An investigation of in vitro cytotoxicity and apoptotic potential of aromatic diselenides

Article abstract of DOI:10.1016/j.bmcl.2014.05.075

A target synthesis of a library of symmetric aromatic diselenides was attempted with the aim of generating anticancer lead compounds. Out of thirteen screened molecules (1-13) against a panel of human cancer cell lines, compound 8 exhibited highest cell growth inhibition in Human leukemia HL-60 cells with IC₅₀ value of 8 μM. Compound 8 had a good pro-apoptotic potential as evidenced from several apoptotic protocols like DNA cell cycle analysis and monitoring of apoptotic bodies formation using phase contrast and nuclear microscopy with Hoechst 33,258. Also, 8 significantly inhibits S phase of the cell cycle and eventually trigger apoptosis in HL-60 cells through mitochondrial dependent pathway substantiated by the loss of mitochondrial potential. A theoretical investigation of DNA binding ability of 8 showed that it selectively bind to minor groove of DNA, where it is stabilized by hydrogen bonding and hydrophobic interactions.

Full text of DOI:10.1016/j.bmcl.2014.05.075

Accepted Manuscript
An Investigation of in vitro Cytotoxicity and apoptotic potential of aromatic
diselenides
Masood Ahmad Rizvi, Santosh Guru, Tahira Naqvi, Manjeet Kumar, Navanath
Khumbhar, Showkat Akhoon, Shazia Banday, Shashank K. Singh, Shashi
Bhushan, G. Mustafa Peerzada, Bhahwal Ali Shah
PII:
S0960-894X(14)00584-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.05.075
BMCL 21689
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 February 2014
11 May 2014
22 May 2014
Please cite this article as: Rizvi, M.A., Guru, S., Naqvi, T., Kumar, M., Khumbhar, N., Akhoon, S., Banday, S.,
Singh, S.K., Bhushan, S., Mustafa Peerzada, G., Shah, B.A., An Investigation of in vitro Cytotoxicity and apoptotic
potential of aromatic diselenides, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/
j.bmcl.2014.05.075
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An Investigation of in vitro Cytotoxicity and apoptotic potential of
aromatic diselenides
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Masood Ahmad Rizvi,a* Santosh Guru,b Tahira Naqvi,a Manjeet Kumar,c Navanath Khumbhar,d
Showkat Akhoon,a Shazia Banday,a Shashank K. Singh,b Shashi Bhushan,b G. Mustafa Peerzadaa
and Bhahwal Ali Shahc*
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aDepartment of Chemistry, University of Kashmir, Hazratbal, Srinagar, 190006, J&K, India.
bDivision of Cancer Pharmacology, CSIR-Indian Institute of Integrative Medicine, Jammu-
180001, India
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cNatural Product Chemistry Division, CSIR-Indian Institute of Integrative Medicine (IIIM),
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Canal Road, Jammu Tawi, 180001, India.
dInstitute of Bio-Informatics and Biotechnology, University of Pune, Ganeshkhind Road, Pune,
411007, India
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* Corresponding authors e-mail address: masoodku2@gmail.com (MAR); bashah@iiim.res.in
(BAS)
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Key words: Diselenides; anti-cancer; apoptosis; DNA-binding; in silico.
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Introduction:
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Metal complexes have been a subject of consistent investigations to develop therapeutics for
treatment of human diseases particularly cancer.1 In this regard, recent investigations ascertain
the dual function of selenium compounds as chemo preventives,2 as well as selective anti tumor
therapeutics, with the induction of apoptosis and inhibition of cell proliferation as the broader
mechanisms in the cancer chemoprevention.3,4 The epidemiological studies suggest a correlation
of increased risk of multi organ cancer with low levels of selenium intake or decreased plasma
levels of selenium.5 Selenium compounds in different chemical architectures target cancer in
different ways and with different efficacies.4 The toxicity concern of inorganic selenium
compounds often limits their use in chemoprevention6 even though they may be equivalent or
more superior chemo preventives to organoselenium compounds.7 In view of low toxicity,
coupled with the fact that human exposure to selenium predominantly occurs through
selenomethionine in food stuff, investigators are focusing on organic forms of selenium for better
chemoprevention. The various pathways that have been considered in the cancer
chemoprevention by organoselenium compounds include: (i) Protective role of selenoproteins
(ii) induction of apoptosis (iii) immune system effects (iv) detoxification of antagonistic metals
(v) inactivation of nuclear transcription factor (vi) regulation of lipoxygenases (vii) reduction of
oxidative stress (viii) induction of Phase II enzymes (ix) inhibition of DNA adduct formation (x)
cell cycle arrest.8 However the significant cellular phenomenon in cancer chemoprevention by
organoselenium compounds is induction of apoptosis.3 Thus, designing organoselenium
compounds can be of interest to potentiate chemotherapy by tackling problems of drug induced
toxicity, drug resistance and other plethora of physiological effects in cancer chemotherapy.9
Therefore, in continuation of our work to develop anticancer lead molecules, 10 we report
synthesis of symmetric aromatic organo diselenides (1-13) and their in vitro cytotoxicity against
a panel of human cancer cell lines. The apoptotic potential of the lead compound, 8 was
investigated by several end point markers of the apoptosis such as DNA damage, loss in
mitochondrial membrane potential and apoptotic bodies formation using phase contrast
microscopy with and without Hoechst staining. Furthermore the DNA binding ability of
synthesized compounds was theoretically investigated using molecular Docking method. The
present study was aimed to highlight the significance of organo diselenides in the discovery of
novel anticancer agents.
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Results and Discussion
Chemistry
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A library of symmetric aromatic diselenides was synthesized according to Bhasin’s synthetic
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methodology.11 The protocol involves generating diselenide anion (Se2 ), by reducing elemental
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selenium with hydrazine hydrate in sodium hydroxide. The diselenide anion (Se2 ) generated in
the reaction mixtures reacts in situ with bromo benzene derivatives to generate the corresponding
diselenides. Initially elemental selenium was stirred with hydrazine and sodium hydroxide in
DMF at room temperature for two hours. The bromo benzene reagents were added to the
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reaction mixture and further refluxed for 4 hrs in DMF around 120 C, to get corresponding
diselenides (scheme-1).
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Scheme-1. Synthesis of symmetric aromatic diselenides
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The reaction of un-substituted as well as methyl and ethyl substituted aryl bromides at
various positions and electron rich 4-methoxy phenyl bromide gave the corresponding products
in good yields (2-7). However, the reaction with electron withdrawing substituent like
bistriflouromethyl substituted phenyl bromide give corresponding diselenide (8) in excellent
yields (86 %). Hydroxy substitution at various positions also provides the corresponding
diselenide in good yields (9-12). Bicyclic system like 1-napthyl bromide also (adaptable to
reaction condition) gave the product in 65% yields (13).
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Biological Activity
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The synthesized diselenides (1-13) were screened for their ability to induce cytotoxicity against
panel of human cancer cell lines viz., human promyelocytic leukemia (HL-60), human epithelial
carcinoma cell line (OVCAR-5), renal cell adenocarcinoma cell line (786-O),
colorectal adenocarcinoma cell line (HT-29), human prostate cancer cell line (PC-3). All the
compounds displayed a range of cytotoxicity towards this cancer cell line, with the compound 8
to be the most cytotoxic (Table 1). Having an impressive cytotoxicity towards leukemia-HL-60
and prostate-PC-3 (IC50 value of 8 and 13 µM) we further explored 8 for its cytotoxicity potential
against breast cancer-MCF-7, pancreatic cancer-MIA-PA-Ca-2 and colon cancer- HCT-116, cell
lines. The IC50 values of 8 in MCF-7, MIA-Pa-Ca-2 & HCT-116 cells were 18, 25 and 27 µM
respectively. Furthermore, the time dependent (6, 12, 24 and 48 h) cytotoxic studies of 8 on HL-
60 cell line indicate the progressive decrease in IC50 values with increasing time (Figure 2).
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Table 1. Inhibition of cell proliferation by compounds 1-13 against a panel of human cancer cell
lines
IC 50 (µM) in different Human Cancer cell lines
Compound
Leukemia
Epithelial
Renal
786-O
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Colorectal
Prostate
PC-3
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HL-60
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OVCAR-5
HT-29
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Figure 2. Time dependent cytotoxic profile of 8 on HL-60 cell line. Data is Mean SD (n= 8
wells), and representative of one of three concordant experiments. Initially, cells were incubated
with MTT solution for 4 hr and optical density of formazon crystals was measured as described
in experimental procedures.
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DNA Cell Cycle Analysis
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The good cytotoxic behavior of 8 towards HL-60 cells (IC50 value of 8µM), encouraged us to
choose this cell line for DNA cell cycle analysis. The cell cycle is a fundamental and ordered
event in which DNA replicates, and homologous chromosomes segregate and get equally
distributed among the daughter cells. Deregulated cell cycle is one of the major hallmarks of
cancer cells. These cells may lose the ability to regulate the cell cycle and control their rate of
proliferation. A rate-limiting step in the cell cycle that is often disturbed in cancer is the
progression of cells through the first gap (G1) phase. Many anticancer drugs act by blocking one
or more stages of the cell cycle and eventually trigger apoptosis. The effect of antiproliferative
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agent on cell cycle progression and DNA damage appears to depend on concentration of the
compound and duration of the treatment. Increased resistance to apoptosis is a characteristic of
many tumor cells. Therefore, apoptosis deficiency is considered to be a major cause of the
therapeutic resistance of tumors in the clinic, since many chemo-and radio therapeutic agents’ act
through the induction of apoptosis. HL-60 cells when exposed to 8 at concentration of 10, 20
and 30 µM for 24 h exhibited an increase in apoptosis percentage in dose dependent manner i.e.,
2, 5 and 24% sequentially (Figure 3). Interestingly, compound delay the s-phase of cell cycle
with concurrent induction of apoptosis in HL-60 cells.
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Figure 3. DNA cell cycle analyses of HL-60 cells exposed to specified concentration of 8 for 24
hr. After treatment cells were stained with Propidium iodide, PI (10µg/mL) to determine DNA
fluorescence and cell cycle phase distribution as described in experimental procedures. Data was
analyzed by Modfit software (Verity Software House Inc., Topsham, ME) for the proportions of
different cell cycle phases. The fraction of cells from apoptosis, G1, S and G2 phases analyzed
from FL2- A vs. cell counts are shown in %.
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Measurement of Mitochondrial Membrane Potential
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The alteration of the mitochondrial function plays a major role in the apoptotic process. The loss
of mitochondrial membrane potential (Ψmt) leads to depolarization of mitochondrial membranes
and mitochondrial dysfunction leading to cell death. We investigated the effects of 8 on the
mitochondrial potential of HL-60 treated cells. This was examined by measuring their ability to
retain Rhodamine 123, a fluorescent dye used to indicate the loss of mitochondrial
transmembrane potential. Under this condition, the control HL-60 cells showed 6% loss of Ψmt,
which increase to 58% after the treatment of 8. It induced the loss of mitochondrial membrane
potential (MMP) in HL-60 cells in a dose dependant manner (Figure 4). The loss of
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mitochondrial membrane potential also releases several pro-apoptotic factors that activate
caspases and finally induces apoptosis.
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Figure 4. Mitochondrial membrane potential analyses was performed on HL-60 cells exposed to
specified concentration of 8 for 24 h. After treatment cells were stained with Rhodamine 123 dye
(200 nm) for 40 min to determine membrane potential as described in experimental procedures.
Data was analyzed by Cell Quest Pro software from BD Biosciences. Data were representative of
one of three similar experiments.
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Apoptotic morphological alteration induce by 8
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The characteristic apoptotic morphological changes of HL-60 treated cells were assessed by the
fluorescent microcopy after staining with Hoechst dye. Because of the integrity of the cell
plasma membrane, Hoechst dye was unable to infiltrate into the HL-60 cells when the cells were
alive or still in the early process of apoptosis, while the dead cells had Hoechst inside and the
nuclei were stained a bright blue color. The results revealed nuclear condensation, membrane
blebbing, nuclear fragmentation and apoptotic bodies (the characteristic of apoptosis) in cells
that had been incubated with 8 (Figure 5). The control cells did not exhibit any of the above
morphological changes, the nuclei were stained a less bright blue and the color was
homogeneous. Chromatin condensation and other apoptotic characters were observed only in the
treated cells. Compound 8 at 20 and 30 µM concentrations significantly induced morphological
characteristics similar to those of apoptotic cells when observed under phase contrast inverted
microscopy as well as nuclear staining through the Hoechst fluorescent dye (Figure 5).
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Figure 5. Effect of 8 on the cellular and nuclear morphology of HL-60 cells. Cells were treated
with 10, 20 & 30µM concentrations of 8 for 24 h, visualized for cellular morphology and
simultaneously stained with DNA binding Hoechst 33258 dye as described in experimental
procedures. Nuclear morphology and apoptotic bodies’ formation were visualized on fluorescent
microscope. Condensed nuclei and the apoptotic bodies are indicated by red arrows. Data are
representative of one of three similar experiments and magnification of the pictures was 30X on
Olympus 1X 70 inverted microscope.
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In Silico DNA Binding study of 8
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Molecular docking and other molecular dynamics simulation algorithms are attractive tools to
theoretically understand the magnitude of binding and mode of interaction in the ligand–receptor
complexes.12 Corroborating experimental results which are a net outcome of all the parameters in
a blend with theoretical calculations can be impressive for the step to step analysis and prediction
of lead compounds in a time and cost effective manner.13 Prompted by a good apoptotic behavior
of 8, we attempted to explore its DNA binding ability through molecular docking studies. In
order to locate the binding site of 8 on DNA molecule, we did initial surface docking. After
optimizing for the grid size of 8 binding site on DNA, re-docking was done using the discussed
procedure (please see experimental procedures). The lowest energy docked complex of DNA-
compound 8 is depicted in Figure 6. The 8 selectively binds to minor groove of DNA, where it is
stabilized by various Vander Waal type and hydrophobic interactions with DNA. One benzene
ring of 8 is interacting with G-C rich region including G22B, G4A and C21B bases whereas other
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benzene ring is stacked with A-T rich region containing nucleotide bases such as A6A, T7A and
T20B (Figure 7). The fluorine atoms of 8 are involved in intermolecular hydrogen bonding
interactions with G22B and G4A of opposite chain bases. Hydrogen bonding interactions such as
F1….HN(2)G22B, F1….HN(2)G4A, F2….HC1’G22B, F5….HC5’G22B, F10….HC(2)A6A,
F11…HC4’T7A, F12….HC1’T20B and F12…HC5’C21B are providing stability to compound 8-
DNA complex. The lowest binding energy of -6.62 Kcal mol-1 and above mentioned hydrogen
bonding interactions are indicative that 8 efficiently binds to the DNA molecule by positioning in
stacked orientation within DNA molecule. The molecular docking results are predictive of the
DNA binding potential of the 8.
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Figure 6: Docked view of compound 8 with DNA dodecamer (1BNA.pdb): a) Ribbon model of
compound 8 in minor groove of DNA. b) Surface area of compound 8 with DNA dodecamer.
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Figure 7: Intermolecular hydrogen bonding interactions between compound 8 and DNA
dodecamer.
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Conclusion
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Our findings showed that all the synthesized diselelenides (1-13) showed promising cytotoxicity
against human cancer cells lines. Compound 8 was found to be the most active and showed pro-
apoptotic effects evidenced by various biological end points like DNA damage with s-phase
delay, loss in mitochondrial membrane potential and apoptotic bodies formation. In silico DNA
binding studies suggested that 8 selectively binds to minor groove of DNA, where it is stabilized
by hydrogen bonding and hydrophobic interactions with DNA. Further studies aimed at the
detailed molecular mode of action of compound 8 are underway. Moreover, efforts are continued
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towards synthesizing library of selenides to develop anticancer leads and thereby a moiety of
therapeutic significance.
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General Experimental Procedures
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1H and C NMR spectra in CDCl3 were recorded on 400 MHz spectrometers with TMS as an
internal standard. Chemical shifts are expressed in parts per million (δ ppm); J values are given
in Hertz. Reagents and solvents used were mostly AR grade. Silica gel aluminum plates were
used for TLC. HRMS were recorded on UHD LC/MS Q-TOF. The RPMI-1640 medium,
Minimum essential medium (MEM), Dulbecco’s Modified Eagles Medium (DMEM), fetal Calf
Serum (FCS), penicillin, streptomycin, L-Glutamine, pyruvic acid, trypsin, gentamycin,
penicillin, sulforhodamine blue (SRB), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl]-tetrazolium
bromide (MTT), NaHCO3, propidium iodide (PI), DNase-free RNase and dimethyl sulphoxide
(DMSO) were purchased from Sigma Aldrich, USA. All other reagents were AR graded and
available locally.
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General procedure for Synthesis of Diselenides: To a rapidly stirred solution of sodium
hydroxide (1.52 g, 38 mmol) and selenium powder (1.98 g, 25 mmol) in dimethylformamide
(100 mL) was added 100% hydrazine hydrate (1 mL, 25 mmol) dropwise at room temperature
(r.t.). The mixture was stirred for two hours. 2-bromo pyridine (3.92 g, 25 mmol) was added
drop-wise to the reaction mixture and refluxed for four hours. After the consumption of whole
reactant as evidenced by TLC, the reaction was stopped and diluted with water. The extraction
was done with ethyl acetate and the organic layer was dried with Na2SO4. The solvent was
removed on a rota-evaporator and the residue was purified by column chromatography to give to
give 1,2-di(pyridine-2-yl)diselane (1) in 75% yields. In a similar manner the other diselenides
were prepared using appropriate amounts of bromo benzene reactants under same procedure.
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1,2-di(pyridine-2-yl)diselane (1): Yield 75 %; Spectroscopic data was in good agreement with
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literature data reported earlier. H NMR (500 MHz, CDCl3): 6.98 (d, 2H, J = 4.7), 7.51 (m, 2H),
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7.75 (d, 2H, J = 7.4), 8.40 (s, 2H); C NMR (100 MHz, CDCl3): 154.8, 149.5, 137.1, 123.5,
120.2; HRMS calcd. for C10H9N2Se2 [M+H] + 316.9091, found 316.90004.
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1,2-diphenylselane (2): Yield 78 %; Spectroscopic data was in good agreement with literature
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data reported earlier. H NMR (500 MHz, CDCl3) δ 7.62 – 7.34 (m, 4H), 7.32 – 6.99 (m, 6H);
13C NMR (100 MHz, CDCl3) δ 132.7, 131.9, 130.8, 127.6; HRMS calcd. for C12H11Se2 [M+H] +
314.9186, found 314.9210.
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1,2-bis(4-methylphenyl)diselane (3): Yield 74 %; Spectroscopic data was in good agreement
with literature data reported earlier.15 1H NMR (500 MHz, CDCl3) δ 7.45 (d, 4H, J = 8.4 Hz),
7.10 (d, 4H, J = 8.4 Hz), 2.32 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 139.9, 133.7, 130.9, 127.9,
23.1; HRMS calcd. for C14H15Se2 [M+H] + 342.9499, found 342.9486.
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1,2-bis(2-ethylphenyl)diselane (4): Yield 78 %; Spectroscopic data was in good agreement with
literature data reported earlier.16 1H NMR (500 MHz, CDCl3) δ 7. 87 – 7.62 (m, 2H), 7.35 – 7.29
(m, 2H), 7.28 – 7.20 (m, 2H), 7.19 – 7.05 (m, 2H), 2.51 – 2.22 (m, 4H), 1.40 – 1.17 (m, 6H); 13C
NMR (100 MHz, CDCl3) δ 148.2, 134.4, 130.8, 127.1, 126.6, 125.6, 27.7, 13.5; HRMS calcd.
for C16H19Se2 [M+H] + 370.9812, found 370.9870.
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1,2-bis(2,6-dimethylphenyl)diselane (5): Yield 75 %; H NMR (500 MHz, CDCl3) δ 7.51 –
7.19 (m, 2H), 7.19 – 6.89 (m, 4H), 2.53 – 2.10 (m, 12H); 13C NMR (100 MHz, CDCl3) δ 142.8,
127.3, 126.4, 23.1; HRMS calcd. for C16H19Se2 [M+H] + 370.9812, found 370.9862.
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1,2-bis(3,5-dimethylphenyl)diselane (6): Yield 83 %; Spectroscopic data was in good
agreement with literature data reported earlier.17 1H NMR (500 MHz, CDCl3) δ 7.34 – 7.29 (m,
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2H), 7.42 – 7.24 (m, 2H), 7.01 – 6.97 (m, 1H), 7.07 – 6.87 (m, 1H), 2.35 – 2.31 (m, 12H); C
NMR (100 MHz, CDCl3) δ 144.8, 138.5, 130.1, 128.5, 21.9; HRMS calcd. for C16H19Se2
[M+H]+ 370.9812, found 370.9810.
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1,2-bis(4-methoxyphenyl)diselane (7): Yield 74 %; Spectroscopic data was in good agreement
with literature data reported earlier.15 1H NMR (500 MHz, CDCl3): 7.52 (d, 4H, J = 8.5), 6.81 (d,
4H, J = 8.5), 3.79 (s, 6H); 13C NMR (100 MHz, CDCl3): 160.1, 138.5, 134.7, 114.5, 55.3;
HRMS calcd. for C16H15Se2 [M+H] + 374.9397, found 374.9405.
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1,2-bis(3,5-bis(trifluoromethyl)phenyl)diselane (8): Yield 86 %; 1H NMR (500 MHz, CDCl3):
7.73-7.91 (m, 4H), 7.99-8.03 (d, 2H, J = 6.9); 13C NMR (100 MHz, CDCl3): 121.4, 122.4, 131.6,
131.7, 132.9; HRMS calcd. for C16H7F12Se2 [M+H] + 586.8681, found 586.8620.
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1,2-bis(4-hydxoxyphenyl)diselane (9): Yield 62 %; Spectroscopic data was in good agreement
with literature data reported earlier.18 1H NMR (500 MHz, CDCl3): 7.36 (m, 4H). 6.69 (m, 4H);
13C NMR (100 MHz, CDCl3): 155.3, 134.8, 122.1, 116.3; HRMS calcd. for C12H11O2Se2 [M+H]+
346.9084, found 346.9101.
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(Diselanediylbis(1,4-phenylene))dimethanol (10): Yield 67 %; Spectroscopic data was in good
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agreement with literature data reported earlier. H NMR (500 MHz, CDCl3): 7.50 (d, 4H, J =
7.3), 7.25(d, 4H, J = 7.7), 4.64 (s, 4H); 13C NMR (100 MHz, CDCl3): 139.8, 133.1, 128.5, 127.4,
64.5; HRMS calcd. for C14H15O2Se2 [M+H] + 374.9397, found 374.9362.
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1,2-bis(2-hydroxyphenyl)diselane (11): Yield 64 %; Spectroscopic data was in good agreement
with literature data reported earlier.15 1H NMR (500 MHz, CDCl3) δ 7.63 – 7.49 (m, 2H), 7.45 –
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7.40 (m, 1H), 7.39 – 7.35 (m, 1H), 7.01 – 6.80 (m, 2H), 6.78 – 6.74 (m, 2H); C NMR (100
MHz, CDCl3) δ 157.3, 135.1, 127.9, 120.8, 115.7, 108.6; HRMS calcd. for C12H11O2Se2 [M+H]+
346.9084, found 346.9110.
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(Diselanediylbis(1,2-phenylene))dimethanol (12): Yield 68 %; H NMR (500 MHz, CDCl3) δ
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7.77 – 7.64 (m, 2H), 7.51 – 7.34 (m, 2H), 7.34 – 7.12 (m, 4H), 4.91 – 4.62 (m, 4H); C NMR
(100 MHz, CDCl3) δ 141.5, 134.8, 134.2, 127.5, 126.6, 125.9, 63.8; HRMS calcd. for
C14H15O2Se2 [M+H] + 374.9397, found 374.9428.
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1,2-bis(naphthalen-1-yl)diselane (13): Yield 65 %; Spectroscopic data was in good agreement
with literature data reported earlier.20 1H NMR (400 MHz, CDCl3): 7.83-7.76 (m, 2H) 7.55-7.48
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(m, 4H) 7.37-7.31 (m, 4H) 7.29-7.25 (m, 2H), 7.21-7.15 (m, 2H); C NMR (100 MHz,CDCl3):
134.2, 131.8, 128.8, 128.3, 126.6, 126.4, 125.6; HRMS calcd. for C20H15Se2 [M+H] + 414.9499,
found 414.9524.
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Cell culture, growth conditions and treatments: Human Prostate Cancer cells PC-3, Breast
Cancer cell MCF-7, Leukemia Cells HL-60, pancreatic cancer cell MIA-Pa-Ca-2 and colon
cancer cells HCT-116 were obtained from European Collection of Cell Cultures (ECACC). The
PC-3, MCF-7 and HCT-116 cells were grown in Minimum essential medium (MEM), MIA-Pa-
Ca-2 cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) and HL-60 Cells were
grown in RPMI-1640 medium supplemented with 10% heat inactivated fetal bovine serum
(FBS), penicillin (100 units/ml), streptomycin (100 µg/ml), L-glutamine (0.3 mg/mL) and
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NaHCO3(8mg/mL). Cells were grown in an atmosphere of 5% CO2 and 95% relative humidity in
a CO2 incubator (Thermocon Electron Corporation, USA) at 37oC. Adherent cultures were
harvested by trypsinaization (0.05% trypsin , 0.02% EDTA in PBS). Soon after cells were ready
to detach, 5ml of medium were added to stop trypsinization. Cells were dispersed gently by
pipetting incomplete growth medium, centrifuged at 1000 rpm, for 5 min. On the other hand,
suspension cultures (HL-60) were harvested by centrifugation for 5 min at 1000 rpm. Different
molecules used in this study were dissolved in DMSO and were delivered to cell cultures in
complete medium. Since DMSO is cytotoxic at high concentrations therefore its final
concentration in the cells was below 1% w/v.
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Cell Proliferation Assay Using MTT: This assay is a quantitative colorimetric method for the
determination of cell survival and proliferation. The assessed parameter is the metabolic activity
of viable cells. Metabolically active cells reduce pale yellow tetrazolium salt (MTT) to a dark
blue water-insoluble formazan, which can be, after solubilization with DMSO, directly
quantified. The absorbance of the formazan directly correlates to the number of viable cells. The
different human cancer cell lines HL-60, PC-3, MIA-Pa-Ca-2, MCF-7 and HCT-116 cells were
plated in 96-well plates at a density of 6000 cell/well in 100µL of medium. The cell cultures
were incubated with different concentrations of test material and incubated for 48 hr. The MTT
dye at a concentration of 2.5mg/mL [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide] was added for 4 hr. The supernatant was aspirated and MTT-formazon crystals
dissolved in 150µL of DMSO; OD was measured at λ540 (reference wavelength, λ620) on an
ELISA reader (BioTek.). Cell growth was calculated by comparing the absorbance of treated
versus untreated cells.21
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Cell cycle analysis: DNA fragmentation constitutes one biochemical hallmark of apoptosis.
Thus, measurement of DNA content makes it possible to identify apoptotic cells, to recognize the
cell cycle Phase specificity, and to quantify apoptosis. For flow cytometry analysis of the relative
nuclear DNA content, the fluorescent dye propidium iodide (PI), which becomes highly
fluorescent after binding to DNA, is most commonly used. After permeabilization, PI binds to
DNA in cells at all stages of the cell cycle, and the intensity with which a cell nucleus emits
fluorescent light is directly proportional to its DNA content. HL-60 cells (2×106 /3ml/well) in 6-
well plate were treated with different concentrations of test material for 24 hr. The cells were
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harvested and centrifuged at 400 g for 5 min. The supernatant discarded and the pellet washed
twice with 2 ml PBS. The cells were fixed overnight in 1 ml chilled 70% ethanol in PBS at 4ºC.
Cell washed twice with 2 ml PBS, cell suspension was incubated with RNase digestion
(400µg/ml) at 37ºC for 1 hr. Finally, cells were stained with PI (10 µg/ml) for 30 min in dark and
analyzed immediately for DNA content on Flow Cytometer. Cell cycle histograms were analysed
using the ModFit LT™ 3.2.1 software packages. In this program, debris and single cell
populations are gated out using two parameter histogram of FL2-A versus FL2-W. The
fluorescence intensity of sub-G0 cell fraction represents the apoptotic cell population.22
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Mitochondrial membrane potential (MMP) assay: HL-60 cells (1x106 cells/2mL/well) were
treated with compound 8 at different concentrations for 24h. Thirty minutes before the end of the
experiment, the cell culture was treated with Rhodamine-123 (200nM) and keep in the dark for
30 mn. Cells were then collected, centrifuged (400g; 4oC; 5min), the pellet was washed with 1
ml of PBS and centrifuge as mentioned earlier. The fluorescence intensity of 10,000 events was
analyzed in FL-1 channel on BD FACSCalibur (Becton Dickinson, USA) flow cytometer. The
decrease in fluorescence intensity because of mitochondrial membrane potential loss was
analyzed in FL-1 channel and the change of in potential membrane (∆ψm) was assessed by
comparing fluorescence.22
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Hoechst 33258 staining of cells for nuclear morphology: HL-60 cells were treated with
indicated concentrations of compound 8 (10, 20 & 30µM) for 24 hr. Cells were centrifuged at
400 g for 5min and washed twice with PBS. Cells were then stained with one milliliter of
staining solution (Hoechst 33258, 10 µg/mL of 0.01M citric acid and 0.45 M disodium
phosphate containing 0.05% Tween 20) under subdued light at room temperature. After staining,
the cells were resuspended in 50µL of mounting fluid (PBS: glycerol, 1/1), 10 µL mounting
solution, containing cells was spread on clean glass slides and covered with the cover slips. The
slides were then observed for any nuclear morphological alterations and apoptotic bodies under
inverted fluorescence microscope (Olympus 1X70, magnification 30X) using UV excitation.23
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In silico study of 8: The crystal structure of DNA dodecamer (1BNA.pdb) having sequence
d(CpGpCpGpApApTpTpCpGpCpG)24 was downloaded from protein data bank (www.rcsb.org)
for molecular docking studies with 8. The geometry optimization of 8 was done by semi-
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empirical PM3 method25 using Spartan Pro 6.1.0 software.26 While as Autodock 4.2 software
was used for Molecular docking studies. 27 Prior to docking studies, DNA structure was refined
by removing non-polar hydrogen atoms and adding Kollman united atom charges and polar
hydrogen atoms. Gasteiger charges and hydrogen atoms were added to the 8 using Autodock
wizard.27 AutoGrid module was used for calculating the grid maps centered on the ligand (8)
binding site of DNA. The grid size was set to 50 A° × 50 A° × 62 A° with a grid spacing 0.375
A°. The step size of 1Å for translation and the maximum number of energy evaluation was set to
2,500,000. The 50 runs were performed. For each of the 50 independent runs, a maximum
number of 2,70,000 LGA operations were generated on a single population of 150 individuals.
The operator weights for crossover, mutation and elitism were maintained as default parameters
(0.80, 0.02, and 1, respectively). The resulting 50 docked conformations were analyzed for
binding energy, intermolecular energy and internal energy using Autodock wizard.27 The DNA-
compound 8 complex with lowest binding energy was used for analysis of hydrogen bonding,
hydrophobic and hydrophilic interactions. Chimera software was used to generate pictorial
presentation of selected docked conformation.28
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Acknowledgments
SG and MK thank CSIR for the award of Senior Research Fellowship.
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Abstract
An Investigation of in vitro Cytotoxicity and apoptotic potential of
aromatic diselenides
Masood Ahmad Rizvi,a* Santosh Guru,b Tahira Naqvi,a Manjeet Kumar,c Navanath Khumbhar,d
Showkat Akhoon,a Shazia Bhanday,a Shashank K. Singh,b Shashi Bhushan,b G. Mustafa
Peerzada,a Bhahwal Ali Shah,c*
A target synthesis of a library of symmetric aromatic diselenides was attempted with the aim of
generating anticancer lead compounds. Out of thirteen screened molecules (1-13) against a panel
of human cancer cell lines, compound 8 exhibited highest cell growth inhibition in Human
leukemia HL-60 cells with IC50 value of 8 µM. Compound 8 had a good pro-apoptotic potential
as evidenced from several apoptotic protocols like DNA cell cycle analysis and monitoring of
apoptotic bodies formation using phase contrast and nuclear microscopy with Hoechst 33258.
Also, 8 significantly inhibits
S phase of the cell cycle and eventually trigger apoptosis in HL-60
cells through mitochondrial dependent pathway substantiated by the loss of mitochondrial
potential. A theoretical investigation of DNA binding ability of 8 showed that it selectively bind
to minor groove of DNA, where it is stabilized by hydrogen bonding and hydrophobic
interactions.
19