Synthesis and evaluation of cytotoxic activities of artemisinin derivatives

Article abstract of DOI:10.1111/cbdd.13016

Artemisinin is a naturally occurring antimalarial agent which has shown potent anticancer activity. In this work, new artemisinin derivatives with the piperazine group were synthesized. The cytotoxic activities of derivatives 5a–5d were evaluated by MTT assay against ten cell lines. The results showed that 5a–5d were more effective in inhibiting cancer cell growth than artemisinin. 5d was the most active against HepG2 and PLC-PRF-5 cells and presented no cytotoxicity on L-02 cells. Hoechst 33342 staining and flow cytometry experiment revealed that 5d could induce HepG2 and PLC-PRF-5 cell apoptosis. Flow cytometry analysis showed that 5d induced the loss of mitochondrial membrane potential (MMP) and increased the levels of intracellular free calcium and reactive oxygen species. 5d also induced cell cycle arrest in G2/M phase in HepG2 cells. According to the results of Western blotting and caspase-3 kit, 5d could significantly increase the content of p53, bax, Apaf-1, and caspase-3 and decrease the protein level of bcl-2, pro-caspase-9, and pro-caspase-3 in HepG2 cells. These findings indicate that 5d activates the mitochondria-mediated apoptotic pathway in HepG2 cells and may merit further investigation as a potential therapeutic agent for hepatocellular carcinoma.

Full text of DOI:10.1111/cbdd.13016

PROF. DEQUN SUN (Orcid ID : 0000-0002-4336-7641)
Article type
: Research Letter
Synthesis and evaluation of cytotoxic activities of artemisinin
derivatives
Qian Sun†, Jin Wang†, Yao Li, Jingjing Zhuang, Qian Zhang, Xiao Sun, Dequn Sun
Marine College, Shandong University at Weihai, No.180, Wenhua West Road, Weihai 264209, P. R. China
 Corresponding author.
Tel.: +86-631-5688537; fax: +86-631-5688303;
e-mail: dequn.sun@sdu.edu.cn
† These two authors contributed equally to this work.
ABSTRACT
Artemisinin is naturally occurring antimalarials which has shown potent anticancer activity.
In the present work, a new kind of artemisinin derivatives with piperazino group were
synthesized. The cytotoxic activities of derivatives 5a-5d were evaluated by MTT assay
against ten cell lines. The result showed that 5a-5d were more effective in inhibiting cancer
cells growth than artemisinin. 5d was the most active against HepG2 and PLC-PRF-5 cells,
and presented no cytotoxicity on L-02 cells. Hoechst33342 staining and flow cytometry
experiment revealed that 5d could induce HepG2 and PLC-PRF-5 cells apoptosis. Flow
cytometry analysis showed that 5d induced the loss of mitochondrial membrane potential
(MMP), and increased levels of intracellular free calcium and reactive oxygen species. 5d
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been through the copyediting, typesetting, pagination and proofreading process, which may
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doi: 10.1111/cbdd.13016
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also induced cell cycle arrest in G2/M phase in HepG2 cells. According to the results of
Western blotting and caspase-3 kit 5d could significantly increased the content of p53, bax,
Apaf-1,caspase-3 and decrease the protein level of bcl-2, pro-caspase-9, 3 in HepG2 cells.
These findings indicated that 5d activates the mitochondria-mediated apoptotic pathway in
HepG2 cells and may merit further investigation as a potential therapeutic agent for
hepatocellular carcinoma.
Keywords: Artemisinin derivatives, Synthesis, Cytotoxic activity, Apoptosis, Cell cycle
Artemisinin (compound 1 Figure 1), an unusual sesquiterpene bearing lactone and
endoperoxide linkage, was an important lead compound for drug design. Derivatives of
artemisinin have been used as antimalarial agents in clinical treatment for many years with
great therapeutic efficacy1, toleration and low toxicity2-5. Dihydroartemisinin (compound 2
Figure 1) exhibited neurotoxicity both in vivo and in vitro6. Artemisinin-like compounds
have been known to be effective against a range of diseases with antitumor activity,
antimalarial activity, antiviral activity and antiangiogenic activity.7, 8
Introduction of additional heteroatoms to the skeleton of artemisinin will afford derivatives
with novel biological properties, and studies on the synthesis method of amino derivatives are
the topic and focus of scholars. Ziffer9,10synthesized 11-azaartemisinin and its alkyl
derivatives with enhanced antimalarial activity. Haynes11 introduced piperazino to C10 of the
artemisinin nucleus to obtain 10-alkylamino derivatives which enhanced antimalarial activity
significantly through suppression of the likely target PfATP6. Sungsik Chohad reported8 that
introducing sulfur atoms or nitrogen atoms with functional groups to an artemisinin moiety
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afforded some new derivatives which inhibit the proliferation of various cancer cell lines in
response to various growth factors. Soomro found12 C10 derivatives were more cytotoxic
towards cancer cells than C9, C11 derivatives.
Artemisinin and its bioactive derivatives are potent anticancer drugs13 that pose minimal
risks to the patient.14 As artemisinin derivatives play very important role in antitumor drug
development. In present work, new artemisinin derivatives were synthesized from artemisinin
(Figure 2) in order to find novel anticancer leading compounds via introducing piperazine
group into a specific position of artemisinin to obtain new 10-substituted
piperazino-artemisinin derivatives. Consequently, the antiproliferative mechanism of the
most active artemisinin derivative on hepatocellular carcinoma cells (HepG-2, PLC-PRF-5)
was explored. All these findings provide new insights which would be value for developing
novel artemisinin derivatives as potential anti-tumor agent.
< insert Figure 1 >
H
H
O
O
O
O
O
O
H
H
H
H
O
O
OH
O
compound 2
compound 1
Figure 1. The structure of artemisinin and dihydroartemisinin
Experimental Section
Chemistry
Melting points were determined in open capillary tubes and were uncorrected. The
products were purified by column chromatography by using silica gel (200-300 mesh). NMR
spectra were run on a Varian-400 at room temperature with TMS as an internal standard and
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CDCl3 as solvents. Mass spectra were recorded with a JEOL MS-D 300 mass spectrometer.
All raw materials were purchased from commercial sources. Reagents were all analytically or
chemically pure. All the solvents and liquid reagents were dried by standard methods in
advance or distilled before use. The synthetic routes were outlined in Figure 2.
< insert Figure 2 >
Figure 2. General synthetic procedures of the artemisinin derivatives
Preparation of dihydroartemisinin 215, 16: Artemisinin (0.4mmol, 112mg) was dissolved in
anhydrous methanol (15ml) and cooled in an ice bath to 0–5℃. To the solution was added
sodium borohydride (4.8equ, 60.53mg) in portion within 30 minutes. The mixture was kept
stirring for approximately 30 minutes at 0–5℃, neutralized to PH 7.0 using acetic acid,
diluted by adding ice water (15ml), the solid was precipitated and was collected by filtration
to yield part of product. The filtrate was concentrated under reduced pressure, solid was
precipitated again by adding ice water, then filtered to yield another part of product. Combing
two parts of precipitate to obtain product 1 as white solid, 87mg, yield: 89%, m.p (146~150℃,
1
reported 153℃ ), HR-MS(ESI) Calcd. for (C15H24O5) (M-OH), 267.1597；Found, 267.1568
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Preparation of compound 311: To a cold stirred solution of dihydroartemisinin 2 (0.35mmol,
0.1g) and triethylamine (1.4mmol, 150mg) in anhydrous dichloromethane (5ml), a cold
solution of trimethylchlorosilane (0.7mmol, 75mg) in anhydrous dichloromethane (2ml) was
added dropwise at 0~5℃. After 5 minutes, the solution was evaporated to dryness under
reduced pressure. The white residue was diluted by petroleum ether (30ml), then the organic
phase was washed with H2O (4×30ml), dried over anhydrous magnesium sulphate and
concentrated to yield compound 3as slight brown slurry in 94% (1.17g), HR-MS (ESI) Calcd.
for (C18H32O5Si) (M+Na), 379.1917；Found, 379.1905
Preparation of compound 411: A cold stirred solution of compound 3 (0.14mmol, 50mg) in
dichloromethane (1ml) was treated drop wise with a cold solution of bromotrimethylsilane
(1.02N) in dichloromethane (4ml) at 0℃. After completion (confirmed by TLC), the reaction
solution was used in the next step without further purification.
Preparation of compound 5a~5d: The above solution （containing 4) was transferred to a
stirred solution of amines (0.98equ) in dichloromethane (2ml) at 0~5℃. The reaction mixture
was stirred overnight at room temperature, then diluted by dichloromethane (20ml), washed
with saturated sodium bicarbonate solution (10ml) and water (3×20ml), dried over anhydrous
magnesium sulphate, concentrated under reduced pressure to yield the crude product, which
was recrystallized to yield the corresponding products 5a-5d.
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Compound 5a: 5a was obtained by recrystallization from dichloromethane/petroleum ether
yield 27%. 1HNMR (400M CDCl3) δ ppm: 0.817(d, J=7.2Hz, 3H, CH3-9), 0.946(d, J=6.4Hz,
3H, CH3-6), 0.962~1.032(m, 2H, 2H-5), 1.244(m, 2H, 2H-7), 1.380(s, 3H, CH3-3),
1.460~1.574(m, 2H, 2H-8), 1.701(m, 2H, 2H-4), 1.854(m, 1H, H-5a), 1.988(m, 1H, H-8a),
2.321(m, 1H, H-6), 2.573(m, 1H, H-9), 2.834(m, 1H, 6-piperazine), 3.173(m, 1H,
6-piperazine), 3.307(s, 1H, 2-piperazine), 3.404(s, 1H, 2-piperazine), 3.471(m, 1H,
5-piperazine), 3.615(m, 1H, 5-piperazine), 4.071(d, J=8Hz, 1H, 10-H), 5.300(s, 1H, 12-H);
13C-NMR (400M): 13.362(9-CH3), 20.224(5-C), 21.530(6-CH3), 24.746(6-CH3),
25.908(3-CH3), 28.543(8-C), 34.220(9-C), 36.249(6-C), 37.376(5a-C), 41.791(5-piperazine),
44.071(4-C), 45.709(8a-C), 51.308(2-piperazine), 51.643(6-piperazine), 80.157(12a-C),
89.563(10-C), 91.519(12-C), 104.004(3-C), 170.537(3-piperazine); HR-MS(ESI) Calcd. for
(C19H31N2O5) (M+H), 367.2233；Found, 367.2214.
Compound 5b17: 5b was obtained by recrystallization from dichloromethane: petroleum ether
yield 29%. HR-MS (ESI) Calcd. for (C20H35N2O4) (M+H), 367.2597；found, 367.2549.
Compound 5c: 5c was obtained by recrystallization from dichloromethane/petroleum ether
yield 30%. 1HNMR (400M CDCl3) δ ppm: 0.821(d, J=7.2Hz, 3H, CH3-9), 0.946(d, J=7.2Hz,
3H, CH3-6), 1.253~1.716(11H), 1.860(m, 1H, 5a-H), 2.016(m, 1H, 8a-H), 2.347(m, 1H, 6-H),
2.609(m, 1H, 9-H), 2.820(m, 2H, 2H-piperazine), 3.102~3.177(m, 6H, 6H-piperazine),
4.065(d, J=10Hz, 1H, 10-H), 5.337(s, 1H, 12a-H), 6.745(m, 1H, 6-Ph), 6.939(s, 1H, 2-Ph),
7.263(m, 1H, 5-Ph); 13C-NMR (400M):13.439(9-CH3), 20.272(5-C), 21.701(6-CH3),
24.828(3-CH3), 25.985(8-C), 28.576(9-C), 29.691(6-C), 34.368(7-C), 36.380(5a-C),
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37.462(4-C), 45.909(8a-C), 47.028(5-piperazine), 49.068(2-piperazine, 6-piperazine),
51.817(3-piperazine), 80.340(12a-C), 90.549(10-C), 9.660(12-C), 103.937(3-C),
115.271(6-Ph), 117.089(2-Ph), 121.696(4-Ph), 130.319(5-Ph), 132.703(3-Ph), 151.038(1-Ph);
HR-MS(ESI) Calcd. for (C25H34Cl2N2O4) (M+H), 497.1974；Found, 497.1918.
Compound 5d: 5d was obtained by recrystallization from dichloromethane/petroleum ether
yield 20%. 1HNMR (400M CDCl3) δ ppm: 0.825(d, J=7.2Hz, 3H, CH3-9), 0.956(d, J=6.4Hz,
3H, CH3-6), 1.269(t, J=7.2Hz, 3H, CH3CH2), 1.336(m, 2H, 2H-5), 1.386(s, 3H, CH3-3),
1.426(s, 4H, 2H-7, 2H-8), 1.525(m, 2H, 2H-4), 1.705(m, 4H, 2-piperazine, 6-piperazine),
1.862(m, 1H, H-5a), 2.017(td, J=12.8Hz, 1H, H-8a), 2.372(dt, J=4, 10.4Hz, 1H, H-6),
2.575(m, 1H, H-9), 2.612(m, 2H, 3-piperazine), 2.951(m, 2H, 5-piperazine), 4.041(d,
J=10.4Hz, 1H, 10-H), 4.071(q, J=7.2Hz, 2H, CH2-O), 5.270(s, 1H, H-12a); 13C-NMR (400M):
13.424(9-CH3), 14.645(5-C), 20.311(CH3CH2), 21.624(6-CH3), 24.742(3-CH3), 25.949(8-C),
26.899(9-C), 28.436(6-C), 34.278(7-C), 36.299(5a-C), 37.383(4-C), 41.791(8a-C),
44.034(2C-piperazine),
45.813(6C-piperazine),
47.217(3C-piperazine),
51.710(5C-piperazine), 61.156(CH2-O), 80.267(12a-C), 90.783(10-C), 91.609(12-C),
103.910(3-C), 155.498(C=O); HR-MS(ESI) Calcd. for (C22H36N2O6) (M+H), 425.2652；
Found, 425.2597.
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Bioactivity assays
Cell culture
All the cells were purchased from the Shanghai Institute for Biological Sciences (SIBS),
Chinese Academy of Sciences (China). HepG2, MCF-7, ZR-75-30 cell lines were maintained
in DMEM supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin. The
L-02 PLC-PRF-5, HCC1428, MDA-MB-231, HUVEC, A549, HCT-15 cells were maintained
in RPMI1640 supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin.
The cells were grown at 37 °C in an environment of 5% CO2.
MTT assays
Cytotoxicity of the artemisinin derivatives 5a-5d were evaluated by MTT assays. Cells
(4×103per well) were plated onto 96-well plates in 100 µL of medium. After incubating for 24
h, the medium was removed and replaced by fresh medium containing derivatives at various
concentrations(5-40 µM) for 24-72 h at 37 °C. Next, the cells were washed with PBS and
then incubated with serum-free medium containing 0.5 mg/ml MTT for 4 h. And then, the
MTT solution was removed and 100 µL/well of DMSO was added to dissolve the formazan.
Optical density of each well was measured by a microplate reader (Bio-Rad 680) at 570 nm.
Cell viability ratio (%) = (A570 sample−A570 blank)/ (A570 control−A570 blank) × 100%.
IC50 values for each cell line were evaluated at a dose of drug causing 50% absorbance
reduction in comparison with DMSO-treated control cells. Each test was conducted in
triplicate.
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Flow Cytometry Analysis of Apoptosis
We used Annexin V-FITC Apoptosis Detection Kit to evaluate 5d-induced apoptosis.
Cells were seeded at a density of 5×104/mL onto 6-well plates. After 24 h incubation, cells
were treated with different concentrations of 5d. Then, cells were washed twice with cold
phosphate buffered saline (PBS) and transferred to individual tubes and centrifuged at 2000
rpm/min for 5 min. The supernatant was removed, and the cells were suspended in 400 µL of
Annexin V-FITC binding buffer and incubated with 5 µL Annexin V-FITC and 5 µL
propidium iodide (PI) for 20 min at room temperature in dark. The apoptotic ratio was
analyzed by flow cytometry (Becton Dickinson， U.S.A.) and Win MDI 2.9 analysis
software.
Hoechst33342 Staining
To certificate apoptosis phenomenon, the Hoechst33342 staining assay was carried out,
2×104 PLC-PRF-5 cells per well were cultured overnight in 24-well plates and treated with
5d at various concentrations 0- 40 μM for 24 h. At the 24-h point, cell morphology was
assessed by Hoechst33342 staining using an Apoptosis and Necrosis Assay Kit (Beyotime
Institute of Biotechnology). Briefly, cells in 24-well plates were trypsinized, washed twice
with PBS (phosphate-buffered saline, pH 7.4), and 100 μl of the cell suspension was
transferred to black 96-well plates. The cells were then stained with Hoechst 33342 (10 ng/ml)
for 20 min at 4℃ in the dark. The condensed or fragmented nuclei of apoptotic cells were
observed using fluorescence microscopy [excitation (Ex), 365 nm and emission (Em), 480
nm].
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Measurement of Mitochondrial Membrane Potential
We evaluated the changes in mitochondrial membrane potential (ΔΨm) in
hepatocarcinoma cells using JC-1 staining. Cells were grown into 6-well plates and then
treated with 5–40 µM 5d for 24 h. Collecting cells and washing two times, suspending cells
with 200 µL PBS. Then, adding JC-1 into cells (10 µg/mL) and incubating 20 min at 37°C,
staining cells were washed with PBS at 2000 rpm/min for 5 min. JC-1 dye enters selectively
into mitochondria and reversibly changes color from green to orange as membrane potential
increase. In a flow cytometric analysis, the green emission was analyzed in fluorescence
channel 1 (FL1) and the orange-red emission in channel 2 (FL2). ΔΨm was expressed by the
ratio of FL2/FL1.
Measurement of ROS Generation
2′,7′-Dichlorofluorescein diacetate (DCFH-DA) was used to quantify the change of
intracellular reactive oxygen species (ROS) levels. DCFH-DA could change into fluorescent
dichlorofluorescein (DCF) when intracellular DCFH reacts with ROS. In order to detect
5d-induced intracellular ROS accumulation, HepG-2 cells were grown in 6-well plates and
then treated with 2.5–10 µM 5d at 37°C for 24 h. After incubation, the culture medium was
removed and the cells were washed three times with PBS. The cells were incubated with
DCFH-DA (3 µM) at 37°C for 20 min. Cellular fluorescence was measured by flow
cytometry with a FACS-SCAN apparatus. Increased values compared to control were
considered to represent the increase of intracellular ROS levels.
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Measurement of Intracellular Free Calcium Levels
The intracellular free calcium levels were quantified by Ca2+indicator dye Fura-3 AM.
Cells pretreated5d (2.5-40 µM)for 24 h were collected and washed by PBS. Then, cells were
cultured with Fura-3 AM (2 µM) for 45 min. Analysis was performed by flow cytometry.
Flow cytometric analysis for measurement of sub-G1 phase
After treatment with various concentrations of 5d, cells were collected and washed with
PBS followed by fixation with 70% ethanol and were incubated at −20ꢀ°C overnight. Cells
were then collected by centrifugation and washed. The pellet was resuspended in PBS and
incubated with RNase A (50ꢀµg/mL) for 30ꢀmin at room temperature, then stained with PI for
10ꢀmin. Then, the samples were analyzed using a FACS Calibur flow cytometry (Becton
Dickinson，U.S.A.).
Caspase-3 activity assay
The activity of caspase-3 was determined using the Caspase-3 activity kit (Beyotime
Institute of Biotechnology, Haimen, China). To evaluate the activity of caspase-3, cell lysates
were prepared after their respective treatment with various designated treatments. Assays
were performed on 96-well microtitre plates by incubating 10 µL protein of cell lysate per
sample in 80 µL reaction buffer (1% NP-40, 20 mM Tris–HCl (pH 7.5), 137 mM Nad and 10%
glycerol) containing 10 µLcaspase-3 substrate (Ac-DEVD-pNA) (2 mM). Lysates were
incubated at 37 °C for 4 h. Samples were measured with an ELISA reader at an absorbance of
405 nm. The detail analysis procedure was described in the manufacturer’s protocol.
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Western blotting
Cells were grown at 5×104 cells/well in 6-well microplates and incubated with NAC,
selenium and cadmium for indicated time. Following treatment, cells were washed with PBS,
and total cells were prepared by scraping in 200 µL of lysis buffer [20 mM Tris–HCl (pH 8.0),
1 mM sodium orthovanadate, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 2 mM
ethylenediaminetetraacetate (EDTA), 1%Triton X-100, 50 mM β-glycerolphosphate, and 10
mg/ml each of aprotinin, leupeptin, and pepstatin]. Fifty micrograms of proteins determined
by Bradford assay were electrophoretically separated using
a
12% sodium
dodecylsulfate-polyacrylamide gel electrophoresis (SDS–PAGE) gel and transferred to
polyvinylidence fluoride (PVDF) membrane and then immunoblotted with the corresponding
antibodies. Immunodetection was performed with enhanced chemiluminescence (ECL)
detection kit (Cell Signaling Technology, Beverly, MA). The protein bands were followed by
densitometry scanning using a ChemilmagerTM5500 Fluorescence system equipped with the
analysis software Alpha-Ease FCTM(Alpha Innotech Corporation, San Leandro, CA 94577,
USA).
Statistical analysis
Results are expressed as means±SD. Data were evaluated with one-way analysis of
variance (ANOVA) procedures among treatment groups. Statistically significant differences
were reported as *p<0.05 or **p<0.01. Data with values of p< 0.05 were generally accepted
as statistically significant.
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Results and discussion
For many years, artemisinin derivatives have been used as antimalarial agents in clinical
treatment with great therapeutic efficacy. And lots of researchers found that artemisinin-like
compounds are effective against a range of diseases with antitumor activity8-10. Thus
artemisinin was an important lead compound for drug design. It has been reported12 that
introduction of additional heteroatom's to C10 position of the artemisinin nucleus can impact
the activity of the derivatives, and this effect is more effective than changing on other
position (such as C9, C11). Piperazine derivatives displayed a broad spectrum of biological
activities, for example, Rengul Cetin Atalay group18found that introduction of 4-substituted
piperazine to the purine ribonucleoside can increase anticancer effects. Richard Rips group
reported that phenols addition with piperazine displayed antihelmintic properties19. Thomas
R. Hemin also found that piperazine derivative (5-phenyl-4-oxo-2-oxazolin-2-yl) piperazineis
a sensitive antimalarial pharmacophoricgroup20. In addition, some research found that
piperazine showed antidepressant activity21, and could act as MC4R antagonists22,
MMPinhibitors23.Many currently used drugs contain a piperazine ring as part of their
molecular structure and some C6-piperazine substituted purine derivatives show potent
anticancer activity18.Thus, we introduce piperazine group into this specific position C10 of
artemisinin to obtain new 10-substituted piperazino-artemisinin derivatives. Artemisinin 1
was reduced to dihydroartemisinin 2 with a yield of 89% using a modified method based on
literature procedure15, 16, which employed sodium borohydride as reductant reagent to
generate hydroxyl in C-10 position, followed by the purification processing of neutralization
with acetic acid and precipitation of pure product in ice water. To remove impurity during
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synthesizing compound 3, modifications based on the procedures reported11 was employed by
careful dropwise addition of trimethylchlorosilane in dichloromethane at 0~5to yield very
pure 3 with high yield (94%). Compound 3 was successfully brominated using
bromotrimethylsilane in anhydrous DCM at 0℃to afford crude 4, which was used in the next
step without further purification. The final amination at 10-C of compound 4 with different
amines in dichloromethane produced desired new artemisinin derivatives 5a, 5b, 5c and 5d.
The obtained target compounds were identified by 1H-NMR, 13C-NMR and HR-MS (see
Supplementary data).
We first performed MTT colorimetric assays to test the cytotoxicity of artemisinin and its
derivatives (5a~5d) on several cancer lines and normal hepatocellular cells. As shown in
Table 1, these four derivatives were generally more effective in inhibiting cancer cells growth
than artemisinin, but presented lower cytotoxicity than doxorubicin. Artemisinin and its
derivatives presented no cytotoxicity on human normal liver cells L-02 (IC50>40 μM). While
regarding to human hepatocellular carcinoma cell lines (HepG2 and PLC-PRF-5), compound
5d which bearing a N-ethoxycarbonyl piperazino group exhibited strongest cytotoxic
activities (the IC50 values were 4.1 μM and 5.7 μM, respectively). Compound 5c was the
second most efficacious compounds on account of its remarkable performance of inhibitory
activity on HepG2 and PLC-PRF-5 cells (IC50 values were 7.2 μM and 12.3 μM,
respectively). And compound 5b also exerted moderate cytotoxic action against HepG2 cells
(IC50=36.1 μM). while, compound 5a have no pronounced inhibition activity against these
two cell lines (IC50>40 μM). The high toxicity to HepG2 and PLC-PRF-5 cells and the low
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toxicity to normal liver cells (L-02) indicated that the three compounds 5d, 5c and 5b, were
worthy of further investigation as anti-hepatocellular carcinoma agents.
Among the human breast cancer cell lines (MCF-7, HCC1428, ZR-75-30, MDA-MB-231),
these four compounds have different inhibition activity. Regarding to MCF-7 cells,
compound 5b and 5d exerted approximate cytotoxic actions with IC50 values in the range of
14.8 and 15.2 µM. Regarding to MDA-MB-231 cells, compound 5d preserved higher
cytotoxicity (IC50=19.2 μM) than 5a (IC50=23.9 μM), and this cell line presented a weak
sensitivity to the other two compounds 5b and 5c (IC50>40). The IC50 values (>40 µM) of
derivatives 5a-5d revealed that they were all tolerated by HCC1428 and ZR-75-30 cells.
Compound 5a, 5b and 5d also have considerably potent antitproliferation activity against
HUVEC cells with IC50 = 27.7, 19.6 and 21.7 µM respectively. Regarding to HCT-15 cells,
compound 5a showed much better activity (IC50 = 8.7 µM) than 5b (IC50 = 24.7 µM). With
regard to A549 cell line, compound 5a and 5d showed analogical activity (IC50=25.9 and
19.9 μM, respectively).
The selectivity index (SI) was calculated by dividing the IC50 value for the normal cell line
(L-02) by the value of the IC50 for cancer cell lines(HepG-2, PLC-PRF-5)24. This value
indicated the degree of selectivity of derivatives to cancer cells. A value of two or more
indicated high specificity. According to the IC50 value of these four compounds above, the
selectivity of 5d against two hepatocellular carcinoma cells lines is significant (SI>7).,And
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according to the different toxicity between artemisinin derivatives and artemisinin, we found
that introducing piperazine group into C10 position of artemisinin could enhance the
anti-proliferative on certain cancer cells in some degree. The structure of compounds 5d,
which showed highest activity against hepatocellular carcinoma cells, inspired us to improve
the oil solubility of piperazine group into C10 position so that we could obtain more active
compounds in future.
Given that 5d exhibited excellent anti-proliferation activity on hepatocellular carcinoma
cells (HepG-2, PLC-PRF-5), we used HepG-2, PLC-PRF-5 cell lines to further investigate
the function of 5d and the mechanisms that underlie such function. Figure 3 shows that 5d
treatment resulted in significant inhibitory effects on the cells growth of the tested
hepatocellular carcinoma cell lines. These effects occurred in a concentration- and
time-dependent manner. The results also indicated that about 50% of the hepatocarcinoma
cells died during treatment with 10 µM 5d for 48 h. However, little cytotoxicity for normal
cells L-02 was observed.
Table 1. cytotoxic activity of artemisinin derivatives and control drugs against ten cell lines
μM
（
）
IC50 Values
Cell line
artemisinin doxorubicin
5a
5b
5c
5d
L-02
HepG2
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
＞
＞
＞
＞
＞
＞
＞
＞
＞
＞
＞
＞
＞
＞
2.4±1.1
0.8±0.3
1.4±0.5
2.0±0.3
2.3±0.7
1.5±0.3
1.8±0.3
2.0±0.2
2.3±0.9
1.5±0.2
＞
＞
＞
＞
＞
36.1±0.7
40
7.2±0.6
4.1±0.3
5.7±0.6
PLC/PRF/5
MCF-7
＞
12.3±1.0
14.8±0.3
＞
＞
＞
＞
＞
＞
＞
15.2±1.6
40
40
40
40
40
40
40
HCC1428
ZR-75-30
MDA-MB-231
HUVEC
HCT-15
40
40
40
40
40
＞
＞
＞
＞
＞
23.9±1.9
27.7±1.2
8.7±0.5
19.2±0.7
21.7±1.1
19.6±1.3
24.7±1.7
40
＞
A549
40
25.9±2.1
＞
19.9±1.3
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The IC50 (concentration of the compound which caused a 50% reduction of survival) values were
calculated from concentration–response curves done in triplicate for each compound. Doxorubicin
was used as positive control. P < 0.05 versus control group (0 µM).
< insert Figure 3 >
Figure 3. Quantification of inhibitory effects compound 5d
P < 0.05 versus control group (0 µM).
5d was further tested to evaluate whether the cytotoxic effect is related to apoptosis. The
quantitative analysis of hepatocarcinoma cell apoptosis was monitored by using FITC
Annexin V and propidium iodide (PI) double staining through flow cytometry (Figure 4A,B).
The representative patterns of cell apoptosis from flow cytometric analysis（Figure 4A）
revealed that the proportion of cells stained with Annexin V increased in a
concentration-dependent manner. The percentages of Annexin V-positive cells were 11.7%,
38%, 45 %, 63.2%, 69.2% (HepG-2), 9.8%, 16%, 39.9%, and 64.9%, 70.7% (PLC-PRF-5)
for cells treated with 0 to 40 µM 5d for 48 h (Figure 4B).The morphological changes were
observed by Hoechst33342 staining in HepG-2 and PLC-PRF-5 cells. 5d treatment induced
marked apoptotic morphological alterations, including cell shrinkage and granular apoptotic
body formation (Figure 4C)
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Figure 4. 5d-induced apoptosis in HepG-2 and PLC-PRF-5 cells.(A) Representative
apoptotic profile of hepatocellular carcinoma cells treated with or without 5d by flow
cytometric analysis. (B) Flow cytometric histograms of 5d induced apoptosis assays. (C)
Hoechst33342 staining of hepatoma carcinoma cells treated or untreated with 5d. P < 0.05
versus control group (0 µM).
The mitochondrial pathway is commonly involved in the cell death stimuli. And the
mitochondrial function disturbance is an important event in apoptosis via the
mitochondria-mediated pathway, and its involves membrane potential decrease, Ca2+
overload and ROS generation24. We examined the effects of 5d on mitochondrial membrane
potential (ΔΨm) in HepG2 cells. At the same time, we measured the DCF-derived
fluorescence, which is an index of ROS accumulation with or without 5d-treated cells by
flow cytometric assay. Results showed that a greater accumulation of ROS was observed in
5d-treated HepG-2 cells than in the control (0 µM) cells. We also used HepG-2 cells to detect
intracellular free Ca2+ levels. 5d induced a gradual increase in intracellular free Ca2+levels in
a concentration-dependent manner (Figure 5C). After a 24 h incubation with 5d (40 µM),
HepG-2 cells exhibited a maximum two-fold increase in Ca2+ levels compared with the
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control group. These results indicate that 5d activates the mitochondria-mediated apoptotic
pathway in hepatocellular carcinoma cells.
< insert Figure 5 >
A
B
C
Figure 5. 5d-induced HepG-2 cells apoptosis correlates with mitochondria-mediated pathway.
(A)The Effects of 5d on mitochondrial membrane potential. (B) The DCF-derived
fluorescence. (C) The intracellular free Ca2+ Levels. *P < 0.05, **P < 0.01 versus control
group (0 µM).
Based on cell cycle analysis (Figure 6), we found that 5d (4 µM) treatment induced
significant G2/M phase arrest. 5d The examination was conducted by JC-1 staining
performed with a flow cytometric procedure. From Figure 5A we can see that 5d caused a
loss of ΔΨm in a concentration-dependent manner on HepG-2 increased the ratio of G2/M
phase approximately 30% in HepG2 cells, and when the 5dconcentration increased to 6ꢀµM,
the sub-G1 phase ratio was increased, and G2/M phase arrest was attenuated. Thus, these
results suggest that 5d promoted cell cycle progression inhibition, and then induced
apoptosis.
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Figure 6. Cell cycle analysis of 5d on HepG2 Cells
To determine whether 5d-induced apoptosis of HepG2 cells involves the activation of a
caspase cascade, HepG2 cells were treated with compound 5d at 0, 2, 4, 6 and 8 μM for 24 h
and total proteins were extracted, and activation of caspases was measured bycaspase-3
activity kit. Table 2 and Figure7 showed that 5d treatment resulted in obvious increasing of
caspase-3 activity, and the up-regulation exhibited a 5dconcentration-dependent pattern.
When the concentration of 5d increased to 8 μM, the content of the caspase-3 in HepG2 cells
was over 5-fold higher than control that in control group.
Table 2. Effect of 5d on the content of the caspase-3 in HepG2
Concentration (μM)
caspase-3
0
2
4
6
8
0.0208±0.0016
0.0243±0.0015
0.0517±0.0022
0.0767±0.0067
0.0969±0.0052
P < 0.05 versus control group (0 µM)
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Figure 7. Effect of 5d on the content of the caspase-3 in HepG2 cells
**P < 0.05 versus control group (0 µM)
As p53 has been described to transactivate the proapoptotic Bcl-2 family member Bax in
25
some cell types , which was shown to induce cytochrome c release when added to isolated
mitochondria26, 27, western blot analysis was used to further detect protein expressions of p53,
bcl-2, bax in HepG2 cells 24h after 5d(0μM, 2μM, 4μM, 6μM, 8 μM) treatment. GAPDH
was used as an internal loading control. As shown in Figure 8, 5d treatment increased p53,
bax protein expression while it decreased bcl-2 protein expression in treated HepG2 cells.
The increased bax:bcl-2 ratio suggests an increased disruption of the nucleus. The
quantitative result for the bax: bcl-2 ratio showed a concentration-dependent effect of 5d.
During apoptosis, cytochrome c is released from the mitochondria, and then acts on Apaf-1
by increasing the binding of Apaf-1 to ATP/dATP in cytoplasm. The activated Apaf-1 forms
an oligomeric apoptosome and cleaves caspase-9 preprotein then stimulates the subsequent
caspase cascade that commits the cell to apoptosis. To confirm whether a mitochondrial
pathway is involved in 5d-induced apoptosis of HepG-2 cells, the activity of apoptosis related
proteins , such as Apaf-1, caspase-9, pro-caspase-9, pro-caspase-3 were chose to test. As
shown in Figure 8, 5d significantly increased the expression of Apaf-1, capase-9 in HepG-2
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cells in a concentration-dependent manner. Meanwhile, the contents of pro-caspase-9 and -3
proteins were decreased respectively. These results indicated that compound 5d can induce
the p53 activity in HepG-2 cells, and then p53 induces mitochondrial cytochrome c release
which can up-regulate Apaf-1 in HepG-2 cells. And these alterations are concurrent with
changes in apoptosis-related genes including caspase-9, -3, Bax, Bcl-2, and pro-caspase-9, 3
suggesting the involvement of an intrinsic mitochondrial pathway in 5d-induced apoptosis in
HepG-2 cells.
Figure 8. Effect of 5d on the content of Apaf-1, pro-caspase-9, pro-caspase-3, p53, bax, bcl-2
in HepG2 cells
*P < 0.05, **P < 0.01 versus control group (0 µM)
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Conclusion
In conclusion, we have developed an efficient synthetic route for the preparation of
10-piperazine artemisinin under mild reaction condition. The four artemisinin derivatives
presented in this paper are generally more toxic against selected cancer cells than artemisinin.
Because hepatocellular carcinoma (HCC) is one of the most frequent primary tumors in
adults with a high mortality rate worldwide.28, 29Compound 5d had good activity against
human hepatocellular carcinoma cell lines (HepG2 and PLC-PRF-5), and presented no
cytotoxicity on human normal liver cells (L-02), 5d might be a valuable potential antitumor
drug against HCC. Flow cytometry analysis showed that 5d induced the loss of mitochondrial
membrane potential (MMP), as well as increased the levels of intracellular free calcium and
reactive oxygen species (ROS). Additionally, 5d also induced cell cycle arrest in G2/M phase
in HepG2 cells. The caspase-3 kit was used to detected the Actived-caspase-3 level in HepG2
cell line., the result demonstrated that 5d could also increase the caspase-3 level in HepG2
cells. Alteration of expression levels in proteins important in the apoptotic cascade was
analyzed by western blotting, and the results showed that compound 5d significantly
increased the content of p53, bax, Apaf-1 and decrease the protein level of bcl-2,
pro-caspase-9, pro-caspase-3 in HepG2 cells. These results indicate that 5dcan induce the p53
activity in HepG-2 cells, and then p53 induces mitochondrial cytochrome c release which can
up-regulate Apaf-1 in HepG-2 cells. And these alterations are concurrent with changes in
apoptosis-related genes including caspase-9, -3, Bax, Bcl-2, and pro-caspase-9, 3 suggesting
the involvement of an intrinsic mitochondrial pathway in 5d-induced apoptosis in HepG-2
cells. These findings indicate that 5d activates the mitochondria-mediated apoptotic pathway
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in hepatocellular carcinoma cells and may merit further investigation as a potential
therapeutic agent for the treatment of hepatocellular carcinoma.
Acknowledgments
This work was funded by the National High-Tech Program of China (863
Program)(2012AA020306).
Conflict of Interest
The authors declare no conflict of interest.
Figure legends
Figure 1. The structure of artemisinin and dihydroartemisinin
Figure 2. General synthetic procedures of the artemisinin derivatives
Figure 3. Quantification of inhibitory effects compound 5d.
P < 0.05 versus control group (0 µM).
Figure 4. 5d-induced apoptosis in HepG-2 and PLC-PRF-5 cells.(A) Representative
apoptotic profile of hepatocellular carcinoma cells treated with or without 5d by flow
cytometric analysis. (B) Flow cytometric histograms of 5d induced apoptosis assays. (C)
Hoechst33342 staining of hepatoma carcinoma cells treated or untreated with 5d. P < 0.05
versus control group (0 µM).
Figure 5. 5d-induced HepG-2 cells apoptosis correlates with mitochondria-mediated pathway.
(A)The Effects of 5d on mitochondrial membrane potential. (B) The DCF-derived
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fluorescence. (C) The intracellular free Ca2+ Levels. *P < 0.05, **P < 0.01 versus control
group (0 µM).
Figure 6. Cell cycle analysis of 5d on HepG2 Cells.
Figure 7. Effect of 5d on the content of the caspase-3 in HepG2 cells
**P < 0.05 versus control group (0 µM)
Figure 8. Effect of 5d on the content of Apaf-1, pro-caspase-9, pro-caspase-3, p53, bax, bcl-2
in HepG2 cells.
*P < 0.05, **P < 0.01 versus control group (0 µM)
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