Interestingly, PNF showed a robust fluorescence that under physiological conditions, PNF can be selectively
response to NaHS, with maximum emission at 598 nm within activated by H
S as a potential anticancer agent.
2
1
h. The fluorescence intensity gradually increased and the
Cell viability experiments were carried out to investigate
saturation point was reached after 8 h treatment with 100 mM PNF’s cytotoxicity towards eleven cancer cell lines, including
NaHS (Fig. S2, ESI†). The change of fluorescence was further A549, A549R, A2780, A2780R, HCT116, A261, U87MG, MCF-7,
confirmed by ultrafast liquid chromatography (UFLC) (Fig. S3, K562, NB-4, and HeLa cells. The prodrug PNF exhibited strong
ESI†). The pH effect studies suggested that PNF had excellent anti-proliferative activity, with IC50 values in the range of
stability and significant fluorescence signal could be released 1.3–8.4 mM (Table S1, ESI†). PNF showed higher biological
between pH 4.5 and 6.0 by NaHS stimulation (Fig. S4 and S5, activity than ANF, especially against drug-resistant cancer cells.
ESI†). Moreover,
enhancement was observed under 0–100 mM NaHS treatment the introduction of the azide group, which increased the
Fig. 1b). An excellent linearity between emission intensity was lipophilicity of PNF and enhanced the cellular uptake rate.
a
concentration-dependent fluorescent The log P of PNF (1.35) was higher than ANF (À0.45) because of
(
observed in the range of 0–50 mM (Fig. 1c). The detection limit Importantly, PNF showed strong cytotoxicity towards cisplatin-
was 0.33 mM, which was much lower than the endogenous resistant cancer cells, such as A549R and A2780R. The anti-
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concentration of H S. These findings suggest that PNF has proliferative potential of PNF against A549R cell lines was
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the capability for detecting the intracellular H S and can be increased 6-folds compared to ANF. Together, these results
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triggered by intracellular H
To examine the selectivity and anti-interference ability of drug-resistant cancer cells (Table S2, ESI†).
PNF towards H S, we subsequently treated various biologically
We assessed the fluorescence response of prodrug PNF in
2
S.
suggest that PNF exhibits potential cytotoxic activity against
2
relevant species with PNF, including inorganic salts, reactive live A549R cells. After pretreatment with PNF, A549R cells were
oxygen species (ROS), reactive nitrogen species (RNS), reactive supplied with NaHS (50, 100, and 200 mM), the strong turn-on
sulfur species (RSS), and other amino acids. We found that PNF fluorescence of PNF was observed (Fig. S8, ESI†). The investiga-
was highly selective for NaHS (Fig. 1d and Fig. S6, ESI†). The tion on the stability of PNF in the culture medium showed that
interference of other biological species was almost negligible there is no non-specific fluorescence recovery interfering with
(
Fig. S7, ESI†). We noted that the emission intensity was the experimental results (Fig. S9, ESI†). In addition, A549R cells
reduced only in the presence of NaHS and FeCl , FeCl , CuCl
were pretreated with DL-propargylglycine (PPG, decreasing
ZnSO
, which may be attributed to the oxidation of NaHS by endogenous H S) or sodium nitroprusside (SNP, stimulating
FeCl and the precipitation of NaHS by FeCl , CuCl , ZnSO
endogenous H
2
3
2
,
4
2
3
2
2
4
,
2
S), and then were further incubated with PNF
1932 | Chem. Commun., 2021, 57, 1931À1934
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10 mM). Negligible fluorescence emission was observed in the
Communication
(
presence of PPG, while a significant enhancement of fluo-
rescence was observed in the presence of SNP. Moreover, fluores-
cence intensity can be further inhibited by co-treatment with PPG
and SNP (Fig. S10, ESI†). These data confirm that PNF can be
applied for imaging exogenous and endogenous H
It is reported that H S can be produced in mitochondria and
To investigate the targeting properties of PNF in
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S.
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lysosomes.
cells, co-localization imaging experiments were performed. PNF
was mainly localized in lysosomes (Pearson’s correlation 0.81)
and partially in mitochondria (Pearson’s correlation 0.22)
(Fig. S11, ESI†). We also tracked the activation process of PNF
in cancer cells by following the cellular fluorescence. A549R
cells were incubated with PNF (5 mM) for 6 h, 12 h, 24 h,
respectively (Fig. S12a, ESI†). The fluorescence intensity of PNF-
treated cells was enhanced in a time-dependent manner, which
was further confirmed by flow cytometry analysis (Fig. S12b,
ESI†). These findings indicated that the fluorescence can be
gradually released from PNF in the H S overexpressing cancer
2
cells. Interestingly, after incubation with PNF for 24 h, we Fig. 3 (a) Representative TEM images of A549R cells treated with PNF (5 mM)
for 24 h, emphasizing the region of mitophagy. (b) Immunoblotting of LC3
observed a distinct fluorescent signal in the nucleus (Fig. S13,
ESI†), suggesting that activated PNF escaped from the
lysosomes to the nucleus. Furthermore, the propidium iodide
and P-glycoprotein in A549R cells treated with PNF for 24 h. (c) The cell
viability of A549R tumor spheroids with different treatments. Tumor spheroids
were treated with PNF (20 mM, or 40 mM) for 48 h. After incubation, the live/
dead cells were stained by AO/EB and then fixed with 4% paraformaldehyde,
(PI) method was performed to study the cell cycle for A549R
cells treated with the prodrug PNF (Fig. S14, ESI†). It was worth the spheroids were finally visualized by a confocal microscope.
noting that PNF treatment caused obviously dose-dependent
cell cycle arrest in the G2/M phase, increasing from B10.4% to mitochondria undergoing progressive degradation (late stage
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B49.6% (Fig. 2a). gH2AX (a biomarker for DNA damage) and of mitophagy). As shown in Fig. 3a, damaged mitochondria,
p53 (tumor suppressor) were also assessed by immunoblotting. the cristae folds on the inner membrane extended to onion-like
PNF treated A549R cells induced obvious increases in both circles, were encapsulated in vacuoles (early stage of
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gH2AX and p53 expression (Fig. 2b), further suggesting that mitophagy), indicating that they were removed from cells
PNF was activated to form ANF escaping from lysosomes to the through auto-phagosomes. Since the encapsulation of mito-
nucleus and induced DNA damage in A549R cells.
chondria in lysosomes is an important hallmark of mitophagy,
Furthermore, we investigated deeply into the anticancer the co-localization of mitochondria and lysosomes was tested.
mechanisms of PNF. Considering that the drug is mainly A remarkable overlap was observed in confocal images of the
localized in the lysosomes, which is closely related to the PNF-treated A549R cells that simultaneously labeled with Mito-
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5
regulation of autophagy. The effect of PNF on A549R cells Tracker Red and LysoTracker Green dyes, whereas no signifi-
was directly examined by transmission electron microscopy cant overlap was observed in the controls (Fig. S16, ESI†). This
(
TEM). As an indicator of autophagy, the formation of auto- was also consistent with the increased levels of LC3-II (Fig. 3b),
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phagic vacuoles was clearly visible in TEM images after treat- a biomarker of autophagosome formation. These results
ment with PNF (Fig. S15, ESI†). We also found that PNF-treated indicate that PNF can induce mitophagy or autophagy of
cells displayed massive multi-membrane structures containing A549R cells. P-glycoprotein (P-gp), known as multidrug resis-
an electron-dense material, which could be identified as tance protein 1 (MDR1), is an important protein in the cell
membrane. Some cancer cells overexpress P-gp, which renders
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these cancer cells resistant to multi-drugs. To investigate
whether the potential anti-proliferative activity of PNF on
A549R cells is related to P-gp, the expression levels of this
protein were determined by immunoblotting. We found that
after treatment with PNF, the expression of P-gp was signifi-
cantly decreased in
a concentration-dependent manner
(Fig. 3b). The observation suggests that the inhibition of
P-glycoprotein by PNF may be closely related to the mechanism
of its potential anti-A549R activity, which has important impli-
cations for the treatment of drug-resistant cancers.
Fig. 2 (a) Effect of PNF on the cell cycle of A549R cells after incubation
for 24 h. (b) A549R cells were treated with the indicated doses of PNF for
2
4 h. The expression levels of p53 and gH2AX in A549R cells were
Multicellular tumor spheroids (MCTSs) are heterogeneous
aggregates of cancer cells with the characteristics of solid
determined by Western blotting. GAPDH was used as a protein loading
control.
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3
0
tumors. Finally, we studied the anti-proliferation activity of Notes and references
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obviously penetrated into the spheres (Fig. S17, ESI†). After
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In summary, we have developed PNF, a fluorogenic small
molecule-based platform that contains an H S-induced reduc-
2
2
tive azide group, which can be activated by overexpressed H S
1
1
1
1
1
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in the cancerous environment. This activation serves to give a
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Conflicts of interest
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3
There are no conflicts to declare.
1934 | Chem. Commun., 2021, 57, 1931À1934
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