A spermine-conjugated lipophilic Pt(iv) prodrug designed to eliminate cancer stem cells in ovarian cancer

Article abstract of DOI:10.1039/c9cc02081k

We developed a spermine-conjugated lipophilic Pt(iv) prodrug that is able to reduce the cancer stem cell population in ovarian cancer. The therapeutic effect is attributed to the hydrophobic tail and cationic spermine head group, the combination of which allows the Pt(iv) prodrug to localize in mitochondria and induce corresponding damage.

Full text of DOI:10.1039/c9cc02081k

View Article Online
View Journal
ChemComm
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: Y. Zheng, M.
Stilgenbauer, A. M. D. S. Jayawardhana, P. Datta, Z. Yue, M. Gray, F. Nielsen, D. Bowers and H. Xiao,
Chem. Commun., 2019, DOI: 10.1039/C9CC02081K.
This is an Accepted Manuscript, which has been through the
Volume 52 Number
1 4 January 2016 Pages 1–216
Royal Society of Chemistry peer review process and has been
accepted for publication.
ChemComm
Accepted Manuscripts are published online shortly after
Chemical Communications
www.rsc.org/chemcomm
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 1359-7345
COMMUNICATION
Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc3N@C2n (2n 68 and 80). Synthesis of the
first methanofullerene derivatives of Sc N@D5h-C80
=
3
rsc.li/chemcomm

Please do not adjust margins
Page 1 of 5
ChemComm
View Article Online
DOI: 10.1039/C9CC02081K
Journal Name
COMMUNICATION
A Spermine-Conjugated Lipophilic Pt(IV) Prodrug
Designed to Eliminate Cancer Stem Cells in Ovarian
Cancer
Received 00th January 20xx,
Accepted 00th January 20xx
Morgan Stilgenbauer,a Amarasooriya M. D. S. Jayawardhanaa, Payel Datta,a Zhizhou Yue,a Michael
DOI: 10.1039/x0xx00000x
Graya, Frederick Nielsen,a David J. Bowers,a Haihua Xiao*b, and Yao-Rong Zheng *a
www.rsc.org/
We developed a spermine-conjugated lipophilic Pt(IV) prodrug that triggering mitochondrial damage.11 Song reported a Ru(II)
are able to reduce the cancer stem cell population in ovarian complex that can effectively eliminate CSCs in the MCF7 breast
cancer. The therapeutic effect is attributed to the hydrophobic tail cancer cell line by targeting mitochondria and endoplasmic
and cationic spermine head group, the combination of which allows reticuli.12 In spite of the recent developments of metallodrugs
the Pt(IV) prodrugs to localize in mitochondria and induce for breast CSCs, metallodrugs that can treat CSCs in ovarian
corresponding damages.
cancer have not been reported.
In this communication, we present the first platinum-based
metallodrug that is able to reduce the CSC population in ovarian
cancer via triggering mitochondrial damage. It was previously
reported that the lipophilic fatty acid-like Pt(IV) prodrugs exhibit
high in vitro and in vivo potency and promising
pharmacokinetics, by virtue of their lipophilic tails.13, 14 In this
work (Fig 1A), we have modified such Pt(IV) prodrugs with the
attachment of a cationic spermine moiety. This modification
endorses the Pt(IV) prodrugs with both cationic and lipophilic
features, therefore facilitating their accumulation in
mitochondria for elimination of CSCs.
We first explored a new design of developing mitochondrial-
damaging Pt(IV) prodrugs. Attachment of cationic moieties to
the lipophilic fatty acid-like Pt(IV) prodrugs is expected to
promote their accumulation in mitochondria, due to the
negative mitochondrial membrane potential (Δψm ~ 150–180
mV). However, the conventional mitochondria-targeted
cationic moiety, triphenylphosphonium, is hydrophobic, likely
leading to much lower solubility of such lipophilic prodrugs.
Alternatively, we sought to use a hydrophilic moiety, spermine,
as shown in Fig 1A. Under physiological conditions, the
conjugated spermine moiety carries a charge of +3. The
synthetic route of the spermine-conjugated lipophilic Pt(IV)
prodrug (1) was described in Scheme S1 of the ESI. Briefly, tri-
Boc-spermine was conjugated to the fatty acid-like Pt prodrug
with a C16 tail via a HATU-catalyzed amide-bond formation
reaction. Boc deprotection was carried out by stirring the
compound in 10% TFA dichloromethane solution at r.t. for 3.5
h. This newly synthesized Pt(IV) prodrug has been characterized
using multinuclear (1H and 13C) NMR spectroscopy, high-
resolution mass spectrometry, and analytical HPLC (Fig S1-3 in
the ESI).
The long-standing challenge of treating ovarian cancer is cancer
recurrence accompanied with drug resistance to the
conventional platinum-based chemotherapy.1 Cancer stem cells
(CSCs) are believed to be the root of this problem.2, 3 CSCs, also
known as tumor-initiating cells, are a small population of cancer
cells that have tumorigenic ability and are responsible for tumor
metastasis, cancer relapse, and development of drug
4
resistance.3, CSCs are highly resistant to chemotherapy,
partially due to their intrinsic features, including enhanced DNA
damage repair ability and overexpression of anti-apoptotic
proteins, drug efflux transporters and detoxifying enzymes.2, 5
Currently, there is no effective clinical treatment for eradicating
CSCs in tumors.
Targeting mitochondria is one of the most promising
therapeutic routes to eradicate CSCs.6, 7 Recent studies indicate
that the CSC population found in various cancer types favor
oxidative phosphorylation in mitochondria for their energy
production, and contain higher mitochondria mass compared to
non-CSCs.7, 8 Increasing attention has been drawn to develop
10
new metallodrugs that target mitochondrial functions.9,
Notably, Suntharalingam and coworkers have developed the
first metallopeptide that can eradicate breast CSCs via
a. Department of Chemistry and Biochemistry, Kent State University, 224 Williams
Hall, Kent, Ohio 44242.
b. Beijing National Laboratory for Molecular Sciences, State Key Laboratory of
Polymer Physics and Chemistry, Institute of Chemistry, Chinese Academy of
Sciences, Beijing 100190, P. R. China.
Email: hhxiao@iccac.ac.cn, yzheng7@kent.edu
Electronic Supplementary Information (ESI) available: Experimental details
regarding the synthesis and characterization of 1, cell culture, the MTT assay,
cellular uptake, and flow cytometric analysis are presented in the ESI. See
DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
ChemComm
Page 2 of 5
COMMUNICATION
Journal Name
View Article Online
DOI: 10.1039/C9CC02081K
Figure 1. Spermine-conjugated lipophilic Pt(IV) prodrugs accumulate in mitochondria: (A) chemical structure of the Pt(IV) prodrug (1); (B)
cytosolic and mitochondrial Pt contents of SKOV3 cells treated with cisplatin (150 μM), 1 (5 μM); (C) images of MDA-MB-231 cells treated
with Mitotracker Green, 1 labeled with Rhodamine B (10 μM), and Hoechst for 1 h at 37 oC 5% CO2. (Scale bar = 20 μm).
Next, accumulation of the Pt(IV) prodrug in mitochondria respectively. As shown in Fig 2A (left panel), the treatment of 1
was confirmed. Graphite furnace atomic absorption (10 μM, 24 h) increased the mitochondrial ROS level as
spectroscopy (GFAAS) was used to measure the Pt contents in compared to the control. Again, the treatment of cisplatin (60
the isolated mitochondria and cytosol of SKOV3 ovarian cancer μM, 24 h) resulted in insignificant change with respect to
cells treated with the different platinum compounds (cisplatin, mitochondrial ROS levels, in line with its mechanism of action.
1). Cisplatin was engaged as a control, since it does not prefer In addition, mitochondrial membrane potential (Δψm), a key
to accumulate in mitochondria. For the SKOV3 cells treated with parameter for assessing mitochondrial functions, was
cisplatin (Fig 1B), a minimal amount of Pt (2.03±0.79 nmol determined using MitostatusTM reagent. As shown in Fig 2A
Pt/106 cells) was found in the mitochondria, while the majority (right panel), the treatment of 1 (10 μM, 24 h) induced the loss
(54.1±8.6 nmol Pt/106 cells) was detected in cytosol. In contrast, of Δψm of the mitochondria of SKOV3 cells. Finally, we used ATP
1 delivered a large amount of Pt content (24.5±2.1 nmol Pt/106 luminescence assays to measure cellular ATP levels, as ATP is
cells) to the mitochondria, which is comparable to the amount mainly produced in mitochondria. As shown in Fig 2B, cisplatin
(61.3±10.0 nmol Pt/106 cells) delivered to the cytosol, as shown (2.5–20 μM, 24 h) does not deplete cellular ATP levels in SKOV3
in Fig 1B. Furthermore, we carried out fluorescence imaging cells. On the other hand, the addition of 1 (2.5–20 μM, 24 h)
studies to pinpoint the mitochondrial accumulation of the Pt significantly suppressed the ATP production (8–79% reduction),
compound in live cells. For visualizing its distribution, the Pt(IV) which is likely attributed to the mitochondrial damage. To
prodrug (1) was labeled with Rhodamine B via the reaction summarize, the spermine-conjugated lipophilic Pt(IV) prodrug
between 1 and Rhodamine B isothiocyanate (see ESI). can damage mitochondria of ovarian cancer cells. Given that
MitoTracker Green FM was used to label mitochondria. We nuclear DNA is a well-known intracellular target for platinum-
already confirmed mitochondrial accumulation of 1-RhB using based anticancer agents, we further analyzed nuclear DNA
GFAAS (Fig S5 in the ESI), which is similar to that of 1. As damage by probing phosphorylation of H2AX (γH2AX), a DNA
expected, co-localization of the different fluorescence signals of damage biomarker. Notably, as shown in Fig S6, the Pt(IV)
1-RhB and MitoTracker Green FM was observed (Fig 1C). In sum, prodrug (1) is able to induce a marked increase in γH2AX as
the combined evidence from both fluorescence imaging and determined by flow cytometry.
GFAAS studies support accumulation of the spermine-
conjugated lipophilic Pt(IV) prodrugs in the mitochondria.
Further experimentation indicated that the spermine-
conjugated lipophilic Pt(IV) prodrug was able to reduce the CSC
Subsequently, we determined the mitochondrial damage population in ovarian cancer. Since mitochondria play a central
triggered by the Pt(IV) prodrug. Mitochondrial ROS levels, a role in supporting cellular metabolism and survival of ovarian
biomarker for evaluating mitochondrial damage, was CSCs, the abovementioned mitochondrial damage triggered by
determined using the MitoSOXTM Red reagent. We used flow the Pt(IV) prodrug is expected to damage and reduce the CSCs
cytometry to measure the mitochondrial ROS levels of the population in ovarian cancer. SKOV3 tumor spheroids were
SKOV3 cells treated with the Pt(IV) prodrug (1) and cisplatin, formed in CSC media in ultra-low attachment flasks or 6-well
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 3 of 5
ChemComm
Please do not adjust margins
Journal Name
COMMUNICATION
spheroids/106 cells for cisplatin-treated group). In summary, the
View Article Online
experimental results indicate that SKOVD3OoI:v1a0r.1ia0n39/cCa9nCcCe0r2c0e8l1lKs
treated with the spermine-conjugated lipophilic Pt(IV) prodrug
exhibit a lower CSC population as compared to the control or
cisplatin-treated group.
Finally, we found that the spermine-conjugated lipophilic
Pt(IV) prodrug exhibits promising in vitro efficacy against
ovarian cancer cells. MTT assays were performed to test in vitro
efficacy of the new Pt agent (1). A panel of human cancer cell
lines, including A2780, A2780cis, SKOV3, MDA-MB-231, and
A549, were employed in the MTT assays. Cancer cells were
treated with cisplatin or 1 for 72 h and cell viability was
evaluated. IC50 values, which represent the concentration
required to inhibit growth by 50%, are shown in Fig 4A. Across
all the tested cell lines, 1 exhibits much higher in vitro potency
as compared to cisplatin. For example, in the A2780cis cell line,
the IC50 value of 1 (1.12±0.18 μM) is 12 times lower than that of
cisplatin (13.19±1.84 μM), and these data have been further
validated using cell counting assays (Fig S7). Resistance factor,
Figure 2. The spermine-conjugated lipophilic Pt(IV) prodrug induced
mitochondrial damage and depleted ATP in ovarian cancer cells: (A)
flow cytometric analysis of mitochondrial ROS level (left, MitoSOX
assays) and membrane potentials (right, MitoStatus assays) of
SKOV3 cells treated with cisplatin (60 μM) and 1 (10 μM) for 24 h at
37 oC 5% CO2; (B) luminescence ATP detection assay of SKOV3 cells
treated with cisplatin and 1 for 24 h at at 37 oC 5% CO2.
plates by following the reported procedure.15 As expected, the
treatment of cisplatin (10 μM, 72 h) enriched the CSCs
population in SKOV3 spheroids, and the enrichment was
confirmed by flow cytometric analysis of CD44 (FL-1), CD117
(FL-2), and CD133 (FL-4) of the treated cells (Fig 3A, middle
16
panel).15,
In contrast, the treatment of 1 (1 μM, 72 h)
significantly decreased the CSC population in the SKOV3
spheroids as shown in Fig 3A (bottom panel). In addition,
spheroid-formation assays were used to further support the
reduction of the CSC population in SKOV3 by 1. In the
experiment, SKOV3 spheroids were treated with cisplatin (5
μM) or 1 (2.5 μM). After 72 h, the SKOV3 cells were harvested,
trypsinized, and re-seeded in ultra-low attachment 96-well
microplates (5000 cells per well). After 72 h, the numbers of
tumor spheroids formed in each well were counted under an
optical microscope. As shown in Fig 3B, the treatment of 1
Figure 3. The spermine-conjugated lipophilic Pt(IV) prodrug is able
to reduce the CSC-like population in ovarian cancer cells: (A) flow
dramatically diminished the capability of SKOV3 cells from cytometric analysis of CSC biomarkers of SKOV3 cells treated with
forming spheroids (10±2 spheroids/106 cells), but in the control
and cisplatin-treated samples, large tumor spheroids were
observed (55±4 spheroids/106 cells for control group and 27±3
cisplatin (10 μM) and 1 (1 μM) for 72 h at 37 oC 5% CO2; (B) spheroid-
formation assays of SKOV3 cells treated with cisplatin (5 μM) and 1
(2.5 μM) for 72 h at 37 oC 5% CO2.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
ChemComm
Page 4 of 5
COMMUNICATION
Journal Name
View Article Online
DOI: 10.1039/C9CC02081K
Figure 4. The spermine-conjugated lipophilic Pt(IV) prodrug overcomes drug resistance and exhibits high potency in vitro: (A) cytotoxicity
profiles of cisplatin and spermine-conjugated lipophilic Pt(IV) prodrug (1) against a panel of human cancer cell lines, resistance factor =
(IC50(A2780cis)/IC50(A2780); (B) representative killing curves of cisplatin (left) and 1 (right) against A2780 (cisplatin sensitive) and A2780cis
(cisplatin resistant) ovarian cancer cell lines.
the fold change in IC50 values [(IC50(A2780cis)/IC50(A2780)],
reflects drug resistance of the Pt compounds tested. As shown
Notes and references
in Fig 4A and 4B, the Pt(IV) prodrug (1) displayed low cross-
resistance as compared to cisplatin, and the resistance factors
are 1.5 for 1 and 8.2 for cisplatin. In summary, the data from the
MTT assays indicate that the newly developed Pt(IV) prodrug
shows superior therapeutic indexes as compared to cisplatin.
In conclusion, we have demonstrated a new chemical design
of a mitochondrial-damaging platinum agent that can reduce
the CSC population and overcome drug resistance in ovarian
cancer. Unlike classic platinum drugs, which mainly target
nuclear DNA, the spermine-conjugated lipophilic Pt(IV) prodrug
tends to accumulate in mitochondria, due to its cationic and
lipophilic structural features. Subsequently, the mitochondrial
damage triggered by the Pt(IV) prodrug reduced the CSC
population in ovarian cancer. In addition to its interesting
mechanism of action, this new platinum agent displays superior
therapeutic indexes as compared to cisplatin. Due to the
different attacking mode, this Pt(IV) prodrug shows low cross-
resistance as compared to cisplatin. In addition, this compound
exhibits much higher in vitro potency than cisplatin. This work
offers a new approach to design Pt anticancer agents to target
the root of cancer relapse and drug resistance in ovarian cancer.
1.
2.
3.
4.
D. Wang and S. J. Lippard, Nat. Rev. Drug Discovery, 2005,
4, 307-320.
M. Dean, T. Fojo and S. Bates, Nat. Rev. Cancer, 2005, 5,
275-284.
P. B. Gupta, C. L. Chaffer and R. A. Weinberg, Nat. Med.,
2009, 15, 1010-1012.
C. T. Jordan, M. L. Guzman and M. Noble, N. Engl. J. Med.,
2006, 355, 1253-1261.
5.
6.
A. Singh and J. Settleman, Oncogene, 2010, 29, 4741-4751.
S. Weinberg and N. Chandel, Nat. Chem. Biol., 2015, 11, 9-
15.
P. Sancho, D. Barneda and C. Heeschen, Br. J. Cancer, 2016,
114, 1305-1312.
G. Farnie, F. Sotgia and M. Lisanti, Oncotarget, 2015, 6,
30472-30486.
S. Marrache, R. Pathak and S. Dhar, Proc. Natl. Acad. Sci. U
7.
8.
9.
S A, 2014, 111, 10444-10449.
10.
S. Wisnovsky, J. Wilson, R. Radford, M. Pereira, M. Chan, R.
Laposa, S. Lippard and S. Kelley, Chem. Biol., 2013, 20,
1323-1328.
K. Laws, G. Bineva-Todd, A. Eskandari, C. Lu, N. O'Reilly and
K. Suntharalingam, Angew. Chem. Int. Ed. Engl., 2018, 57,
287-291.
B. Purushothaman, P. Arumugam, H. Ju, G. Kulsi, A. A. S.
Samson and J. M. Song, Eur. J. Med. Chem., 2018, 156, 747-
759.
Y.-R. Zheng, K. Suntharalingam, T. C. Johnstone, H. Yoo, W.
Lin, J. G. Brooks and S. J. Lippard, J. Am. Chem. Soc., 2014,
136, 8790-8798.
M. Miller, Y. Zheng, G. Suresh, C. Pfirschke, H. Zope, C.
Engblom, R. Kohler, Y. Iwamoto, K. Yang, B. Askevold, N.
Kolishetti, M. Pittet, S. Lippard, O. Farokhzad and R.
Weissleder, Nat. Commun., 2015, 6. 8692.
J. Cole, S. Joseph, C. Sudhahar and K. Dahl, J. Vis. Exp., 2014,
91, 51891.
11.
12.
13.
14.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
Y.-R. Z thanks the financial support provided by the startup
fund, Farris Family Innovation Fellowship, LaunchPad Award
provided by Kent State University. M.G. thanks the financial
support provided by the SURE fellowship program supported by
the Division of Research and Sponsored Programs, Kent State
University.
15.
16.
L. Ma, D. Lai, T. Liu, W. Cheng and L. Guo, Acta Biochim.
Biophys. Sin., 2010, 42, 593-602.
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 5 of 5
ChemComm
View Article Online
DOI: 10.1039/C9CC02081K
The spermine-conjugated lipophilic Pt(IV) prodrug is designed to induce mitochondrial damage
and eliminate ovarian cancer stem cells.