Probing the Anticancer Action of Oridonin with Fluorescent Analogues: Visualizing Subcellular Localization to Mitochondria

Article abstract of DOI:10.1021/acs.jmedchem.6b00408

Oridonin (1) is a complex ent-kaurane diterpenoid exhibiting remarkable antitumor activity. However, the detailed mechanism or cellular target that underlies this activity has not yet been identified. Herein, we report an efficient approach for exploring the anticancer mechanism of oridonin through development of the potent fluorescent analogues. A series of novel fluorescent oridonin probes linked with coumarin moieties were designed, synthesized, and characterized. Fluorescence microscopy and confocal imaging studies suggested that fluorescent oridonin probe 17d was rapidly taken up into tumor cells and the mitochondrion was the main site of its accumulation. Moreover, we confirmed that cytochrome c played an important role in oridonin induced mitochondrion-mediated apoptosis and α,β-unsaturated ketone is the active moiety of oridonin, which is crucial to its uptake, localization, and cytotoxicity. Our results provide new insights on the molecular mechanism of oridonin and would be useful for its further development into an antitumor agent.

Full text of DOI:10.1021/acs.jmedchem.6b00408

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Article
Probing the Anticancer Action of Oridonin with Fluorescent
Analogues: Visualizing Subcellular Localization to Mitochondria
Shengtao Xu, Shanshan Luo, Hong Yao, Hao Cai, Xiaoming Miao, Fang Wu, Dong-
Hua Yang, Xiaoming Wu, Weijia Xie, Hequan Yao, Zhe-sheng Chen, and Jinyi Xu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00408 • Publication Date (Web): 18 Apr 2016
Downloaded from http://pubs.acs.org on April 22, 2016
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Probing the Anticancer Action of Oridonin with
Fluorescent Analogues: Visualizing Subcellular
Localization to Mitochondria
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Shengtao Xu†,#, Shanshan Luo‡,#, Hong Yao†, Hao Cai†, Xiaoming Miao§,$, Fang Wu∆,*, Dong-
Hua Yang¶, Xiaoming Wu†, Weijia Xie†, Hequan Yao†, Zhe-Sheng Chen¶, Jinyi Xu†,*
†State Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, China
Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, P. R. China
‡Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai 201203, P. R.
China
§State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai
University, Tianjin 300071, P. R. China
∆Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems
Biomedicine, Shanghai Jiao Tong University, Shanghai, 200240, P. R. China
¶College of Pharmacy and Health Sciences, St. John's University, 8000 Utopia Parkway, Queens,
New York, NY 11439, USA
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ABSTRACT
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Oridonin (1) is a complex ent-kaurane diterpenoid exhibiting remarkable anti-tumor activity.
However, the detailed mechanism or cellular target that underlies this activity has not yet been
identified. Herein, we report an efficient approach for exploring the anticancer mechanism of
oridonin through development of the potent fluorescent analogues. A series of novel fluorescent
oridonin probes linked with coumarin moieties were designed, synthesized and characterized.
Fluorescence microscopy and confocal imaging studies suggested that fluorescent oridonin probe
17d was rapidly taken up into tumor cells, and the mitochondrion was the main site of its
accumulation. Moreover, we confirmed that cytochrome c played an important role in oridonin
induced mitochondrion-mediated apoptosis and α, β-unsaturated ketone is the active moiety of
oridonin, which is crucial to its uptake, localization, and cytotoxicity. Our results provide new
insights on the molecular mechanism of oridonin and would be useful for its further development
into an anti-tumor agent.
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INTRODUCTION
In the last few decades, natural products have remained an important source for anticancer drug
discovery1,2. Isodon species are widely distributed plants, and more than six hundred
diterpenoids have been isolated from this species over the past thirty years3. Many Isodon
diterpenoids have been tested in laboratory assays against tumor cells and a few compounds have
shown potent antiproliferative activity with low toxicity, especially those with an ent-kaurane
skeleton such as oridonin (1)4, eriocalyxin B (2)5, longikaurin A (3)6, nodosin (4)7 and so on
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(Figure 1, 1-5)8. The unique skeletons as well as their potent anti-tumor activities have made
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these diterpenoids promising candidates as anticancer agents9.
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Figure 1. Structures of typical natural ent-kaurane diterpenoids.
Oridonin (1) has been attracting increasing attention in recent years by cancer biologists due to
its remarkable anti-tumor activity10-13. Evidence has suggested that oridonin exerts extensive
antineoplastic activities against various human cancer cells, while exhibiting relatively low
toxicity14. This unique and safe anticancer pharmacological profile of oridonin stimulated
interest in the research of its mechanism of anticancer action and structural modifications.
Previous studies have revealed that oridonin could induce cell cycle arrest, apoptosis, and
autophagy15 by regulating a series of transcriptional factors16,17. For instance, Bu et al. reported
that oridonin could induce apoptosis via p53- and caspase-dependent induction of p38 MAPK in
SW1990 pancreatic cancer cells18. Xu et al. revealed that in pancreatic cancer BxPC-3 cells,
oridonin could up-regulate p53 and down-regulate CDK1, which led to cell cycle arrest in the
G2/M phase19. Additionally, Tommasi employed a mass spectrometry-based chemical
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proteomics approach to uncover target proteins of oridonin and suggested HSP70 1A as a
potential target for oridonin in Jurkat cells20.
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In spite of the preclinical success of oridonin, its detailed anticancer mechanism, mode of action
and intracellular target remain elusive. Further development of oridonin for cancer therapy was
hampered largely due to these weaknesses. Therefore, expanding the scarce knowledge of the
antineoplastic molecular mechanism and potential binding proteins of oridonin for further
rational new drug design is an unmet need. More importantly, a systematic approach to target
elucidation would facilitate both the introduction of new drugs into clinical practice and
undesired targets identified21. However, discovering these functional targets and clarifying the
mechanisms of bioactive natural products have proven to be especially perplexing due to the
structural complexity of natural products, low abundance of targets in vivo, and limitations of
bioanalytical methods22.
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Despite these obstacles, a number of remarkable strategies have been developed to tackle these
challenges23,24. Fluorescence imaging is such a strategy that has been widely used to visualize
cell events and associated phenotypes at many levels, from molecules to complete organisms25,26
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In recent years, natural products linked to fluorescent organic dyes for fluorescence imaging has
become a powerful tool to explore biological phenomena and provide insights into their
mechanisms of action at many levels27,28. Reports for the use of bioactive fluorescent probes
from natural products to investigate their modes of action and unveil the cellular processes
including cellular uptake, localization or specific interactions have already been developed
(Figure 2, 6-11)29-34. In these cases, the application of fluorescent natural product probes has
greatly contributed to the investigations of their modes of action and provided tools for
visualization of their targets.
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Figure 2. Structures of reported fluorescent natural product probes.
This perspective led us to develop fluorescent oridonin analogues that retain their cytotoxicity,
and yet has properties that facilitate its visualization within cells. We predicted that these novel
fluorescent oridonin probes could provide insight to understand the subcellular localization,
anticancer mechanism and structure-activity relationships (SARs) of oridonin, and ultimately
accelerate the development of oridonin as cancer therapeutics. Herein, we describe the design
and synthesis of a series of novel fluorescent oridonin analogues and the application of these
probes in revealing the anticancer action of oridonin.
RESULTS AND DISCUSSION
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Design and Synthesis of Fluorescent Oridonin Probes
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Fluorescent probes of natural products are typically generated by the attachment of a fluorophore
to natural product via a proper linker, leaving the binding interactions unaltered. However, these
interactions are often affect by linker type, length, and attachment point. Fortunately, our
previous SARs analysis has revealed that the modification on 14-hydroxyl group of oridonin did
not diminish its antiproliferative activity against a panel of human cancer cell lines35. With this in
mind, fluorescent oridonin probes were designed and synthesized by connecting 14-hydroxyl
group of oridonin with different coumarin moieties through linkers of various lengths.
Specifically, highly fluorescent N, N-dialkyl-7-aminocoumarin derivatives (14, 21, Scheme 1, 2)
were employed as fluorescent moieties, because these fluorescent dyes has no toxicity and offer
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synthetic flexibility36,37
.
As depicted in Scheme 1, fluorescent oridonin probes (17a-e) were synthesized from
commercially available oridonin (1) in 5 steps. 7-(Diethylamino)coumarin-3-carboxylic acid (14)
was firstly synthesized by the reaction of substituted salicylaldehyde (12) with ethyl
propanedioate by using piperidine as the catalyst in methanol, followed by the hydrolysis of the
corresponding coumarin ester (13) with 10% NaOH aqueous solution at room temperature in 56%
overall yield over two steps. Since the length of the linkers may influence the hydrophobicity of
the labeled molecules, diverse diols and anhydride handles were employed to further extend the
variability of linker’s length. For example, compound 15c was obtained by the treatment of 14
with 1,4-butanediol (10 equiv) in the presence of DMAP and EDCI, followed by flash column
chromatography purification in 85% yield. Then, compound 15c was treated with glutaric
anhydride at room temperature to give the acid 16d, which was further coupled with oridonin (1)
in the presence of EDCI and DMAP in dry dichloromethane to deliver probe 17d in 80% yield.
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Using similar methodology, reaction of oridonin with intermediates 16a-c and 16e gave other
fluorescent probes 17a (in 91% yield), 17b (84%), 17c (88%), and 17e (72%), respectively.
Interestingly, fluorescent side chains at C-14 position of oridonin can be highly regio-selectively
synthesized from oridonin without any protection, which was consistent with previous reports38.
In addition, the structure of compound 17d was unambiguously identified by 2D NMR
experiments, whereby HMBC correlations were observed between the C14-H methine resonance
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(δH 5.88, 1H, s) of oridonin to C resonances at δC 171.3 of the linker (Please see Supporting
Information).
7-(Dimethylamino)coumarin-4-acetic acid (21) was obtained in 29% overall yield by hydrolysis
of the corresponding coumarin ester (20), which was formed by the reaction between 3-
dimethylaminophenol (18) and diethyl-1,3-acetonedicarboxylate (19) in the presence of zinc
chloride (Scheme 2). Fluorescent probe 24c was prepared in 89% yield via ester condensation
reaction between oridonin and compound 21 directly in the presence of EDCI and DMAP in dry
dichloromethane. The other targeted fluorescent probes (24a-b) could be synthesized from
intermediates 23a-b, which were prepared using the same methods described above.
It has been widely accepted that the α,β-unsaturated ketone system in the D-ring of ent-kaurane
diterpenoids is a key pharmacophore for their antiproliferactive activity, and destruction of this
ketone system could counteract their anticancer effects. Thus, reductive oridonin (26) and its
corresponding fluorescent probe (27) were prepared as negative controls as shown in Scheme 3.
Reduction of the double bond between C-16 and C-17 of oridonin was accomplished by catalytic
hydrogenation on 10% Pd/C in methanol at room temperature, then flash chromatography of the
evaporated residue, eluting with dichloromethane/methanol (40:1) afforded compound 26 in 94%
yield. The preparation of its fluorescent probe 27 was carried out by coupling compound 16d to
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the C-14 position of 26 in 82% yield. On the other hand, compound 25 was employed as a
positive control, which was synthesized in 80% yield through selective esterification of oridonin
at 14-hydroxyl group with n-caprylic acid (Scheme 3).
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Scheme 1. Synthesis of fluorescent oridonin probes 17a-ea
aReagents and conditions: (a) Ethyl propanedioate, piperidine, MeOH, reflux, over night; (b) 10%
NaOH, rt, 24 h; (c) Corresponding diols, DMAP, EDCI, CH2Cl2, rt, 8-48 h; (d) Glutaric
anhydride or succinic anhydride, DMAP, CH2Cl2, rt, 48-72 h; (e) EDCI, DMAP, CH2Cl2, rt, 0.5-
2 h.
Scheme 2. Synthesis of fluorescent oridonin probes 24a-ca
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aReagents and conditions: (a) MeOH, ZnCl2, reflux, over night; (b) 10% NaOH, rt, 24 h; (c)
Corresponding diols, DMAP, CH2Cl2, rt, 12-18 h; (d) Glutaric anhydride or succinic anhydride,
DMAP, CH2Cl2, rt, 48-72 h; (e) EDCI, DMAP, CH2Cl2, rt, 0.5-1 h.
Scheme 3. Synthesis of oridonin analogues 25, 26 and fluorescent oridonin probe 27a
aReagents and conditions: (a) n-caprylic acid, EDCI, DMAP, CH2Cl2, rt, 2 h; (b) 10% Pd/C,
MeOH, rt, 1 h; (c) EDCI, DMAP, CH2Cl2, rt, 0.5 h.
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Cytotoxicity
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We investigated the antiproliferative activities of nine novel fluorescent oridonin probes
including 17a-e, 24a-c, 27, and two oridonin analogues 25, 26 against human hepatocellular
carcinoma Bel-7402 cells by using MTT assay. Oridonin (1) was also tested for comparison, and
the data was summarized in Table 1. Oridonin and its 14-OH modified derivative 25 showed
good cytotoxicity against Bel-7402 cells; however, analogue 26, whose α,β-unsaturated ketone
moiety was destructed, displayed poor antiproliferative activity. Almost all the fluorescent
probes exhibited slightly lower antiproliferative activity compared with the parent oridonin,
except probe 17d. The probes containing 3-substituted coumarin (17a-e) maintained their
cytostatic activity better than probes containing 4-substituted coumarin (24a-c). We also found
that the length of spacer joining the coumarin skeleton and oridonin had an effect on the
antiproliferative activities of the probes. Compound 17d with eleven-atom spacer expressed
better inhibitory effect on growth than its analogues 17a-c and 17e. Although compound 24c is
more potent than 24a, giving its short spacer which may interfere with the binding of natural
product to its target and affect its mode of action, compound 24a was more suitable for further
studies.
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Based on the above preliminary results, compounds 1, 17d, 24a, 26, and 27 were selected to
further test their in vitro antiproliferative activities on three human cancer cell lines. As shown in
Table 2, fluorescent probes 17d and 24a had cytotoxicity comparable to, or even stronger than
that of oridonin (1). However, compound 26 showed lower activity compared to that of oridonin,
especially in A549 cell line (IC50: 67.0 μM vs 10.4 μM), and its corresponding probe 27 also had
no obvious activity as expected. These results suggested that α,β-unsaturated ketone moiety is
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crucial to the inhibitory activity of oridonin and its derivatives, and the coumarin moiety
possesses low cytotoxicity.
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Table 1. Growth inhibitory effects and fluorescence properties of target and reference
compounds.
b
Inhibition rate (%)a
CH2Cl2
Stocks
shift
Compd
100 μM
10 μM
1 μM
λexc (nm) λem (nm)
(nm)
94.6%
9.5%
94.8%
3.7%
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0.7%
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4.9%
2.7%
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1.4%
1.1%
9.3%
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57.5%
2.1%
1.8%
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87
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15a
17a
17b
17c
17d
17e
24a
24b
24c
25
94.4%
94.6%
94.6%
93.8%
94.6%
93.3%
89.2%
92.4%
94.7%
57.2%
37.2%
72.4%
85.0 %
89.9%
94.0%
70.1%
46.4%
18.7%
94.5%
92.7%
4.2%
26
3.7%
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aGrowth inhibitory effects of compounds 1, 17a-e, 24a-c, 25-27 on human hepatocellular
carcinoma Bel-7402 cells. MTT assays, Bel-7402 cells were treated with varying concentrations
of indicated compounds for 72 h. Values are the mean of three independent experiments.
bFluorescence properties were evaluated in CH2Cl2 with compounds at a concentration of 1×10-6
M, λexc: maximum excitation, λem: maximum emission.
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Table 2. IC50 values of oridonin (1), its synthetic analog (26), and fluorescent derivatives (17d,
24a, and 27) on inhibiting proliferation of human cancer cells (μM)a.
Compd HepG2
A549
Helab
15.2±1.0 10.4±0.4 17.8±0.9
1
2.6±0.1
8.8±0.6
5.1±0.1 2.0±0.1
11.2±0.9 4.6±0.2
17d
24a
26
70.6±4.3 67.0±7.1 52.7±3.9
144.1±9.5 78.0±5.2 79.6±6.3
27
aMTT assays: cells were incubated with indicated compounds for 72 h, (means±SD, n=3);
bHepG2 (liver hepatocellular carcinoma cell), A549 (Human lung adenocarcinoma cell), Hela
(human cervical cancer cell).
Fluorescent Properties
As shown in Table 1, the fluorescent properties of target compounds including the excitation and
emission of probes were evaluated in dichloromethane at a concentration of 1×10-6 M by using
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spectrofluorophotometer. All the probes exhibited significant differences between maximum
excitation (λexc) and emission (λem) wavelength, namely stokes shift. The probes bearing 3-
substituted coumarin (17a-e, 27) displayed fluorescence excitation peaks at about 425 nm and
the emission wavelengths varied in the range of 454 to 459 nm, expressing a shift toward the UV
region compared with probe 15a. It was encouraging to see larger stokes shift in these probes
than 15a, making them more appropriate for the imaging studies. We speculated that this might
be attributed to the appended oridonin moiety. The probes bearing 4-substituted coumarin (24a-c)
had larger stokes shift (~80 nm) with λexc at ~380 nm and λem at ~460 nm. Preliminary studies on
the fluorescent property showed that all these probes were suitable for imaging studies.
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Uptake Profiles of Probes
The MTT assay showed that fluorescent probes 17d and 24a had significant antiproliferative
activities comparable to that of oridonin. However, no obvious antiproliferative activities were
observed on probes 15a and 27. Uptake profiles of these probes were detected to uncover the
relationships between antiproliferative activities and uptake using fluorescence microscopy. As
shown in Figure 3A, bright fluorescence staining inside the cells was observed when probes 17d
and 24a were incubated at a concentration of 5 μM for 1 h in HepG2 cells. Then, cells stained
with naked coumarin dye (15a) were used as a control to exclude the possibility that the uptake
of the fluorescent oridonin probes was driven by coumarin dye itself. As shown in Figure 3Ac,
no fluorescence could be detected from cells treated with 50 μM 15a; as a matter of fact, even at
a concentration of 100 μM, no obvious fluorescence could be observed after one-hour staining
(Supporting Information, S1). Unexpectedly, even at a concentration up to 20 μM, no
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fluorescence staining from probe 27 could be detected after one-hour incubation (Figure 3A d).
The lack of fluorescence from controls 15a and 27 in live cells suggest that the uptake and
localization was indeed due to the natural product’s activity, and α,β-unsaturated ketone moiety
is crucial to the uptake of oridonin.
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In parallel, we performed time- and concentration-based uptake studies of probe 17d in HepG2
cells. The minimum concentration of probe 17d was optimized to 1 μM (data not shown) and the
uptake of probe 17d at this concentration was observed quickly as shown in Figure 3B. Notably,
distinguishable fluorescence could be detected in HepG2 cells in as early as 5 min after
incubation with probe 17d, and the same phenomenon occurred when the A549 cells were
incubated with 17d (Supporting Information, S2).
Figure 3. Uptake of probes in HepG2 cells. (A) Uptake of indicated probes in HepG2 cells. (a) 5
µM 17d for 1 h; (b) 5 µM 24a for 1 h; (c) 50 µM 15a for 1 h; (d) 20 µM 27 for 1 h. (B) Time-
course uptake of 1 µM probe 17d in HepG2 cells, images were collected using fluorescence
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microscopy. (a) HepG2 cells were treated with 17d for 0 min; (b) 5 min; (c) 10 min; (d) 15 min;
(e) 30 min; (f) 1 h. Bars denote 50 μm.
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Intracellular Distribution of Probes 17d and 24a in Cancer Cells
To examine whether the types of linked coumarins would affect the uptake or localization of
oridonin probes, we simultaneously investigated the difference between probes 17d and 24a, that
were linked with different coumarin dyes. Both A549 and HepG2 cells were treated with 1 μM
17d or 24a for 12 h, washed with media, and imaged by a confocal laser scanning microscope. In
HepG2 cells, probe 17d appeared more effective than 24a, stronger fluorescence signal could be
detected from 17d (Supporting Information, Figure S3). But no obvious difference could be
observed in A549 cells (Figure 4), the fluorescence signals of both 17d and 24a could be
detected within the cytoplasm of cells presented as spots adjacent to the nuclear. Thus, the next
subcellular localization studies were reasonably carried out in A549 cells.
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Figure 4. Live-cell imaging of probes. A549 cells were treated with 1 μM probe 17d (a) or 24a
(b) for 12 h. Images were collected with excitation of probes at λex= 405 nm and emission at
λem= 460 nm, cells were imaged at 40x with an inverted confocal microscope (LSM710 NLO,
Zeiss). DIC: differential interference contrast field. Bars denote 10 μm.
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Subcellular Localization Studies
To further detail the subcellular localization of fluorescent oridonin probe 17d in A549 cells, we
co-stained 17d-treated cells with a panel of organelle-specific dyes. Fortunately, confocal
microscopy of cells co-stained with MitoTracker Red (mitochondria specific dye) confirmed the
localization of 17d in mitochondria. In particular, the overlap of red fluorescence from the red
mitochondrial stain and the green fluorescence from probe 17d provided compelling evidence in
support of this conclusion (Figure 5a). Notably in this localization study, probe 17d was
absorbed quickly after 1 h incubation and localized to mitochondria with a Pearson’s coefficient
value (Rr) of 0.979. However, the Rr value decreased to 0.843 when cells were treated with 17d
for 3 h, and only partial overlap of these two fluorescent probes could be observed at 12 h. After
24 h, no co-localization could be observed and the fluorescence became dispersed, which may be
attributed to the cytotoxicity of probe.
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Figure 5. Time-course of co-localization of probe 17d with MitoTracker (mitochondria specific
dye) in A549 cells. Images were collected using confocal microscopy (LSM710 NLO, Zeiss,
60x): (a) A549 cells incubated with 1 μM 17d for 1 h, right panel: cellular localization of 17d,
middle panel: overlay of two images, left panel: cellular localization of MitoTracker; A549 cells
incubated with 1 μM 17d for 3 h (b), for 12h (c), for 24h (d). Images were collected with
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excitation of probe 17d at λex= 405 nm and emission at λem= 460 nm, MitoTracker at λex= 579
nm and emission at λem= 600 nm. Bars denote 10 μm.
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Mitochondrial Membrane Potential Assay
The mitochondrion is an essential organelle that plays a crucial role in energy production; its
dysfunction would induce irreversible cell apoptosis in a short time39. Inspired by the
observations in which the fluorescent oridonin probe provided direct visual evidence for co-
localization with mitochondria in A549 cells, we speculated that these fluorescent oridonin
probes may reduce mitochondrial membrane integrity and thereby affect cell proliferation40.
Thus, commercially available fluorescent probe JC-1 was used to measure the mitochondrial
membrane potential (MMP). In healthy cells, JC-1 aggregates in the mitochondria and
spontaneously forms complexes that fluoresce red. In apoptosis or necrotic cells, JC-1 exists in
the monomeric form and stains the cytosol green. As shown in Figure 6a, treatment of A549 cells
with 1 μM 17d induced the dissipation of MMP in a time-dependent manner, as indicated by a
decrease in red fluorescence emission and an increase in green fluorescence emission. The ratio
of fluorescent intensity of red to green was determined and used as an indicator of cell healthy
status. The ratio began to decrease after 12 h treatment of 1 μM 17d, and after 48 h treatment, the
ratio decrease dramatically from 1.496 to 0.312. However, no significant changes of MMP could
be observed after the treatment of 20 μM 27 (Figure 6b).
Previous investigations indicated that oridonin could induce apoptosis through several apoptotic
pathways, especially mitochondria-dependent apoptosis41. Therefore, we detected the influence
of oridonin on MMP using JC-1 probe. Briefly, A549 cells were first incubated with or without
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10 μM oridonin for 36 h, then dyed with fluorescent JC-1. As shown in Figure 6c, oridonin
treatment in A549 cells induced the dissipation of MMP, as indicated by a change of
fluorescence intensity from red to green in treated cells. Collectively, these results suggested that
mitochondrial pathway directly participated in apoptosis induced by oridonin.
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Figure 6. Compounds 17d and 1 induced loss of mitochondrial membrane potential (MMP) in
A549 cells analyzed by JC-1 fluorescence. (a) Independent experiments with 10000 cells per
treatment condition reveal a decrease of photometric ratio of red to green fluorescence after 17d
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treatment, and no significant change could be observed after 27 treatment (b). Data was
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represented as mean ± SD of three independent experiments, *P<0.05, **P<0.01, ***P<0.001. (c)
Oridonin induced mitochondrial depolarization in A549 cells. After treatment with 10 μM 1 for
36 h, the fluorescence intensity changed significantly from red to green in treated cells. The
upper panel represents the normal group, lower panel depicts the loss of MMP after the treatment
of oridonin.
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Immunofluorescence Co-localization Analysis of Cytochrome c
Cytochrome c is widely believed to be localized uniquely in the mitochondrial intermembrane
space under normal physiological conditions. The release of cytochrome c from mitochondria to
the cytosol after the dissipation of MMP, where it activates the caspase family of proteases, is
believed to be a primary trigger leading to the onset of apoptosis42. Thus, we detected the
localization of cytochrome c in A549 cells after the treatment of oridonin with
immunofluorescence co-localization analysis. As shown in Figure 7, in control groups, the
green-stained cytochrome c by a fluorescently labeled secondary antibody were located in the
mitochondria which were stained red by Mitotracker Red, resulting in a bright orange
fluorescence. However, when A549 cells were treated with 3 or 5 μM oridonin for 24 or 48 h,
part of the orange fluorescence was quenched and green color appeared outside, indicating
leakage of cytochrome c from mitochondria into the cytosol. We also found that the intensity of
green light was proportional to both incubating time and concentration of oridonin. The
combination of these observations suggested the release of cytochrome c from mitochondria to
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cytosol in response to treatment with oridonin, which might be the critical step in the process of
oridonin-mediated apoptosis.
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Figure 7. Immunofluorescence co-localization analysis reveals the release of cytochrome c from
mitochondrion to cytosol during the apoptostic process. Cells were incubated with primary
antibody against cytochrome c followed by treatment with Alexa Fluor 488-conjugated (green)
goat anti-rabbit secondary antibody. The mitochondria were stained with Mitotracker Red (red),
and cell nuclei were stained with DAPI (blue). Images were collected using confocal microscopy
(LSM710 NLO, Zeiss): (a) A549 cells incubated without oridonin, right panel: merged images,
left panel: enlarged view of representative cells; (b) A549 cells incubated with 3 μM 1 for 24 h,
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48 h (c); 5 μM 1 for 24 h (d), 48 h (e). Yellow arrow: cytochrome c located in mitochondria;
white arrow, cytochrome c released from mitochondria to cytosol. Bars denote 10 μm.
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Structure-Activity Relationships Studies
Above experiments clarified that the loss of cancer cell viability was associated with the
decreased mitochondrial membrane potential and the release of cytochrom c. Further studies
were conducted to explore which unique pharmacophore of oridonin can initiate these biological
responses. We compared the localization of probe 17d when pre-incubated with oridonin (1), its
14-OH modified derivative (25), and α,β-unsaturated ketone reduction product (26), respectively.
When A549 cells were pre-treated with oridonin or its analogues, both 1 and 25 could block the
uptake of probe 17d effectively as shown in Figure 8a and 8b. However, no significant changes
in both fluorescence intensity and localization of 17d were noted when A549 cells were pre-
treated with 20 μM 26 for 24 h. This observation suggests that oridonin and its fluorescent
probes act similarly in A549 cell and probe 17d might share a common target with oridonin or its
derivative 25. More importantly, the active α,β-unsaturated ketone could bind tightly to the target
in an irreversible manner43,44. Indeed, α,β-unsaturated ketone moiety is a common and
structurally important functionality that is widespread in various naturally occurring products,
especially in ent-kaurane diterpenoids45,46. As shown in Figure 1, almost all the ent-kaurane
diterpenoids that exhibit substantial antitumor activity share this common moiety, and this result
provides direct evidence that the ent-kaurane diterpenoids might share a common mode of action.
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Figure 8. Confocal fluorescent images detailing the impact of compounds 1, 25 and 26 on the
subcellular localization of probe 17d: A549 cells were incubated with 20 μM oridonin (1) (a), 1
μM 25 (b), 20 μM 26 (c) for 24h, then 1 μM probe 17d for 1h. Images were collected with
excitation of probe at λex= 405 nm and emission at λem= 460 nm, cells were imaged at 40x with
an inverted confocal microscope (LSM710 NLO, Zeiss). DIC: differential interference contrast
field. Bars denote 10 μm.
CONCLUSION
In this experiment, nine novel fluorescent analogues were designed and synthesized to visualize
the anticancer mechanism of action of oridonin. Most of the target probes displayed satisfactory
antiproliferative activities and optimal fluorescent properties. Further fluorescent and biological
profiles were examined by using typical probes (17d, 24a, and 27) to investigate the mode of
action of oridonin in cancer cell lines. It was demonstrated that oridonin fluorescent probe 17d
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could be absorbed quickly by cancer cells, and α,β-unsaturated ketone moiety was the key
pharmacophore for the anticancer activity and uptake of oridonin. Moreover, time-based
subcellular localization studies of probe 17d in A549 cells were performed, and the co-
localization analysis illustrated the mitochondria-specific localization of fluorescent oridonin
probe 17d, which could be developed as a specific mitochondria biomarker. The subsequent
studies on mitochondrial physiology indicated that the mitochondrial pathway is involved in
oridonin-mediated apoptosis and that cytochrome c plays an important role in the apoptotic
process.
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In summary, we report an effective strategy for fluorescent labeling of oridonin, which may be
used for studies on other ent-kaurane diterpenoids for which the anticancer action and targets are
currently unknown. Further progress on identification of cellular targets of oridonin and other
ent-kaurane diterpenoids will be reported in due course.
EXPERIMENT SECTION
Chemistry
General Information
Commercially available reagents and solvents were used without further purification. Column
1
13
chromatography was carried out on Merck silica gel 60 (200-400 mesh). H, C and 2D NMR
spectra were recorded with 300 MHz spectrometers in the indicated solvents (TMS as internal
standard). Chemical shifts were reported in parts per million (ppm, δ) downfield from
tetramethylsilane. Proton coupling patterns are described as singlet (s), doublet (d), triplet (t),
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quartet (q), double doublet (dd), multipet (m) and broad (br). Low- and high-resolution mass
spectra (LRMS and HRMS) were measured on Agilent QTOF 6520. Melting points were taken
on XT-4 micro melting point apparatus and uncorrected. The purity of all tested compounds was
characterized by HPLC analysis and individual compounds with a pruity of ≥95% were used for
subsequent experiments (see the Supporting Information). HPLC was performed using
Phenomenex Gemini C18 (4.6×150mm, 5µm) and samples were detected using a wavelenght of
240 nm.
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1H NMR and 13C NMR Spectra of the Representive Compounds and Probes
Synthesis of Probe 17d
Compound 1 (100 mg, 0.27 mmol) was dissolved in dichloromethane, then EDCI, DMAP and
coumarin intermediates 16d (120 mg, 0.27 mmol) were added. The reaction mixture was stirred
at room temperature for 1 h. Then the mixture was washed with 10% HCl. The organic layer was
washed with brine, dried over anhydrous Na2SO4. After flash chromatography
(dichloromethane/methanol 150:1), compound 17d was obtained as a yellow solid (171 mg,
80%); mp 130-131 °C ; 1H NMR(CDCl3, 300 MHz), δ (ppm) 8.42 (1H, s), 7.37 (1H, d, J = 8.94
Hz), 6.62 (1H, dd, J = 8.97, 2.34 Hz), 6.46 (1H, d, J = 2.16 Hz), 6.05 (1H, s), 6.07 (1H, d, J =
10.50 Hz), 5.88 (1H, s), 5.51 (1H, s), 4.20-4.34 (3H, m, 20-CH2), 4.05-4.15 (4H, m), 3.73 (1H,
m), 3.42-3.52 (5H, m), 3.17 (1H, d, J = 9.81 Hz), 2.60 (1H, m), 2.29-2.37 (4H, m), 2.29 (1H, m),
1.97 (1H, m), 1.90 (2H, m), 1.85-1.95 (4H, m), 1.73 (1H, m), 1.68 (1H, m), 1.58 (1H, m), 1.44
(1H, m), 1.30 (1H, m), 1.21-1.24 (2H, m), 1.24 (6H, t, J = 7.14 Hz), 1.88 (6H, s); 13C
NMR(CDCl3, 75 MHz), δ (ppm) 206.5, 172.7, 171.3, 164.2, 158.4, 158.2, 152.9, 149.9, 149.2,
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131.1, 120.0, 109.5, 108.8, 107.7, 96.7, 96.1, 76.4, 74.1, 73.4, 64.5, 64.0, 63.4, 61.9, 59.7, 54.6,
45.0, 41.3, 38.7, 33.7, 33.5, 33.0, 32.5, 30.5, 30.0, 29.6, 25.4, 25.3, 21.6, 19.8, 19.8, 12.4; MS
(ESI) m/z: 792.4 [M-H]-, 794.6 [M+H]+ HR-MS (ESI) m/z: calcd for C43H56NO13 [M+H]+
794.3746, found 794.3765.
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Synthesis of Probe 24a
Following the procedure described for the preparation of compound 17d, compound 24a was
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synthesized as a pale yellow solid (yield 73%); mp 147-148 °C ; H NMR (CDCl3, 300 MHz): δ
(ppm) 1.09 (s, 3H), 1.11 (s, 3H), 1.26 (m, 4H), 1.61 (m, 7H), 1.97 (m, 1H), 2.22 (m, 1H), 2.57
(m, 4H), 3.07 (s, 6H, N(CH3)2), 3.15 (m, 1H), 3.50 (m, 1H), 3.74 (s, 2H), 4.05 (d, J = 8.7 Hz,
1H), 4.28 (m, 5H), 5.52 (s, 1H), 5.90 (s, 1H), 6.04 (m, 1H), 6.09 (s, 1H), 6.15 (s, 1H, Ar-H), 6.57
(s, 1H, Ar-H), 6.66 (d, J = 9.0 Hz, 1H, Ar-H), 7.43 (d, J = 9.0 Hz, 1H, Ar-H); 13C NMR (CDCl3,
75 MHz): δ (ppm) 206.0, 171.3, 170.3, 168.5, 161.3, 155.4, 152.5, 149.4, 147.7, 124.8, 119.7,
110.2, 108.6, 107.9, 97.8, 95.6, 75.7, 73.8, 72.9, 62.8, 62.5, 61.7, 61.5, 59.0, 54.2, 40.8, 39.6,
38.2, 37.3, 33.2, 32.1, 30.0, 29.8, 29.4, 29.1, 21.2, 19.3; MS (ESI) m/z: 736.3 [M-H]-, 738.5
[M+H]+; HR-MS (ESI) m/z: calcd for C39H48NO13 [M+H]+ 738.3129, found 738.3131.
Synthesis of Compound 25
Compound 1 (200 mg, 0.55 mmol) was dissolved in dichloromethane, then EDCI, DMAP and n-
caprylic acid (79 mg, 0.55 mmol) were added. The reaction mixture was stirred at room
temperature for 2 h. Then the mixture was washed with 10% HCl. The organic layer was washed
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with
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(dichloromethane/methanol 200:1), compound 25 was obtained as a white solid (216 mg, 80%);
mp 69-71 °C ; 1H NMR (CDCl3, 300 MHz): δ (ppm) 6.24 (d, J = 9.4 Hz, 1H), 6.13 (s, 1H), 5.81
(s, 1H), 5.48 (s, 1H), 4.37 (s, 1H), 3.30, 4.07 (dd, JA = JB = 9.6 Hz, each 1H), 3.75 (m, 1H), 3.50
(m, 1H), 3.18 (d, J = 9.6 Hz, 1H), 2.62 (m, 1H), 2.27 (m, 3H), 2.01 (m, 1H), 1.54 (m, 8H), 1.24
(m, 10H), 1.10 (s, 6H), 0.82 (m, 3H); 13C NMR (CDCl3, 75 MHz): δ (ppm) 206.7, 172.1, 149.8,
120.0, 96.2, 76.7, 73.8, 73.1, 63.5, 61.8, 59.9, 54.6, 41.3, 41.2, 38.6, 34.5, 33.7, 32.5, 31.5, 30.5,
30.3, 29.8, 29.6, 24.8, 24.6, 22.5, 21.6, 13.8; MS (ESI) m/z: 491.3 [M+H]+; HR-MS (ESI) m/z:
calcd for C28H42NaO7 [M+Na]+ 513.2823, found 513.2841.
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Synthesis of Compound 26
Compound 1 (200 mg ) was dissolved in methyl alcohol (10 mL), then 10% Pd/C 20 mg was
added to this solution. The mixture was stirred under hydrogen atmosphere at room temperature
for 1 h. The reaction mixture was filtered and the solvent was evaporated under reduced pressure.
Flash chromatography of the residue, eluting with dichloromethane-methanol (40:1), gave
compound 26 (188 mg, 94%) as a white solid; mp 206-207 °C ; 1H NMR (DMSO-d6, 300 MHz):
δ (ppm) 0.96 (s, 3H), 0.99 (s, 3H), 1.03(d, J = 7.2 Hz, 3H), 1.06 (m, 1H), 1.19 (m, 2H), 1.29 (m,
1H), 1.47 (m, 4H ), 1.62 (m, 1H), 1.88 (m, 1H), 2.00 (m, 1H), 2.32 (m, 1H), 2.91 (m, 1H), 3.43
(m, 1H), 4.06, 3.81 (dd, JA = JB = 10.8 Hz, each 1H, 20-CH2), 4.06 (d, J = 4.8 Hz, 1H), 4.79 (s,
1H), 5.73 (d, J = 11.1 Hz, 1H), 6.09 (s, 1H), 6.73 (s, 1H); 13C NMR (DMSO-d6, 75 MHz): δ
(ppm) 224.6, 96.7, 96.5, 73.2, 73.0, 71.4, 62.7, 61.1, 60.2, 52.6, 44.7, 38.3, 38.2, 33.3, 32.4, 29.2,
21.4, 19.0, 18.2, 10.1; MS(ESI)m/z: 367.8 [M+H]+; HR-MS (ESI) m/z: calcd for C20H30NaO6
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[M+Na]+ 389.1935, found 389.1939. The β-orientation of 17-CH3 has been confirmed by
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Synthesis of Probe 27
Following the procedure described for the preparation of compound 17d, compound 27 was
synthesized as a yellow solid; (yield 82%); mp 126-127 °C ; 1H NMR (CDCl3, 300 MHz): δ (ppm)
1.01 (s, 3H), 1.06 (m, 3H), 1.14 (s, 3H), 1.16 (t, J = 7.2 Hz, 6H, N(CH2CH3)2), 1.52 (m, 7H),
1.85 (m, 5H), 1.90 (m, 4H), 2.21 (m, 1H), 2.34 (m, 4H), 2.58 (m, 1H), 2.78 (m, 1H), 3.41 (q, J =
7.2 Hz, 4H, N(CH2CH3)2), 3.60 (m, 1H), 4.19, 3.96 (dd, JA = JB = 10.8 Hz, each 1H, 20-CH2),
4.08 (m, 2H), 4.27 (m, 2H), 5.72 (d, J = 11.4 Hz, 1H), 5.90 (d, J = 1.2 Hz, 1H), 6.39 (d, J = 2.4
Hz, 1H, Ar-H), 6.54 (dd, J = 9.0, 2.4 Hz, 1H, Ar-H), 7.30 (d, J = 9.0 Hz, 1H, Ar-H), 8.35 (1H, s,
Ar-H); 13C NMR (CDCl3, 75 MHz): δ (ppm) 221.9, 172.1, 170.7, 158.0, 152.5, 130.6, 125.0,
109.1, 107.2, 96.2, 95.4, 76.5, 73.8, 72.9, 64.1, 63.6, 63.0, 61.0, 60.2, 59.5, 54.0, 53.0, 45.6, 44.6,
40.5, 38.2, 38.0, 36.6, 33.2, 33.1, 32.6, 32.0, 31.7, 29.8, 29.2, 24.9, 24.8, 20.8, 19.3, 13.4, 11.9,
9.3; MS (ESI) m/z: 794.4 [M-H]-, 796.6 [M+H]+; HR-MS (ESI) m/z: calcd for C43H58NO13
[M+H]+ 796.3903, found 796.3907.
Cytotoxicity Assay
The effect of oridonin and its derivatives on the cytotoxicity of cancer cells was determined by
MTT assay. Briefly, the MTT assay was performed in 96-well plates using HepG2, A549 and
Hela cells. These cells (5×104 cells/mL) at the log phase of their growth cycle were added to
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each well (100 µL/well), then treated in the presence or absence of test compounds, and
incubated for 72 h at 37 °C in a humidified atmosphere of 5% CO2. After terminating the
treatment, 20 µL of MTT solution (5 mg/mL) was added to each cultured medium and cells were
incubated for another 4 h. Then, DMSO was added to each well (150 µL/well). After 10 min at
room temperature, the OD value of each well was measured on a Microplate Reader (BIO-RAD
Instruments Inc NO.550) at the wavelength of 490 nm. In these experiments, the negative
reference agent was 0.1% DMSO, and paclitaxel at the concentration of 10 mg/mL was used as
the positive reference.
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Fluorescent Cell Staining
For fluorescence observation, A549 cells were seeded in Lab-Tek four-chambered slides (Nalge
Nunc International, Naperville, IL, USA) and incubated with various concentrations of
fluorescent oridonin derivatives for different time. After incubation, cells were washed with PBS
for three times and antifade mounting medium was added. Images were obtained with an
inverted confocal microscope (LSM710 NLO, Zeiss). Each experiment was repeated 3 times and
selected images were presented in the manuscript.
Subcellular Localization Studies
A549 cells were seeded in Lab-Tek four-chambered slides (Nalge Nunc International, Naperville,
IL, USA), pretreated with 1 μM 17d for 1, 3, 12, 24 h, washed with PBS, and incubated with
MitoTracker (Life Technologies Inc., USA, MitoTracker® Red CMXRos, 200 nM) for another
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