Mitochondrial heat shock protein-guided photodynamic therapy

Article abstract of DOI:10.1039/c9cc06411g

Mitochondria targeting sensitizers are continuing to gain importance in photodynamic therapy (PDT). Members of the 90 kDa heat shock protein (Hsp90) family, including TRAP1 (Hsp75), are overexpressed in cancer cells and help to control the antiapoptotic protein activity. The present work introduces an Hsp90 inhibitor-mitochondria targeting indocyanine dye conjugate (IR-PU) for high PDT efficacy.

Full text of DOI:10.1039/c9cc06411g

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Mitochondrial heat shock protein-guided
photodynamic therapy†
Cite this: Chem. Commun., 2019,
55, 12631
Ajesh P. Thomas,‡a An-Jung Lee,‡b L. Palanikumar,a Batakrishna Jana,a
Kibeom Kim,a Sangpil Kim,a Haewon Ok,a Jihoon Seol,a Dongseok Kim,a
Received 19th August 2019,
Accepted 19th September 2019
Byoung Heon Kang*b and Ja-Hyoung Ryu
*
a
DOI: 10.1039/c9cc06411g
rsc.li/chemcomm
Mitochondria targeting sensitizers are continuing to gain importance in chaperones facilitating tumor growth and propagation.7 Among the
photodynamic therapy (PDT). Members of the 90 kDa heat shock different HSP90 paralogues, Hsp90 and TRAP1 (Hsp75) have been
protein (Hsp90) family, including TRAP1 (Hsp75), are overexpressed in found to be overexpressed in the cytoplasm and mitochondria of
cancer cells and help to control the antiapoptotic protein activity. The cancer cells, respectively.8 The overexpression of these proteins in
present work introduces an Hsp90 inhibitor-mitochondria targeting cancer helps to maintain the stability and activity of multiple client
indocyanine dye conjugate (IR-PU) for high PDT efficacy.
proteins involved in antiapoptotic and pro-tumorigenic pathways,
ultimately resulting in the attenuation of therapeutic efficacy.7a,9
Photodynamic therapy (PDT) has emerged as a potential thera- PDT efficacy can be improved by inhibiting HSP90s.10 There are
peutic tool for treating various cancers, mainly due to its reports demonstrating various attempts to target HSP90s, where
noninvasive nature and the precise spatiotemporal control.1 co-delivery of HSP90 inhibitor and photosensitizer were imple-
Controlled generation and deactivation of short-lived cytotoxic mented using nanocarrieres.6a,11 However, low tumor delivery
agents within a cell on irradiation of a prodrug or photosensitizer efficiency and unwanted accumulation in internal organs are
is the key step in PDT. A combination of photosensitizer (PS) or still drawbacks of this approach.12
drug, light and oxygen work together to generate highly reactive
Herein, we introduce a heat shock protein inhibitor-photo-
singlet oxygen (1O2) by a triplet–triplet energy transfer from the sensitizer molecular conjugate, IR-PU for the selective accumulation/
sensitizer to triplet ground state oxygen.2 PDT’s advantages over targeting, and the simultaneous inhibition of Hsp90 and TRAP1 in
conventional cancer treatments (chemotherapy or radiotherapy), both the cytoplasm and mitochondria along with PDT (Fig. 1).
include its switchable nature, minimal side effects and the possi- Previously, we found that IR-Pyr showed better photostability and
bility of imaging of cancerous tissue during the therapy.3 However, higher mitochondrial accumulation than the parent dye IR-780,
some factors reduce PDT efficacy and originate from limitations of leading to the death of tumor cells.13 In addition, we showed that
the photosensitizers used, light penetration depth, and the hypoxic mitochondria targeting-HSP inhibitor, a purine-derivative, directly
conditions around tumor tissues.4 Recently, targeting mitochon- induced mitochondrial dysfunction, resulting in improved anti-
dria; vital organelles for cell survival, as they play a central role in cancer activity.8a Thus, the rationale behind the design is that
energy production and apoptotic pathways, have been recognized IR-PU binds Hsp90 family proteins such as Hsp90 and TRAP1 that
as an efficient strategy in various therapeutic techniques particularly are overexpressed in many cancer cells, leading to selective
PDT.5 Apart from these, low selectivity to cancer cells against normal accumulation in cancer cells and concomitant inhibition of
healthy cells, and inherent resistance to the therapeutic efficacy that their molecular chaperon activity, and mitochondria-targeted
originates from the overexpression of antiapoptotic agents during PDT can rapidly damage the biological functions of the organelles
the therapy become more critical.6
The 90 kDa heat shock proteins (HSPs) are antiapoptotic Hsp90/TRAP1 inhibition and PDT for superior anticancer efficacy.
under photo activation. As a result, there is a synergistic effect of
proteins that are overexpressed in cancer cells and act as molecular
Synthesis of IR-PU (Scheme S1, ESI†) was achieved by a
multi-step synthetic strategy, where in the last step IR-Pyr13 and
PU-SH were conjugated to each other by stirring in dimethyl
a Department of Chemistry, School of Natural Sciences, Ulsan National Institute of
Science and Technology (UNIST), Ulsan-44919, South Korea
b Department of Biological Sciences, School of Natural Sciences, Ulsan National
Institute of Science and Technology (UNIST), Ulsan-44919, South Korea.
E-mail: jhryu@unist.ac.kr, kangbh@unist.ac.kr
1
formamide (DMF) for 24 h and confirmed by H NMR, 13C NMR
and ESI-MS (Fig. S1–S15, ESI†). The electronic absorption spectrum
of the compound showed bands in a very broad range, between
280 nm and 850 nm, with an absorption maximum at 799 nm in
DMSO (Fig. S16, ESI†). The absorption from 280 to 500 nm,
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c9cc06411g
‡ These authors contributed equally to this work.
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Fig. 1 The synthetic scheme for IR-PU and a schematic representation illustrating mitochondrial HSP guided PDT.
peaking at 300 nm, indicates the successful conjugation of the Enhanced SOSG fluorescence intensity is a direct indication of
purine derivative with the IR dye. The emission spectrum in increased singlet oxygen generation in the medium. A compara-
DMSO exhibited a peak at 815 nm when excited at 770 nm. The tive analysis with IR-Pyr revealed higher generation of 1O2 by
photo and dark stability of IR-PU was compared with IR-780 IR-PU. Additionally, fluorescence measurement without irradia-
(a well-studied indocyanine derivative used in PDT) and IR-Pyr tion or with irradiation but without IR-PU (only SOSG) produced
(a water soluble indocyanine dye) in phosphate-buffered saline no fluorescence, confirming the photo-excited generation of 1O2
(PBS) and DMSO.14,15 The analysis indicated that the photo- (Fig. 2b). The superiority of IR-PU over IR-Pyr to generate 1O2 could
stability of IR-PU in PBS is better in comparison with IR-780, be due to greater triplet population in IR-PU than IR-Pyr upon
possibly due to a change in its electronic properties upon photoexcitation.
chemical modification (Fig. 2a).
The PU-H71 moiety of IR-PU is a competitive inhibitor
Further, the photo-induced singlet oxygen generation and against ATP binding pocket of Hsp90 and TRAP1. The binding
the Hsp90/TRAP1 inhibition efficacy of the conjugate were affinities of IR-PU to Hsp90 and TRAP1 were measured by
1
investigated. The ability to generate O2, which is the primary fluorescence polarization using a probe, PU-H71-FITC3.16 The
criterion for PDT, was investigated using singlet oxygen sensor data indicated that IR-PU has a high binding affinity for TRAP1
green (SOSG). IR-PU in PBS was mixed with an equimolar and moderate affinity for Hsp90 (Fig. 2c and d), but less when
solution of SOSG and the change in SOSG fluorescence inten- compared with PU-H71 (a known HSP inhibitor). The control
sity at 504/530 nm was monitored at different time intervals compound IR-Pyr (without HSP inhibitor) didn’t show any
both before and after irradiation with laser light (808 nm, 200 mW). affinity. The results imply that IR-PU has a high affinity towards
Fig. 2 (a) Photostability of IR-PU in PBS. (b) Comparison of singlet oxygen generation efficiency between IR-Pyr and IR-PU in PBS using SOSG.
(c) Affinities for TRAP1, and (d) affinities for HSP90 in comparison with PU-H71 and IR-Pyr. (e) Confocal images showing mitochondria co-localization of
IR-PU compared with Mitotracker Green in HeLa cells. (f) MitoSOX fluorescence in HeLa cells upon incubation with IR-PU followed by 2 min irradiation
using 808 nm (200 mW) (left) and before irradiation (right). (g) Mitochondrial depolarization by IR-PU in HeLa cells monitored by the change in
fluorescence of TMRM both before irradiation (left) and 2 h after irradiation with 808 nm laser. (h) NCI-H460 cells were incubated with 10 mM IR-PU or
IR-Pyr for the indicated time and analyzed with a fluorimeter. (i) NCI-H460 cells were incubated with 10 mM IR-PU or IR-Pyr with or without 200 mM
novobiocin for 30 min for 2 h and analyzed with a fluorimeter. (n = 3, P value = 0.013).
12632 | Chem. Commun., 2019, 55, 12631--12634
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the HSP proteins. The pyridinium groups in IR-PU, which is
known to target mitochondria, should enhance mitochondrial
accumulation. Mitochondrial co-localization was investigated
by confocal microscopic analysis in HeLa cell lines in comparison
with mitotracker green that showed high accumulation in mito-
chondria as shown in Fig. 2e. The Pearson coefficient for mito-
chondrial localization was calculated to be 0.73, which is higher
when compared to the widely used indocyanine dye IR-780
(Pearson coefficient 0.61).4b
ROS (1O2) generation in HeLa cells was studied by following
the increased fluorescence intensity of the ROS-sensitive dye
MitoSOX Red upon laser irradiation at 808 nm (200 mW, 2 min)
using confocal microscopy. HeLa cells were incubated with IR-PU
for 4 h before replacement with fresh media and irradiation. The
intense red fluorescence after irradiation indicates the generation
1
of O2 (Fig. 2f and Fig. S17, ESI†). The signal increased slightly
Fig. 3 (a) Fluorescence image of IR-PU accumulating in HeLa, NCI-H460,
and normal hepatocyte cells (scale bar 10 mM). (b) MTT assay to compare
without irradiation, indicating generation of reactive oxygen
species, probably due to Hsp90/TRAP1 inhibition by the PU-H71
moiety. Mitochondrial membrane depolarization is one of the early
hallmarks of cellular apoptosis during PDT. We expected the
synergistic effect of PDT and Hsp90/TRAP1 protein inhibition would
greatly destabilize mitochondrial membranes and, ultimately, lead
photo and dark toxicity of IR-PU with control molecules,
P value:
**** o0.0001, *** = 0.0002. (c) Western blot analysis showing the
changes in different protein levels during PDT with IR-PU and IR-Pyr.
to a large population of apoptotic cells. To test this, we incubated showed no significant toxicity, even when compared with IR-Pyr
TMRM-labeled HeLa cells with IR-PU. Confocal imaging after (Fig. S21, ESI†).
irradiation showed decreased fluorescence intensity of the dye
We used Western blot analyses to investigate the inhibition
compared to the initial intensity (Fig. 2g and Fig. S18, ESI†), of Hsp90 and TRAP1 in NCI-H460 cells. IR-PU strongly inhibited
indicating mitochondrial membrane depolarization due to PDT Hsp90 considering the decreased level of Hsp90 client proteins
and TRAP1 inhibition within mitochondria.
(chk1) and induction of heat shock response (elevation of Hsp70).
The HSP inhibitor driven selective accumulation of IR-PU Further, there was a slight decrease of TRAP1 client protein sorcin in
was demonstrated using cellular accumulation studies of the the IR-PU treated sample. The control experiment with IR-Pyr did
probe in different cancer cell-lines (HeLa and NCI-H460) and not show any signs of HSP90 and TRAP1 inhibition as expected. The
hepatocyte normal cell line. The result showed high accumulation mechanism of cell death was investigated using Western blot and
of IR-PU in both HeLa and NCI-H460 cancer cell line compared to FACS analysis using annexin V and propidium iodide. Cleaved
hepatocyte normal cell line after 6 h incubation (Fig. 3a), attributed caspase3 and PARP1 were detected (Fig. 3c) after PDT with IR-PU,
to the affinity of PU-H71 moiety of IR-PU towards the overex- indicating apoptosis-mediated cell death, which was further con-
pressed HSPs in the cancer lines. Further, in cellulo cytotoxicity was firmed by FACS analysis (Fig. S22, ESI†). After 2 min irradiation with
tested in NCI-H460 (human lung cancer cell line) and mouse 808 nm laser the late apoptotic population increased from 4% to
primary hepatocytes (normal liver cells) to check the synergistic 21%, which was much higher than with IR-Pyr under similar
effect of Hsp90/TRAP1(human lung cancer cell line) and mouse conditions. Treatment with the caspase inhibitor Z-VAD decreased
primary hepatocytes (normal liver cells) to check the synergistic the apoptotic population significantly (5%), confirming the apopto-
effect of Hsp90/TRAP1 inhibition and mitochondria targeted PDT. tic mechanism of PDT with IR-PU. Overall, these results justify the
The results are compared with IR-Pyr (both in dark and after photo approach of using an HSP inhibitor-PDT sensitizer conjugate as an
excitation) and PU-H71. The cytotoxicity analyses proved that IR- efficient therapeutic tool.
PU have a concentration dependent phototoxicity in NCI-H460 cell
The in vivo effect of IR-PU was investigated in an NCI-H460
lines after irradiation with an IC50 of less than 6 mM (Fig. S19, tumor xenograft model. NCI-H460 cells were injected subcuta-
ESI†). The phototoxicity of IR-PU was significantly higher than that neously into both sides of 9 weeks nude mice. After several days,
of IR-Pyr (Fig. 3b), which can be attributed to the higher cellular tumor-bearing mice were randomly grouped and intraperitoneally
accumulation of the drug, driven by Hsp90 binding that also injected with 10 mg kgÀ1 of IR-Pyr or IR-PU dissolved in 20%
induces additional toxicity due to the protein inhibition. This Chremophore in PBS. In vivo fluorescence imaging was performed
was confirmed by cellular imaging of NCI-H460 cells by novobiocin by setting the excitation at 760 nm and emission at 830 nm, which
(a competitive Hsp90 binder) pretreatment decreased IR-PU shows the accumulation of IR-PU in the mouse tumor site (Fig. 4a–c)
accumulation, corroborating HSP’s role in elevated cellular and fluorescence signals of IR-PU in tumor site were significantly
accumulation (Fig. 2h, i and Fig. S20, ESI†). PU-H71 exhibited higher than those of IR-Pyr (Fig. 4d), indicating IR-PU’s superior
a lower cytotoxic effect compared to IR-PU, confirming the cancer-targeting ability due to HSP-binding moiety.
synergistic effect of both HSP inhibition and PDT with IR-PU.
To check the antitumor effect of PDT with IR-PU, NCI-H460
Remarkably, cell viability analysis using IR-PU in normal cells implanted nude mice were treated with vehicle (DMSO) and IR-PU.
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National Research Foundation of Korea (2017R1A2B4003617,
2016R1A5A1009405, and 2019R1A2C2086618).
Conflicts of interest
Authors declare no competing financial interest.
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In conclusion, we have designed and synthesized a NIR dye-
HSP inhibitor conjugate, IR-PU that produces enhanced therapeutic
effects. The cancer-specific accumulation of the conjugate, with the
added synergistic effect on PDT from Hsp90/TRAP1 inhibition,
confers promising therapeutic efficacy and cancer selectivity to the
conjugate. HSP-directed cancer cell mitochondrial accumulation of
IR-PU and the PDT efficacy were demonstrated by a series of
experiments. The apoptosis-mediated cell cytotoxicity of the conju-
gate was high in the in NCI-H460 cancer cell line, and the results
from in vivo experiments confirm the therapeutic promise of IR-PU.
This work was supported by the Korea Foundation for the
Advancement of Science & Creativity (KOFAC), and funded by the
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