Combination of betulinic acid with diazen-1-ium-1,2-diolate nitric oxide moiety donating a novel anticancer candidate

Article abstract of DOI:10.2147/OTT.S154412

Background: Betulinic acid (BA) is a complex lupane triterpenoid with unique antineoplastic activity. However, its antiproliferative activity is far from satisfaction. In order to improve its anticancer efficacy, betulinic acid was conjugated with a nitric oxide (NO)-releasing moiety to get a novel hybrid, BA-78. Methods: The antiproliferative activity of BA-78 against 6 cell lines and the ability of releasing nitric oxide were determined. The pro-apoptosis mechanism of BA-78 was investigated as well. Results: BA-78 exhibited time-dependent release of NO, and it displayed higher antiproliferative potential than BA through increasing apoptosis and inducing cell cycle arrest at G1 phase. Western blotting results showed that BA-78 increased the expression of Bax, Bid, Bad and cytochrome C and reduced the level of anti-apoptosis proteins including Bcl-2 and Bcl-xl. Conclusion: Our study revealed that novel compound BA-78, possessing betulinic acid and nitric oxide (NO)-releasing moiety, could be developed as an antitumor agent.

Full text of DOI:10.2147/OTT.S154412

OncoTargets and erapy
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Open access Full Text article
O r i g i n a l r e s e a r c h
combination of betulinic acid with diazen-1-ium-
1,2-diolate nitric oxide moiety donating a novel
anticancer candidate
This article was published in the following Dove Press journal:
OncoTargets andTherapy
laiyin Zhang1
shuangxing hou2
Bo li3
Jianjian Pan4
liping Jiang5
guiying Zhou2
hong gu6
caixing Zhao6
huiping lu6
Fenfen Ma6
Background: Betulinic acid (BA) is a complex lupane triterpenoid with unique antineoplastic
activity. However, its antiproliferative activity is far from satisfaction. In order to improve its
anticancer efficacy, betulinic acid was conjugated with a nitric oxide (NO)-releasing moiety to
get a novel hybrid, BA-78.
Methods: The antiproliferative activity of BA-78 against 6 cell lines and the ability of releasing
nitric oxide were determined. The pro-apoptosis mechanism of BA-78 was investigated as well.
Results: BA-78 exhibited time-dependent release of NO, and it displayed higher antiprolifera-
tive potential than BA through increasing apoptosis and inducing cell cycle arrest at G1 phase.
Western blotting results showed that BA-78 increased the expression of Bax, Bid, Bad and
cytochrome C and reduced the level of anti-apoptosis proteins including Bcl-2 and Bcl-xl.
Conclusion: Our study revealed that novel compound BA-78, possessing betulinic acid and
nitric oxide (NO)-releasing moiety, could be developed as an antitumor agent.
1Department of Pharmacy, linyi
People’s hospital, linyi, 2Department
of neurology, shanghai Pudong
hospital, Fudan University Pudong
Medical center, 3Department of
hand surgery, huashan hospital,
Fudan University, 4Department
of general Medicine, Fudan
Keywords: lupane triterpenoid, nitric oxide, anticancer, apoptosis, NONOate, Bcl-2 family
Introduction
Natural products have always been the main source of anticancer drugs, especially in the
treatment of solid tumors, against which targeted therapies have shown disappointing
efficiency.1 Betulinic acid (BA) (Figure 1), isolated from the root of Pulsatilla chinensis
(Bge) has long been used in traditional treatments.2 BA exhibits multiple biological
activities, including anti-tumor, anti-HIV and antiangiogenesis,3,4 among which, its
unique, safe and remarkable anticancer pharmacological profile is of particular interest.5
It is generally accepted that the apoptotic events induced by BA were associated with
apoptosis-related proteins.6 It activates the mitochondrial pathway of apoptosis, which
results in the release of cytochrome c (Cyt-c).7 However, the development of BA for
cancer therapy is hampered largely by its modest efficacy and broad range of phar-
macological activity. Therefore, it is highly desirable to develop novel derivatives of
BA to improve its cytotoxic potency while reducing its side effects.
University, 5Department of
Physiology and Pathophysiology,
shanghai Medical college, Fudan
University, 6Department of Pharmacy,
Pudong hospital, Fudan University,
shanghai, china
correspondence: huiping lu;
Fenfen Ma
Nitric oxide (NO) is a gas signaling molecule, which plays multiple roles in physi-
ological conditions or under pathologic processes.8 Studies have shown that high level
of NO cannot only induce apoptosis of tumor cells, but can also sensitize tumor cells to
chemotherapy.9 However, limited by the gas property of NO, NO donors are alterna-
tively used as surrogates for NO in anticancer studies. Diazeniumdiolates (NONOates)
are considered as reliable sources of NO,10 as shown in Figure 2, NONOate prodrugs
could form the parent anion under physiological conditions (pH 7.4, 37°C), which
Department of Pharmacy, Pudong
hospital, Fudan University,
2800 gongwei road, huinan Town,
shanghai 201399, china
Tel +86 216 803 6288
Fax +86 215 198 0045
email lhp310@126.com;
cdc343cdc001@163.com
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http://dx.doi.org/10.2147/OTT.S154412

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29
Materials and methods
Materials
20
27
30
26
19
Mouse melanoma cells (B16F10), human breast cancer cells
(MCF-7), human colon cancer cells (HCT-116), human ade-
nocarcinomic alveolar basal epithelial cells (A549), human
hepatocarcinoma (HepG2) and human embryonic lung fibro-
blast (Helf) were purchased from American Type Culture
Collection (Manassas, VA, USA). 3-[4,5-dimethylthiazol-2-
yl]-2,5-diphenyl-tetrazolium bromide (MTT) and propidium
iodide (PI) were purchased from Sigma Chemical Co.
(St Louis, MO, USA). The Annexin V-fluorescein isothio-
cyanate (FITC) apoptosis detection kit was purchased from
BD Parmingen (San Diego, CA, USA). Griess reagent was
purchased from Beyotime (Jiangsu, China). Primary anti-
bodies against Bax, Bcl-xl, Bak, Bid, Bad, Bcl-2, Cyt-c and
β-actin were purchased from Cell Signaling Technology,
(Boston, MA, USA) and fluorescent secondary antibodies
(goat-anti-rabbit or goat-anti-mouse) were purchased from
Life Technologies (Grand Island, NY, USA). All cell culture
reagents, including no-essential amino acids and the bicin-
choninic acid (BCA) protein assay kit were purchased from
Life Technologies.
OH
28
25
23
O
3
HO
Figure 1 chemical structure of betulinic acid.
further decomposes to release 2 molecules of NO. Recently,
abundant NONOate-based hybrids have been developed as
anticancer agents, including PABA/NO (2), JS-K (3), 5-Fu/
NO (4), and oridonin/NO (5) (Figure 2).11–14 It was observed
that the derivatives releasing higher amount of NO showed
superior antiproliferative activity, suggesting that NO was a
component of the cytostatic effects of such kind of hybrids.
Encouraged by these findings, we designed and synthe-
sized a novel hybride (BA-78) containing BA and NONOate
moieties to improve the antiproliferative activity of natural
BA. Herein, we report the synthesis, in vitro antiproliferative
activity and NO release ability of BA-78, and evaluate its
possible mechanism of action.
chemistry
All commercially available solvents and reagents were used
without further purification. Flash column chromatography
1
was carried out on 200–300 mesh silica gel. H nuclear
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Figure 2 Mechansim of nO release from diazeniumdiolate and the structures of PaBa/nO (2), Js-K (3), 5-Fu/nO (4), and oridonin/nO (5).
Abbreviation: nO, nitric oxide.
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Combination of betulinic acid and its anticancer efficacy
magnetic resonance (NMR) and 13C NMR spectra were to afford product 10 (51%) as a white solid. 1H-NMR (CDCl3,
recorded with a Bruker AV-300 spectrometer in the indicated 300 MHz): δ 0.73, 0.84, 0.90, 0.97, 1.68 (each 3 H, s), 1.59
solvents (trimethylchlorosilane [TMS] as internal standard). (m, 4 H), 1.98 (2 H, m), 2.24 (2 H, m), 3.01 (1 H, m), 3.49
Mass spectra were obtained using FTMS-2000. The synthetic (4 H, t), 3.81, 4.01 (each 1 H, m), 4.59, 4.72 (each 1 H, m), 5.80
method and physicochemical date of compounds 6 and 9 have (2 H, s); 13C-NMR (CDCl3, 75 MHz): δ 15.9, 16.6, 18.1,
been disclosed in previous reports, the NMR spectrums of 19.3, 20.8, 15.4, 26.2, 29.7, 29.8, 30.6, 31.1, 32.3, 37.0, 38.3,
compounds 7, 8, and 10 are available in Figures S1–S3.15,16 38.5, 39.2, 40.6, 40.8, 42.1, 42.4, 45.1, 46.9, 49.2, 50.5, 56.3,
67.2, 72.1, 96.5, 109.6, 152.8, 173.4, 179.7; mass spectrom-
O2-(Methylthiomethyl)-1-(pyrrolidin-1-yl)
diazen-1-ium-1,2-diolate (7)
etry (MS) electrospray ionization (ESI) m/z [M+H]+ 642.4,
high resolution mass spectrometer (HRMS) (ESI) m/z calcu-
lated for C37H63N4O6 [M+NH4]+ 659.4742, found 659.4738.
The sodium NONOate 6 (6.88 g, 45.0 mmol) was added to
a suspension of sodium carbonate (4.78 g, 45.0 mmol) and
dimethylformamide (DMF) (100 mL) at room temperature,
and this mixture was stirred for 10 min. Chloromethyl methyl
sulfide (3.72 mL, 45.0 mmol) was added dropwise, and the
reaction was allowed to proceed at room temperature for 4 h
with stirring. Ethyl acetate (200 mL) was added to quench the
reaction, the solids were filtered off, the organic phase was
washed with water (5×60 mL), and dried with Na2SO4. The
solvent was removed in vacuo to give a liquid residue, which
was purified by silica gel column chromatography using
EtOAc/hexane (1:4, v/v) as the eluent. Compound 7 was
obtained as a canary yellow liquid (3.80 g, 45%): 1H NMR
(300 MHz, CDCl3): δ (ppm) 5.15 (s, 2 H), 3.53–3.48 (m, 4 H),
2.21 (s, 3 H), 1.91–1.86 (m, 4 H).
Determination of in vitro antiproliferative
activity
The viability of 5 human cancer cell lines (B16F10, MCF-7,
HCT-116, A549, and HepG2) treated with BA, BA-78, and
Cisplatin, was determined using the MTT assay. Briefly,
cells were seeded in 96-microwell plates, incubated at 37°C
in a humidified incubator with 5% CO2 for 24 h prior to the
experiments. After removing the medium, 100 μL of fresh
medium containing the test compounds at 5 concentra-
tions (ie, 10−4, 10−5, 10−6, 10−7, and 10−8 M) were added to
microtiter plates and cells were incubated with each com-
pound at 37°C for another 72 h. At the end of the incubation,
the medium was discarded, cells were washed with PBS and
100 μL of MTT buffer was added to each well. Cells were
incubated with MTT for at least 4 h at 37°C and the viabilities
were proportioned to the value of optical densities, which
was quantitatively measured by spectrophotometry (Biorad,
Nazareth, Belgium) at 570 nm. The dose–response curve (not
shown) was created by plotting the percent viability against
the log of concentrations of the corresponding compound for
each cell line. The molar concentrations that caused 50% cell
killing (IC50) were determined.
O2-chloromethyl-1-(pyrrolidin-1-yl)
diazen-1-ium-1,2-diolate (8)
A solution of compound 7 (273 mg, 1.44 mmol) in dry
dichloromethane (30 mL) was cooled to 0°C and sulfuryl
chloride (1.44 mL, 1.0 M solution in dichloromethane) was
added dropwise, the ice bath was removed and the reaction
mixture was stirred at room temperature for another 2 h.
The solvent was evaporated to afford compound 8 in nearly
quantitative yield, which was used without further purifica-
tion. 1HNMR (300 MHz, CDCl3): δ (ppm) 5.80 (s, 2 H), 3.63
(m, 4 H), 1.95 (m, 4 H).
Detection of nO production
NO was indirectly determined by measuring nitrite content
in the culture supernatant using Griess reagent. Briefly,
B16F10 cells (2×104 cells/mL) were seeded in a 96-well
Ba-78
To a solution of 0.1 g, 9 mmol in anhydrous DMF was added plate and incubated at 37°C for 24 h followed by incubation
Cs2CO3 (1 eq.) and compound 8 (1.2 eq.). The reaction was with 10 μM of BA-78 for different times (ie, 10, 30, 60 min,
stirred at room temperature for 6 h. The reaction mixture 3, 6, 12, 24, and 48 h). For each well, 50 μL of supernatants
was diluted with 20 mL of ethyl acetate, and the organic layer was collected in a tube, the collection was incubated with
was separated. The aqueous layer was extracted with ethyl 25 μL of sulfanilic acid for 10 min at room temperature
acetate (2×10 mL). The combined organic layer was dried in the dark. After the reaction, 25 μL of N-(1-naphthyl)
over anhydrous sodium sulfate, and the solvent was evapo- ethylenediamine was added into the tube and the plate was
rated under reduced pressure. The crude material was purified incubated for another 10 min at room temperature in the dark.
by flash column chromatography (1:1 hexane/ethyl acetate) The absorbance was read at 550 nm by a spectrophotometry
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(BioTek Instruments, Inc., Waltham, MA, USA). The amount Technology) overnight at 4°C. After the membranes were
of NO was quantified according to the standard curve washed, they were then incubated with fluorescent secondary
prepared under the same condition and expressed as NO antibodies for 60 min at room temperature. The membranes
released/micromole of BA-87.
were washed and proteins were detected with LI-COR
Odyssey Infrared Imaging System.
Determination of apoptosis by flow
cytometry
cell cycle analysis
B16F10 cells (5×105 cells/mL) were seeded in 6-well plates 5×104 B16F10 cells were seeded into 6-well plates and
and treated with BA-78 at different concentrations (ie, 0, incubated overnight. After incubation with various concen-
0.25, 0.5, and 1.0 μM) for 36 h. The cells were then washed trations of BA-78 for 48 h, cells were harvested, washed
twice and digested with trypsin. After centrifugation, the with cold PBS and fixed with 70% ethanol in PBS at −20°C
supernatants were removed and cells were resuspended in for 12 h. Subsequently, the cells were resuspended in PBS
500 μL of 1× binding buffer to obtain a concentration of containing 100 μg/mL RNase and 50 μg/mL PI and incubated
1×106 cell/mL. Annexin V-FITC 5 μL and PI 10 μL were at 37°C for 30 min. Cell cycle distribution of nuclear DNA
added and cells were incubated at room temperature for was determined by flow cytometry on a FC500 cytometer
another 15 min in the dark. The stained cells were analyzed by (Beckman Coulter).
a FC500 cytometer (Beckman Coulter, Brea, CA, USA).
statistical analysis
Data were expressed as mean ± standard error of the mean.
Western blot analysis
B16F10 cells were incubated with BA-78 at different con- Data with normal distribution were analyzed by t-test or one-
centrations (ie, 0, 0.25, 0.5, and 1.0 μM) for 48 h. Cells were way analysis of variance non-parametric analysis followed
washed with cold PBS twice and lysed on ice for 10 min, and by the Bonferroni post hoc test. Data with abnormal distribu-
lysates were centrifuged at 13,000 g for 15 min. The protein tion were analyzed by Kruskal–Wallis test (GraphPad 5.0).
concentration in the supernatant was determined using BCA P,0.05 was considered statistically significant.
protein assay. Equal amounts of protein were loaded and
Results
separated using sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) and transferred to poly- chemistry
vinylidene fluoride (PVDF) membrane. Membranes were As shown in Scheme 1, the synthesis of NONOates was car-
blocked for 1 h at room temperature with 5% bovine serum ried according to the literature in the presence of nanometer-
albumin and then were incubated with primary antibodies sized TiO2.16 Briefly, reaction of pyrrolidine with NO gas
against Bax, Bad, Bid, Bak, Bcl-xl, Bcl-2, cytochrome c, at room temperature under atmospheric pressure afforded
caspase-3 and β-actin (all purchased from Cell Signaling O2-sodium-1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolates (6)
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Scheme 1 synthetic route for hybride (Ba-78).
Notes: reagents and conditions: (A) MeOna, MeOh, nanometers-sized TiO2, rt, 54 h, 78%; (B) clch2sch3, DMF, na2cO3, rt, 3 h 45%; (C) sO2cl2, DcM, rt, 2 h, 98%;
(D) ac2O, DMaP, pyridine, rt, 2 h, 92%; (E) 8, cs2cO3, DMF, rt, 6 h, 51%.
Abbreviations: DcM, dichloromethane; DMaP, dimethylaminopyridine; DMF, dimethylformamide.
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Combination of betulinic acid and its anticancer efficacy
in 78% yield. The sodium salts were alkylated with chlo- hybrids.17 Using Griess assay, we tried to characterize the NO
romethyl methyl sulfide to afford O2-(methylthiomethyl)- release in cancer cells and identify the correlation between
1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolates (7), which NO production level and cytotoxicity of BA-87. As shown
were subsequently reacted with sulfuryl chloride in dichlo- in Figure 3A, ~80% of nitrite/nitrate was detected in the
romethane to afford the O2-chloromethyl-protected NONOate supernatant of B16F10 cell culture subjected with BA-78
(8). This preparative approach of NONOates under atmo- after 12 h, suggesting that NO might be a key factor in the
spheric pressure using easily available apparatus promoted cytotoxicity of such hybride. Furthermore, the cytotoxicity
the following synthesis of BA-78. Next, acetylated BA (9) of components 8, 9, and their equimolar combination (8+9)
was prepared by using acetic anhydride in 92% yield, which were tested against B16F10 cells (Figure 3B). As expected,
subsequently reacted with NONOate (8) to afford the target the IC50 values of components 8, 9, and their combination
compound 10 (BA-78) in yield of 51%.
were significantly higher than that of BA-87 (Figure 3C).
Previous results suggested that the antiproliferative activity
of BA-78 was attributed to the synergic effects of BA and
NO donor moieties.
cytostatic effects of Ba-78 against
human cancer cells
With BA-78 in hand, we first evaluated its cytotoxicity on
cancer cell by MTT method. As shown in Table 1, BA-78
displayed preferred inhibitory efficacies than BA against
the selected malignant cell lines, with IC50 values ranging
from 0.89 to 7.44 μM. The most sensitive cancer cell line was
B16F10, against which the IC50 value of BA-78 was 0.89 μM,
which were even lower than that of positive control cisplatin.
This is in line with reports that BA is more potent against
melanoma. In addition, similar experiments were performed
with the Helf and human normal liver cells (L02). The results
showed that BA-87 had little inhibiting effects on the growth
of Helf and L02 cells; the IC50 values on Helf and L02 cells
were much higher than that on cancer cells. From the growth
response curves (Supplementary materials) of BA-78 on
these cells at a concentration of 1 μM, it can be inferred that
the growth of Helf and L02 cells were not affected at all,
while the growth of tumor cells were affected significantly.
These data suggested that BA-78 has selective cancer cell
killing properties, and will be safe for cancer treatment.
Ba-78 increased level of cell apoptosis
High level of NO is toxic and able to induce apoptosis of
tumor cell.18 To investigate whether BA-87 can induce
cell apoptosis, B16F10 cells were incubated with 0, 0.25,
0.5, and 1.0 μM of BA-78 and Annexin V-FITC/PI double
staining was applied to test cell apoptosis at the end of the
incubation. Annexin V is a phospholipid-binding protein with
strong affinity for phosphatidylserine, which appears on the
cell surface as a general indicator of apoptosis. As shown in
Figure 4, BA-78 dose dependently increased the percentage
of Annexin V-positive cells (right quadrants, Q2+Q3) ranging
from 8.27% (0.25 μM) to 22.15% (1 μM), which were sig-
nificantly higher than the basement apoptosis rate in control
cells (only 4.25%). Table 2 shows the percentages of early
apoptosis cells (Q3) and advanced apoptosis cells (Q2).
Ba-78 reduced level of anti-apoptosis
proteins and increased level of pro-
apoptosis proteins
The previous flow cytometry results indicated the pro-
Nitrogen oxide-releasing efficacy of
Ba-78
The NO-releasing efficacy has been proven to be an important apoptosis activity of BA-78. We further evaluated the results
component for the antiproliferative activity of NO-based by detecting the expression of apoptosis-related proteins.
Table 1 antiproliferative activities of Ba-78 against different cell lines
Compounds
Cell lines (IC50,a µM ± SEM)
B16F10
MCF-7
HCT-116
A549
HepG2
Helf
L02
Ba
26.98±2.50
0.89±0.14
11.73±0.95
17.53±1.72
0.92±0.09
7.83±0.52
27.3±3.28
1.53±0.24
9.45±1.04
38.30±5.90
7.44±0.98
12.67±1.85
30.40±5.14
4.32±0.73
4.77±0.62
61.99±8.61
17.76±0.88
21.75±3.03
96.32±7.99
27.21±3.18
9.17±0.83
Ba-78
cisplatin
Notes: aMTT methods, cells were incubated with indicated compounds for 72 h (mean ± seM, n=3). ic50 represents the concentration that caused 50% cell killing.
Abbreviations: a549, human adenocarcinomic alveolar basal epithelial cells; B16F10, mouse melanoma cells; Ba, betulinic acid; Ba-78, synthetic route for hybride;
HCT-116, human colon cancer cells; Helf, human embryonic lung fibroblast; HepG2, human hepatocarcinoma; L02, liver cells; MCF-7, human breast cancer cells;
seM, standard error of the mean.
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Figure 3 Time-dependent production of nO in B16F10 cells.
Notes: (A) B16F10 cells were incubated with 10 μM of Ba-78 for 48 h, and concentrations of nO in cultural medium were determined at different time points. Data
represents level of nO produced by per micromole of Ba-78; (B) structure of components 8 and 9, and Ba-78; (C) comparison of antiproliferative activity of Ba-78 with
its components 8, 9, and the equimolar mixture of 8 and 9. Data were average of 3 independent experiments. **P,0.01 versus control group, ***P,0.001 versus control
group. ic50 represents the concentration that caused 50% cell killing.
Abbreviations: B16F10, mouse melanoma cells; Ba-78, synthetic route for hybride; nO, nitric oxide.
The Bcl-2 family of proteins determines cell fate by regu-
Ba-78 induced cell cycle arrest at
lating the integrity of the mitochondrial outer membrane
g1 phase
(MOM). It includes adaptor proteins only containing BH3
Because mitochondrial dysfunction has a close relation-
domain such as Bim and Bid, which initiate apoptosis. The
ship with events such as apoptosis, necrosis and cell cycle
second group are prosurvival proteins (Bcl-2 and Bcl-xL),
arrest, we further analyzed the effect of BA-78 on cell
which inhibit the activation of a pro-apoptosis group con-
sisting of Bax and Bak. The latter constitute the pore-like
structures in the MOM, from which Cyt-c could be released
and initiate mitochondrial apoptosis pathway.19 As shown
in Figure 5A and B, the expression of pro-apoptotic pro-
teins increased with the incubation of BA-78, while the
expression of antiapoptotic proteins profoundly decreased.
Furthermore, the cytosolic expression of Cyt-c, which plays
a key role in the caspase-dependent apoptotic pathway,
also increased after the treatment of BA-78. These results
suggested that apoptotic pathway was involved in BA-78
induced cell death.
cycle of B16F10 cells. B16F10 cells were incubated with
different concentrations of BA-78 (eg, 0, 0.25, 0.5 and
1.0 μM) for 36 h, and after staining with PI, the cell cycle
was analyzed by flow cytometry. As shown in Figure 6,
BA-78 dose dependently increased the number of B16F10
cells in G1 phase, suggesting that BA-78 could induce cell
arrest at G1 phase. Accordingly, the number of cells in G2
phase reduced from 18.45% in control group to 4.84% in
1 μM of BA-78 treatment group as displayed in Table 3,
indicating that BA-87 redirected cells from mitotic phase
to quiescent stage. Taken together, these data showed that
the anti-proliferation activity of BA-78 against cancer
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Combination of betulinic acid and its anticancer efficacy
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Figure 4 effect of Ba-78 on the induction of apoptosis in B16F10 cells.
Note: Flow cytometric analysis was applied to determine the level of apoptosis in control group, Ba-78 at 0.25, 0.5 and 1.0 μM.
Abbreviations: B16F10, mouse melanoma cells; BA-78, synthetic route for hybride; FITC-A, fluorescein isothiocyanate-annexin.
cell was at least partially dependent on cell cycle arrest at a number of structural modifications of BA have been carried
out to improve its efficacy.21 The positions of C-1, C-2, C-3,
C-4, C-20 and C-28 are the diversity centers of BA, and some
derivatives resulted from structural modifications at these
positions exhibited potent anticancer activities.22–24 Rational
design of pharmacological agents by connecting NO donors
takes account of specific modifications of known molecules
with the purpose of optimizing their efficacy and safety.25–28
NO-releasing derivatives of BA have been reported previ-
ously, a series of compounds were synthesized by connecting
BA with furoxan NO donors, and they exhibited obvious
cytotoxicity against human hepatocellular carcinoma cells
in vitro.29 In the present study, we successfully prepared a
G1 phase.
Discussion
BA is a naturally occurring pentacyclic triterpenoid, which
has been used as an anticancer agent.20 In recent years,
Table 2 Flow cytometry analysis of apoptotic B16F10 cells
Group
Q1 (%)
Q2 (%)
Q3 (%)
Q4 (%)
control
Ba-78
0.11
0.20
0.05
0.12
1.87
5.10
4.47
14.80
2.38
3.17
6.39
7.35
95.60
91.50
89.10
77.70
0. 25 μM
0.50 μM
1.0 μM
Abbreviations: B16F10, mouse melanoma cells; Ba-78, synthetic route for hybride.
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Combination of betulinic acid and its anticancer efficacy
B16F10 cells
Control
0.25 µM
500
500
400
300
200
100
0
400
300
200
100
0
0
20
40
60
80
100
0
20
40
60
80
100
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500
400
300
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100
0
500
400
300
200
100
0
0
20
40
60
80
100
0
20
40
60
80
100
PI
PI
Figure 6 cell cycle arrest induced by Ba-78.
Note: Flow cytometric analysis of control group, Ba-78 at 0.25, 0.5 and 1.0 μM.
Abbreviations: B16F10, mouse melanoma cells; Ba-78, synthetic route for hybride; Pi, propidium iodide.
novel NO-releasing derivative of BA (BA-78) using another displaying antithrombotic, vasorelaxant, and cytostatic
kind of NO donors, NONOate. As shown in Figure 2, a activities.30 The nitrite measured after culture of tumor cells
number of NONOates-based compounds have been devel- with BA-78 may be generated by at least 2 different modes.
oped as anticancer agents. NONOates release NO spontane- These are as follows: 1) oxidation of NO released from the
ously under physiological conditions (pH 7.4, 37°C) with drug by molecular oxygen, and 2) oxidation of NO by tumor
a range of half-lives from a few seconds to several days, cell catalase. Both processes lead to the generation of nitrite.
BA showed moderate cytotoxicity on cancer cells used in our
study (17.53–38.30 μM), and the connection with NONOate
Table 3 The influence of cell cycle progression in B16F10 cells by
dramatically increased the antiproliferative efficacy of
Ba-78 at different concentrations
BA-78 (0.89–7.44 μM). We attributed this improvement
Group
G1 (%)
S (%)
G2 (%)
mainly to the NO-releasing ability of BA-78. However, it
should be noted that NO can both promote and inhibit tumor
progression and metastasis, this dual nature of NO is greatly
dependent on the dose and duration of NO exposure and
cellular sensitivity.31
negative control
Ba-78
40.23
43.18
46.29
52.06
41.32
40.94
43.76
43.10
18.45
15.88
9.95
0.25 μM
0.50 μM
1.0 μM
4.84
Abbreviations: B16F10, mouse melanoma cells; Ba-78, synthetic route for hybride.
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Low concentration of NO can promote invasion and metas- excellent antiproliferative ability in melanoma B16F10 cells
tases in different tumor models, and high concentration of NO with an IC50 as low as 0.89 μM. Notably, NO release assay
can inhibit tumor growth and metastases.32,33 NO alone shows a suggested that the potent cytotoxicity of BA-78 was partly
high IC50 concerning its cytostatic efficacy, while the reported attributed to NO produced from NONOate. The anticancer
anticancer candidates with NO-releasing residue have much mechanism studies further revealed that the antiproliferative
lower IC50 (from hundreds nanomoles to several micromoles). activity of BA-78 was ascribed to the induction of mito-
It has been reported by Heigold et al that NO induced apopto- chondrial apoptosis and cell cycle arrest in the G1 phase.
sis selectively in NOX-expressing transformed cells through Taken together, these findings provide a preliminary basis
formation of peroxynitrite in early stages of carcinogenesis.34 for the rational design and mechanism study of NO-based
Later, it was found that at late stages of tumor progression, derivative of BA.
membrane-associatedcatalaseprotectsfromapoptosisinduced
by exogenous NO.35 There is a delicate balance between oxi-
Acknowledgments
dation of NO/decomposition of peroxynitrite by tumor cell
protective catalase and NO-mediated inhibition of catalase.36
Besides, it should be kept in mind that the intracellular NO is
not always correlated with those obtained in simple phosphate
buffer for the sake of cell permeability and tumor microenvi-
ronment. In addition, our study showed that BA-78 exerted
much higher inhibitory activity against tumor growth than
NO, it seems that other features of BA-87 besides NO release
might contribute to its antitumor activity. Many research by
us and others have verified these assumptions.
This work was supported by grants from the Outstanding
Leaders Training Program of Pudong Health Bureau of
Shanghai (NO PWR12015-06, NO PWRd2017-11) and
by a program of Shanghai Pudong Hospital (NO 201602),
Fudan University. Shanghai Key Laboratory of Bioactive
Small Molecules, Department of Pharmacology, School
of Pharmacy and Institutes of Biomedical Sciences, Fudan
University, Shanghai, China. Laiyin Zhang is the first author
for this study.
BA was reported to induce apoptosis by damaging the
mitochondrial membrane, leading to Cyt-c release into
cytosol, which, in turn, regulates the downstream caspase
activation.37,38 In the present study, we also found the increase
in Cyt-c expression in cytosol. Besides, there is a direct
relationship between antiproliferative activity of BA and
depression of antiapoptotic Bcl-2 family proteins.39 Bcl-2
family proteins serve as critical regulators of apoptosis.40,41 As
shown in Figure 5, the expression of pro-apoptotic proteins
increased with the incubation of BA-78, while the expres-
sion of anti-apoptotic proteins significantly decreased. This
observation was in line with previous reports. We therefore
concluded that BA-78 induced apoptosis through mitochon-
drial apoptotic pathway.
Disclosure
The authors report no conflicts of interest in this work.
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