Synthesis and in vitro antitumor activity of novel acylspermidine derivative N-(4-aminobutyl)-N-(3-aminopropyl)-8-hydroxy-dodecanamide (AAHD)against HepG2 cells

Article abstract of DOI:10.1016/j.bioorg.2019.102937

Naturally occurring polyamines like Putrescine, Spermidine, and Spermine are polycations which bind to the DNA, hence stabilizing it and promoting the essential cellular processes. Many synthetic polyamine analogues have been synthesized in the past few years, which have shown cytotoxic effects on different tumours. In the present study, we evaluated the antiproliferative effect of a novel, acylspermidine derivative, (N-(4-aminobutyl)-N-(3-aminopropyl)-8-hydroxy-dodecanamide)(AAHD)on HepG2 cells. Fluorescence staining was performed with nuclear stain (Hoechst 33342)and acridine orange/ethidium bromide double staining. Dose and the time-dependent antiproliferative effect were observed by WST-1 assays, and radical scavenging activity was measured by ROS. Morphological changes such as cell shrinkage & blebbing were analyzed by fluorescent microscopy. It was found that AAHD markedly suppressed the growth of HepG2 cells in a dose- and time-dependent manner. It was also noted that the modulation of ROS levels confirmed the radical scavenging activity. In the near future, AAHD can be a promising drug candidate in chalking out a neoplastic strategy to control the proliferation of tumour cells. This study indicated that AAHD induced anti-proliferative and pro-apoptotic activities on HCC. Since AAHD was active at micromolar concentrations without any adverse effects on the healthy cells (Fibroblasts), it is worthy of further clinical investigations.

Full text of DOI:10.1016/j.bioorg.2019.102937

Accepted Manuscript
Synthesis and in vitro antitumor activity of novel acylspermidine derivative N-
(
4-aminobutyl)-N-(3-aminopropyl)-8-hydroxy-dodecanamide (AAHD) against
HepG2 cells
Abdulrahman L. Al-Malki, Syed S. Razvi, Furkhan Ahmed Mohammed, Mazin
A. Zamzami, Hani Choudhry, Taha A. Kumosani, Khadijah S. Balamash,
Fawzia A. AlshibiLi, Khalid O. Abualnaja, Wesam H. Abduaal, Mustafa A.
Zeyadi, Maryam A. AlZahrani, Mahmoud Alhosin, Tadao Asami, Said S.
Moselhy
PII:
S0045-2068(19)30001-X
https://doi.org/10.1016/j.bioorg.2019.102937
102937
DOI:
Article Number:
Reference:
YBIOO 102937
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
1 January 2019
13 March 2019
15 April 2019
Please cite this article as: A.L. Al-Malki, S.S. Razvi, F. Ahmed Mohammed, M.A. Zamzami, H. Choudhry, T.A.
Kumosani, K.S. Balamash, F.A. AlshibiLi, K.O. Abualnaja, W.H. Abduaal, M.A. Zeyadi, M.A. AlZahrani, M.
Alhosin, T. Asami, S.S. Moselhy, Synthesis and in vitro antitumor activity of novel acylspermidine derivative N-
(
4-aminobutyl)-N-(3-aminopropyl)-8-hydroxy-dodecanamide (AAHD) against HepG2 cells, Bioorganic
Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.102937
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis and in vitro antitumor activity of novel acylspermidine derivative N-(4-
aminobutyl)-N-(3-aminopropyl)-8-hydroxy-dodecanamide (AAHD) against HepG2 cells.
1
,2,3
1
4
Abdulrahman L. Al-Malki
, Syed S. Razvi , Furkhan Ahmed Mohammed , Mazin A.
1
,5,6
1,2,5,6
1,2,7
1
Zamzami , Hani Choudhry
, Taha A. Kumosani
,Khadijah S.Balamash , Fawzia A.
1
1,2,3
1
1
AlshibiLi , Khalid O.Abualnaja
, Wesam H. Abduaal , Mustafa A. Zeyadi , Maryam
1
1,5,6
1,8*
1,2,3,9*
A.AlZahrani , Mahmoud Alhosin
, Tadao Asami
and Said S. Moselhy
1
Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah, Saudi
Arabia.
2
Experimental Biochemistry Unit, King Fahd Medical Research Center, King Abdulaziz
University, Jeddah, Saudi Arabia.
3
Bioactive Natural Products Research Group, King Abdulaziz University, Jeddah, Saudi Arabia.
Department of Biology, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia.
Cancer Metabolism and Epigenetic Unit, Faculty of Science, King Abdulaziz University,
4
5
Jeddah, Saudi Arabia.
6
Cancer and Mutagenesis Unit, King Fahd Medical Research Center, King Abdulaziz
University, Jeddah, Saudi Arabia.
7
Production of Bioproducts for Industrial Applications Research Group, King Abdulaziz
University, Jeddah, Saudi Arabia.
8
Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo, Tokyo 113–
8
657, Japan.
9
Biochemistry Department, Faculty of Science, Ain shams University, Cairo, Egypt.
*
Corresponding authors:
Prof. Tadao Asami
Graduate School of Agricultural and Life Sciences,
The University of Tokyo
Bunkyo, Tokyo 113–8657, Japan.
Email: asami@mail.ecc.u-tokyo.ac.jp
Prof. Said Salama Moselhy
Faculty of Sciences,
Ain Shams University
Email: moselhy6@hotmail.com.
1

Abstract:
Naturally occurring polyamines like Putrescine, Spermidine, and Spermine are polycations
which bind to the DNA, hence stabilizing it and promoting the essential cellular processes. Many
synthetic polyamine analogues have been synthesized in the past few years, which have shown
cytotoxic effects on different tumours. In the present study, we evaluated the antiproliferative
effect of a novel, acylspermidine derivative, (N-(4-aminobutyl)-N-(3-aminopropyl)-8-hydroxy-
dodecanamide) (AAHD) on HepG2 cells. Fluorescence staining was performed with nuclear
stain (Hoechst 33342) and acridine orange/ethidium bromide double staining. Dose and the time-
dependent antiproliferative effect were observed by WST-1 assays, and radical scavenging
activity was measured by ROS. Morphological changes such as cell shrinkage & blebbing were
analyzed by fluorescent microscopy. It was found that AAHD markedly suppressed the growth
of HepG2 cells in a dose- and time-dependent manner. It was also noted that the modulation of
ROS levels confirmed the radical scavenging activity. In the near future, AAHD can be a
promising drug candidate in chalking out a neoplastic strategy to control the proliferation of
tumour cells. This study indicated that AAHD induced anti-proliferative and pro-apoptotic
activities on HCC. Since AAHD was active at micromolar concentrations without any adverse
effects on the healthy cells (Fibroblasts), it is worthy of further clinical investigations.
Keywords: Polyamine analogues; ROS; Antiproliferative; Anticancer; Hepatocellular
carcinoma; HepG2
2

1
. Introduction
Hepatocellular carcinoma (HCC) is the sixth most common type of cancer worldwide with a
reputation of third deadliest cancers causing 600,000 deaths every year globally (1,2). In the
regions of East Asia and sub-Saharan Africa, the incidence rate is much higher with 20 per
1
00,000 persons (3). This could be primarily due to the Hepatitis B virus infections, which can
be timely controlled by efficient immunization drives. However, the incidence rate of HCC has
raised alarmingly in the USA in the recent few years with a 5-year survival rate of 15% (4). The
burgeoning obesity epidemic, chronic liver disease and cirrhosis are the contributing factors in
such high incidence of HCC in the USA (4). Despite the technological improvements in the
development of novel therapies, currently, there is just single FDA approved drug—Sorafenib to
treat HCC that acts as a pan-kinase inhibitor (5). Hence, it is highly desirable to develop effective
therapeutic approaches which can be employed to treat HCC.
Polyamine metabolism and the biosynthetic pathway have been studied extensively for the
last three decades. Naturally occurring polyamines like Spermine, Spermidine and Putrescine
play a crucial role in the proliferation and maintenance of healthy cells (6,7). However, the
demand of these biogenic polyamines rises manifold in the rapidly increasing HCC cells. In
order to maintain adequate supply, the import of polyamines from the external sources increases
with decreased specificity (8). Hence synthetic polyamine analogues can be supplied from the
external source that has a similar structure as biogenic polyamines but differs in the function
leading to cell death and apoptosis.
3

Many synthetic analogues have been developed and tested in a panel of diverse cancer cell
lines. Over the last few years, the development of polyamine analogues as chemotherapeutic
agents against different cancers has fascinated many researchers (7,9,10). Many polyamine
analogues have been extensively studied as LSD1 inhibitors leading to modulation of the
chromatin (11,12). Moreover, many previous studies have also evaluated polyaminobenzamides
(PABAs) and polyaminohydroxamic acid (PAHA) derivatives as HDAC inhibitors (13,14). In
our earlier study, we demonstrated the pro-apoptotic effect of the novel acylspermidine
analogues on Jurkat and MCF7 (15). But the off-target impact and the dose-limiting cytotoxicity
still remains a challenge in the way of designing new therapeutic strategies.
Building upon the data obtained on acylspermidine analogues from our earlier studies, we
evaluated the in vitro anticancer effect of a novel acyl spermidine derivative-N-(4-aminobutyl)-
N-(3-aminopropyl)-8-hydroxy-dodecanamide (AAHD) on HepG2 cells.
2
. Materials and Methods
2
2
.1.
Chemical Synthesis of the Acylspermidine derivative-AAHD
.1.1. N1,N8-bis-tert-butoxycarbonyl-N4-(8-hydroxylauroyl)spermidine
Under
the
nitrogen
atmosphere,
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (665 mg, 3.47 mmol) was added to a solution of N1,N8-bis-tert-
butoxycarbonylspermidine (1.00 g, 2.89 mmol), 8-hydroxylauric acid (626 mg, 2.89 mmol) and
dimethylaminopyridine (424 mg, 3.47 mmol) in dichloromethane (20mL), and stirred for 2 days
at room temperature. Then, a solution of 10% (w/w) citric acid (10 mL) was added to the
reaction mixture and stirred for 10 min.
The reaction mixture was extracted with dichloromethane (10 mL×3), dried with Na SO and
2
4
concentrated in vacuo. The residue was purified with silica gel column chromatography (n-
4

1
8
4
hexane:
ethyl
acetate
=
1:1)
to
give
N ,N -bis-tert-butoxycarbonyl-N -(3-
hydroxylauroyl)spermidine as a colorless oil (1.05 g, 1.94 mmol, 67.1%), which was sufficiently
pure for further use.
4
2
.1.2. N -(8-hydroxylauroyl) spermidine
1
8
4
Under nitrogen atmosphere, N ,N -bis-tert-butoxycarbonyl-N - (8-hydroxylauroyl) spermidine
1.05 g, 1.94 mmol) was dissolved into 50% (v/v) trifluoroacetic acid solution in
(
dichloromethane (10 mL) and stirred for 20 min. After the addition of methanol (30 mL), the
solvent was removed in vacuo. Then 1 M NaOH (10 mL) was added and the aqueous solution
was extracted with dichloromethane (10 mL×3). The combined organic layers were dried with
4
Na SO and concentrated in vacuo to give N -(3-hydroxylauroyl) spermidine as a colourless oil
2
4
(182 mg, 0.53 mmol, 18.3%). The general scheme of the synthesis has been described in Fig. 1.
1
2
.2.
H-NMR data of AAHD
1
H-NMR (500 MHz, CDCl ) δ 3.57 (m, 1H, CH(OH)), 3.43-3.24 (m, 4H, CH NCH ), 2.75-
3
2
2
2
.66 (m, 4H, 2×CH NH ), 2.40-2.20 (m, 2H, COCH2), 1.70-1.50 (m, 8H,
2 2
NCH CH CH CH NH , NCH CH CH NH ), 1.34 (m, 10H, (CH ) , (CH) CH ), 0.91 (t, J = 7.0
2
2
2
2
2
2
2
2
2
2 3
2
3
Hz, 3H, CH ). The data was generated by using the NMR Instrument JNM ECA500Ⅱ, Jeol Ltd,
3
Tokyo, Japan.
2
.3.
Cell Culture and Treatment
Human liver hepatocellular carcinoma cell line (HepG2) and baby hamster kidney cell line
(BHKSINLU2, BHK-21) were purchased from ATCC (CRL-11997 and CRL-12071
respectively). The cells were maintained in Dulbecco’s Eagle’s Modified Medium (DMEM)
TM
(
HyClone , catalog # SH30022.01) supplemented with 10% Fetal Bovine Serum (FBS),
5

TM
TM
(
HyClone , catalog # SH30910.03HI) and 1% Penicillin-streptomycin antibiotics (HyClone ,
o
catalog # SV30010) in a humidified incubator with 5% CO2 and 37 C.
2
.4. Hoechst 33258 and Acridine Orange/Ethidium Bromide staining
The fluorescent staining was performed as described earlier (16). Briefly, the HepG2 cells
4
were seeded in clear flat-bottom 24-well plates at a density of 2 x 10 cells/well. After treating
the cells with the desired concentrations for suitable time durations, the spent media was
removed and a fresh 200 µl solution of Hoechst 33258 Stain (Sigma-Aldrich) was added and
incubated for 30 minutes at room temperature. For Acridine orange (AO) and Ethidium Bromide
(EtBr) staining (Sigma-Aldrich), after the desired treatment conditions, 5µl of AO and 5µl of
EtBr were added to the media and incubated for 20min. After incubation, the cells were directly
observed under a fluorescence microscope at 400X magnification (Leica, Wetzlar, Germany).
2
.5.
Cell viability assay (WST-1)
The antiproliferation of HepG2 cells was done by using WST-1 (Sigma-Aldrich, USA) reagent
according to the manufacturer’s instructions. Briefly, the HepG2 cells were seeded on a
3
transparent, flat-bottom 96 well plate at a density of 6 x 10 cells/well and incubated for 24 h and
then treated with different concentrations (0.05mM, 0.1mM, 0.2mM, 0.3mM) for required time
periods. After incubation for the desired time durations, 10 µl of the WST-1 reagent was added
to the wells and incubated for additional 4 h at 37°C. Finally, the absorbance was recorded at 450
nm by microplate ELISA reader (ELx800TM Biotek, USA); Gen5 software (Biotek, USA. The
control/un-treated cells were considered as 100% viable, and the cell viability was calculated as
reported earlier (17).
2
.6. Assessment of mitochondrial potential
6

The mitochondrial potential (ΔΨm) was measured by using 5,5′,6,6′-tetrachloro-1,1′,3,3′-
tetraethyl-benzamidazolylcarbocyanine iodide (JC-1; Sigma Aldrich-molecular probe T3168)
dye using the manufacturer’s instructions (16,18). In brief, the HepG2 cells were seeded in 96-
3
well plate at a density of 10 cells/well and incubated for 24 h. Then the cells were treated with
desired concentrations for required time periods. The treated cells were trypsinized and
suspended in 1mL PBS and 10 μg/mL JC-1 dye was added and incubated for 15 min. Finally, the
red and green fluorescence was observed with a flow cytometer at an excitation wavelength of
4
88 nm. The red fluorescence was observed at 585 nm and the green at 530 nm.
.7. Wound healing assay
2
4
The HepG2 cells were seeded in a clear 6-well flat bottom plate at a density of 2×10
cells/well. The spent media was replaced with fresh DMEM media after an incubation of 24 h.
After incubation, the cells were treated with different concentrations (0.1, 0.2 and 0.3mM) of
AAHD for 24 h. Then, a scratch was made on the monolayered cells with a 10 µl pipette tip in
each well. Phase contrast microscope (Leica, Wetzlar, Germany) with 500X magnification was
used to record the images at 0 h and the images of the same scratch were taken after an
incubation of 24 h (19)(20).
2
.8. Statistical Analysis
The data is represented as the mean ± S.E.M of 3 independent experiments performed in
triplicate. The significance of the treated and the untreated samples was calculated by student’s
t’ test using GraphPad Prism 6 software (GraphPad, San Diego, USA). The statistical
significance is denoted by *, p < 0.05; **, p < 0.01 and ***, p < 0.001.
. Results
‘
3
7

The scheme of synthesis of the novel acylspermidine derivative-AAHD has been
1
summarized in Fig. 1 and the H-NMR data can be viewed under section 2.2. in the material and
method section.
3
.1.
AAHD altered the morphology of the nucleus as detected by fluorescent staining
To observe the morphological alterations of the nucleus and the cytoplasm, we treated
HepG2 cells with different concentrations of AAHD and subsequently stained them with
fluorescent stains like Hoechst 33342 and AO/EtBr dyes. When the cells were observed under
4
00x magnification, it revealed that the cells with compromised membranes showed deep blue
stained nuclei, while the intact cells were blue in colour on staining with Hoechst 33342 (Fig
A). Moreover, when the cells were stained with AO/EtBr stain, the untreated cells showed
2
green nuclei while the treated cells showed orange/red colour nuclei (Fig 2B) indicating the
breach of the cell membrane. There was a considerable shrinkage of the cell membrane and
blebbing which could be a possibility of an initiated cell death process.
3
.2.
AAHD reduced the cell viability of HepG2 in a dose-dependent manner
Thereafter, we performed an antiproliferative study on HepG2 cells to assess the cytotoxic
potential of the AAHD in a dose- and time-dependent manner through WST-1 assay.
Interestingly, as shown in the Fig 3A, there was a decrease in the cell viability starting from 0.05
mM (88.2%), which significantly decreased to 71.8%, 39.4% and 30.7% at a concentration of
0
.1mM (p < 0.01), 0.2mM (p < 0.001) and 0.3mM (p < 0.001) respectively. In contrast to the
tumor cells, the normal BHK cells showed slight inhibitory effect with the cell viability of
9
0
0.6%, 81.5%, 82.6% and 80.7% with the corresponding AAHD concentrations of 0.05mM,
.1mM, 0.2mM, 0.3mM respectively (Fig 3B). The IC value of AAHD on HepG2 cells was
5
0
8

found to be 0.176 mM and, while the IC value of AAHD on normal BHK cells was calculated
5
0
as 0.421 mM.
3
.3.
The antiproliferative effect of AAHD on HepG2 was time-dependent
We next investigated the time-dependent antiproliferative impact of AAHD on HepG2
cells through WST-1 assay. The cell viability decreased to 87.6% after a time duration of 3 h,
after a span of 6 h, it was 70.6% (p < 0.01). When we further increased the time of incubation for
9
4
h, the cell viability was significantly reduced (p < 0.01) to 68% which also further decreased to
6.3% (p < 0.001) at 12 h, as depicted in Fig 3C. The decrease in the cell viability from 6 h to 9
h was minimal (1.4%), but altogether, the result showed that the sequential program of cell death
could have been activated. Thymoquinone was used as a positive control to compare the
cytotoxic effect of AAHD (Fig. 3D).
3
.4.
in HepG2 cells
It has been well documented that the accumulation of ROS and mitochondrial damage
AAHD induced the production of ROS and decreased the mitochondrial potential
inside the tumor cells triggers apoptosis leading to activation of a nonreversible cascade of cell-
death events. To investigate the role of mitochondria and ROS levels in cell death, we measured
the ROS levels and evaluated the magnitude of the decrease in the mitochondrial potential
(ΔΨm). As the concentration of AAHD increased, there was a significant increase in the ROS
levels with the increase in the concentration of the drug ranging from 0.05mM to 0.3mM as
shown in the Fig 4A. Interestingly, there was a significant decrease (p < 0.05) in the ΔΨm
commencing from the initial dose of 0.05mM as shown in the Fig 4B. However, the decrease in
the ΔΨm was not much prominent from 0.05mM (OD-27.6) to 0.1mM (OD-26); but was quite
significant from 0.2mM (O.D-22.6; p < 0.01)) to 0.3mM (O.D-12.6; p < 0.001).
9

3
.5. AAHD inhibits wound healing in HepG2 cells.
Finally, we sought to investigate the potential of AAHD to impede the movement of
HepG2 cells and prevent the healing of wound-induced in the form of a scratch. It was observed
that the scratch/wound which was quite apparent at 0 h (Fig. 5A) was healed in the control group
after an incubation of 24 h as depicted in Fig 5B. Whereas, when AAHD was used to treat
HepG2 cells with concentrations of 0.1, 0.2 and 0.3mM (Fig. 5C, 5D & 5E respectively), the
wound healing was inhibited significantly. This demonstrates the potential of AAHD in
controlling the cell migration and could be a candidate to regulate metastasis in hepatocellular
carcinoma which needs to be further investigated.
4
. Discussion
Natural polyamines like spermidine, spermine and their precursor putrescine play a vital
role in the regular maintenance and cell proliferation of the eukaryotic cell. However, in cancer
and other hyperproliferative diseases, the requirement and the metabolism of polyamines is
dysregulated. Therefore, the polyamine pathway can be an attractive therapeutic target for
designing an anticancer chemotherapeutic drug regimen. In our previous in vitro study with other
polyamine analogues, we demonstrated the pro-apoptotic effect of the acylspermidine analogues
on MCF7 and Jurkat cells (17). It is quite evident from the nuclear and cytoplasmic staining
results that AAHD induces chromatin condensation accompanied by the formation of perinuclear
apoptotic bodies. Moreover, the dose- and time-dependent antiproliferative assays demonstrate a
sequential decrease in the cell viability suggesting a possible activation of the apoptotic cascade.
Several previous studies with different synthetic polyamine analogues have demonstrated both
caspase-dependent and caspase-independent mechanisms (21–24). Interestingly, the analogue
AAHD showed minimal cytotoxicity towards the non-carcinogenic, healthy BHK cells
1
0

expressing its specific action on tumour cells. There is considerable evidence that the polyamine
analogues specifically target the mitochondria as potential sites of activity (25,26). Hence, we
evaluated the modulatory effect of the AAHD on the ΔΨm. It was observed that with the
increase in the concentration, the ΔΨm decreased gradually which could be due to the
depolymerization of the mitochondrial membrane. Many studies have implicated the role of
cytochrome c in the apoptotic cell death with tumour cells treated with specific polyamine
analogues (27,28). The significant loss of ΔΨm and accumulation of ROS leads to the cell death
which is a prominent biomarker to be measured to know the pathological state of a cell (29). Our
results are in line with the above mentioned previous studies reported on the role of a decrease in
the ΔΨm and hence resulting in the release of the pro-apoptotic factors which could be further
confirmed by protein and gene studies. However, it may be possible that the mitochondrial
pathway would not be the primary target of all the polyamine analogues, as the antiproliferative
effect occurs prior to the initiation of mitochondrial activity. Moreover, the cells lacking
mitochondria also show positive antiproliferative response with the polyamine analogues (30).
Hence it could be professed that the acylspermidine analogue-AAHD targets mitochondria and
initiates cell death which must be further confirmed by molecular studies. Additionally, the
results obtained from the cell migration assay have shown that this derivative could also inhibit
the migration of cancer cells significantly which needs to be further studied.
In conclusion, the acylspermidine analogue-AAHD could be an excellent
pharmacological lead in the development of novel chemotherapeutic drug targeting
hepatocellular carcinoma. AAHD could also be a part of the current treatment regimens as it
shows minimal toxicity towards non-cancerous cells. We recommend the future ‘Omics’
approach in deciphering the actual mechanism and pathways involved which could give us a
1
1

clear understanding of the mechanism of action and the gene alteration pattern induced by
AAHD.
Conflict of interest
The authors declare that they have no financial conflict of interest.
Consent
All the authors have carefully read the manuscript and have approved it for publication.
Acknowledgements
This project was funded by the Deanship of Scientific Research (DSR), at King Abdulaziz
University, Jeddah, under grant no (RG- 22-130- 39). The authors, therefore, acknowledge with
thanks DSR for technical and financial support.
References:
1
2
3
4
5
.
.
.
.
.
Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, et al. Cancer statistics in China,
015. CA Cancer J Clin. 2016;66(2):115–32.
Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin. 2016;66(1):7–
0.
2
3
El-Serag HB. Epidemiology of viral hepatitis and hepatocellular carcinoma.
Gastroenterology. 2012 May;142(6):1264–1273.e1.
Singh S, Singh PP, Roberts LR, Sanchez W. Chemopreventive strategies in hepatocellular
carcinoma. Nat Rev Gastroenterol Hepatol. 2014 Jan;11(1):45–54.
DeWaal D, Nogueira V, Terry AR, Patra KC, Jeon S-M, Guzman G, et al. Hexokinase-2
depletion inhibits glycolysis and induces oxidative phosphorylation in hepatocellular
carcinoma and sensitizes to metformin. Nat Commun [Internet]. 2018;9(1):446. Available
1
2

from: http://www.nature.com/articles/s41467-017-02733-4
6
7
.
.
Moschou PN, Roubelakis-Angelakis KA. Polyamines and programmed cell death. J Exp
Bot. 2014 Mar;65(5):1285–96.
Wang Y, Zhang J, Li M, Li M, Xie S, Wang C. Synthesis and evaluation of novel
amonafide–polyamine conjugates as anticancer agents. Chem Biol Drug Des [Internet].
2
017 May 1;89(5):670–80. Available from: https://doi.org/10.1111/cbdd.12888
8
9
.
.
Burns MR, Graminski GF, Weeks RS, Chen Y, O’Brien TG. Lipophilic lysine-spermine
conjugates are potent polyamine transport inhibitors for use in combination with a
polyamine biosynthesis inhibitor. J Med Chem. 2009;52(7):1983–93.
Murray-Stewart T, Ferrari E, Xie Y, Yu F, Marton LJ, Oupicky D, et al. Biochemical
evaluation of the anticancer potential of the polyamine-based nanocarrier Nano11047.
PLoS One [Internet]. 2017 Apr 19;12(4):e0175917. Available from:
https://doi.org/10.1371/journal.pone.0175917
1
0. Dai F, He H, Xu X, Chen S, Wang C, Feng C, et al. Synthesis and biological evaluation of
naphthalimide-polyamine conjugates modified by alkylation as anticancer agents through
p53 pathway. Bioorg Chem [Internet]. 2018;77:16–24. Available from:
http://www.sciencedirect.com/science/article/pii/S0045206817307988
1
1
1. Mould DP, McGonagle AE, Wiseman DH, Williams EL, Jordan AM. Reversible
inhibitors of LSD1 as therapeutic agents in acute myeloid leukemia: clinical significance
and progress to date. Med Res Rev. 2015 May;35(3):586–618.
2. Milelli A, Marchetti C, Turrini E, Catanzaro E, Mazzone R, Tomaselli D, et al. Novel
1
3

polyamine-based Histone deacetylases-Lysine demethylase 1 dual binding inhibitors.
Bioorg Med Chem Lett [Internet]. 2018 Feb [cited 2018 Feb 18]; Available from:
http://linkinghub.elsevier.com/retrieve/pii/S0960894X18301392
1
1
1
3. Varghese S, Gupta D, Baran T, Jiemjit A, Gore SD, Casero RA, et al. Alkyl-substituted
polyaminohydroxamic acids: A novel class of targeted histone deacetylase inhibitors. J
Med Chem. 2005;48(20):6350–65.
4. Varghese S, Senanayake T, Murray-Stewart T, Doering K, Fraser A, Casero RA, et al.
Polyaminohydroxamic acids and polyaminobenzamides as isoform selective histone
deacetylase inhibitors. J Med Chem. 2008;51(8):2447–56.
5. Razvi SS, Choudhry H, Moselhy SS, Kumosani TA, Hasan MN, Zamzami MA, et al.
Synthesis, screening and pro-apoptotic activity of novel acyl spermidine derivatives on
human cancer cell lines. Biomed Pharmacother [Internet]. 2017 Sep 1 [cited 2017 Oct
7
];93:190–201. Available from:
http://www.sciencedirect.com/science/article/pii/S0753332217311514?via%3Dihub
1
6. Mohammed FA, Elkady AI, Syed FQ, Mirza MB, Hakeem KR, Alkarim S. Anethum
graveolens (dill) – A medicinal herb induces apoptosis and cell cycle arrest in HepG2 cell
line. J Ethnopharmacol [Internet]. 2018;219:15–22. Available from:
http://www.sciencedirect.com/science/article/pii/S0378874118305750
1
1
7. Razvi SS, Choudhry H, Moselhy SS, Kumosani TA, Hasan MN, Zamzami MA, et al.
Synthesis, screening and pro-apoptotic activity of novel acyl spermidine derivatives on
human cancer cell lines. Biomed Pharmacother. 2017;93.
8. Elkady AI. Crude alkaloid extract of Rhazya stricta inhibits cell growth and sensitizes
1
4

human lung cancer cells to cisplatin through induction of apoptosis. Genet Mol Biol. 2013
Mar;36(1):12–21.
1
2
9. Hasan MN, Choudhry H, Razvi SS, Moselhy SS, Kumosani TA, Zamzami MA, et al.
Synthetic Strigolactone Analogues Reveal Anti-Cancer Activities on Hepatocellular
Carcinoma Cells. Bioorg Med Chem Lett [Internet]. 2018;28. Available from:
https://www.sciencedirect.com/science/article/pii/S0960894X18301057
0. Shan RENF, Zhou YUNFEI, Peng AIFEN, Jie ZHIG. Inhibition of Aurora-B suppresses
HepG2 cell invasion and migration via the PI3K/Akt/NF-κB signaling pathway in vitro.
Vol. 8, Experimental and Therapeutic Medicine. 2014. p. 1005–9.
2
2
1. Damiani E, Wallace HM. Polyamines and Cancer. Methods Mol Biol. 2018;1694:469–88.
2. Li M, Wang Y, Ge C, Chang L, Wang C, Tian Z, et al. Synthesis and biological evaluation
of novel alkylated polyamine analogues as potential anticancer agents. Eur J Med Chem.
2
018 Jan;143:1732–43.
2
2
2
3. Gurkan AC, Arisan ED, Yerlikaya PO, Ilhan H, Unsal NP. Inhibition of autophagy
enhances DENSpm-induced apoptosis in human colon cancer cells in a p53 independent
manner. Cell Oncol (Dordr). 2018 Jun;41(3):297–317.
4. Thomas TJ, Thomas T. Cellular and Animal Model Studies on the Growth Inhibitory
Effects of Polyamine Analogues on Breast Cancer. Med Sci (Basel, Switzerland). 2018
Mar;6(1).
5. Vertino PM, Beerman T a, Kelly EJ, Bergeron RJ, Porter CW. Selective cellular depletion
of mitochondrial DNA by the polyamine analog N1,N12-bis(ethyl)spermine and its
1
5

relationship to polyamine structure and function. Mol Pharmacol. 1991;39(4):487–94.
2
2
6. Bergeron RJ, Neims AH, McManis JS, Hawthorne TR, Vinson JRT, Bortell R, et al.
Synthetic Polyamine Analogues as Antineoplastics. J Med Chem. 1988;31(6):1183–90.
7. Hegardt C, Johannsson OT, Oredsson SM. Rapid caspase-dependent cell death in cultured
human breast cancer cells induced by the polyamine analogue N1,N11-
diethylnorspermine. Eur J Biochem [Internet]. 2002;269(3):1033–9. Available from:
http://dx.doi.org/10.1046/j.0014-2956.2001.02744.x
2
8. Huang Y, Greene E, Murray Stewart T, Goodwin AC, Baylin SB, Woster PM, et al.
Inhibition of lysine-specific demethylase 1 by polyamine analogues results in reexpression
of aberrantly silenced genes. Proc Natl Acad Sci [Internet]. 2007;104(19):8023–8.
Available from: http://www.pnas.org/cgi/doi/10.1073/pnas.0700720104
2
3
9. Joshi DC, Bakowska JC. Determination of Mitochondrial Membrane Potential and
Reactive Oxygen Species in Live Rat Cortical Neurons. J Vis Exp [Internet]. 2011 May
2
3;(51):2704. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3143685/
0. Albanese L, Bergeron RJ, Pegg AE. Investigations of the mechanism by which
mammalian cell growth is inhibited by N1N12-bis(ethyl)spermine. Biochem J. 1993
Apr;291 ( Pt 1:131–7.
Figure legends:
Figure 1: The overall scheme of synthesis in the preparation of AAHD is represented as a flow chart with
‘
R’ moiety as an acyl side chain.
1
6

Figure 2: (A) The HepG2 cells were treated with different concentrations of AAHD (0.2mM & 0.3mM)
and Hoechst 33342 stain was used to observe the alterations in the morphology. There was a considerable
change in the morphology of the cells as compared to the control group. (B) Nuclear and morphological
changes are depicted after Acridine Orange/Ethidium Bromide staining. The significant changes are
indicated by red arrows.
Figure 3: (A) Dose-dependent effect of AAHD on cell viability of HepG2. HepG2 cells were treated with
several concentrations of AAHD for 24 h. The cell viability was assessed using WST-1 assay. (B) Dose-
dependent effect of AAHD on cell viability of BHK (non-cancerous) cells. (C) Time-dependent effect of
AAHD on cell viability of HepG2. (D) Thymoquinone was used as a positive control to assess the dose-
dependent effect on HepG2. The values here are represented as mean + S.E.M. of two experiments;
statistically significant (unpaired ‘t’ test; two-tailed): *, p < 0.05; **, p < 0.01; ***, p < 0.001; versus the
corresponding untreated control.
Figure 4: (A) AAHD induced the production of ROS in HepG2 cells as a consequence of the decrease in
the mitochondrial potential. (B) The mitochondrial potential of AAHD-treated HepG2 cells was
decreased significantly with the increase in the concentration. The values are represented as mean +
S.E.M. of two experiments; statistically significant (unpaired ‘t’ test; two-tailed): *, p < 0.05; **, p<0.01;
***, p<0.001; versus the corresponding untreated control.
Figure 5: Inhibitory effect of AAHD on HepG2 cell migration. (A) The scratch is quite
prominent in the control at 0 h. (B) The wound scratch has been healed in the control with the
migration of cells after 24 h. (C) AAHD reduced the migration of cells when used at a
concentration of 0.1mM after 24 h (D) Effect of AAHD at a concentration of 0.2mM after 24 h.
(E) Maximum inhibition of cell migration was observed after an incubation of 24 h with AAHD
at a concentration of 0.3mM.
1
7

1
8

1
9

2
0

2
1

Highlights

Novel acylspermidine derivative- N-(4-aminobutyl)-N-(3-aminopropyl)-8-hydroxy-
dodecanamide (AAHD) showed anticancer effect on hepG2 cells.
AAHD demonstrated dose- and time-dependent decrease in the cell viability.
AAHD triggered the production of ROS and decreased the mitochondrial potential
exerting a pro-apoptotic effect.



The Hoechst 33342 staining and Acridine orange/Ethidium bromide staining showed
significant morphological changes in the nuclei of the HepG2 cells.
2
2

Graphical abstract
2
3