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Biological cytotoxicity evaluation of spiro[azetidine-2, 3’-indole]-2’, 4(1’H)-

dione derivatives for anti-lung and anti-breast cancer activity

Article in Der Pharmacia Lettre · July 2011

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Der Pharmacia Lettre, 2011: 3 (5) 236-243
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ISSN 0975-5071
USA CODEN: DPLEB4

Biological cytotoxicity evaluation of spiro[azetidine-2, 3’-indole]-2’, 4(1’H)-

dione derivatives for anti-lung and anti-breast cancer activity
Prakash S. Sukhramani *1, Poonam S. Sukhramani 1, Sonal R. Tirthani 2, Sarav A. Desai 3,
Maulik P. Suthar 4
1
Veerayatan Institute of Pharmacy, Bhuj-Mandvi Road, Jakhania, Mandvi, Kutch, Gujarat, India
2
Sir P.T. Science College, College Campus, Modasa, Sabarkantha, Gujarat, India
3
Pioneer College of Pharmacy, Sayajipura, Vadodara, Gujarat, India
4
S. K. Patel College of Pharmaceutical Education & Research, Ganpat University, Kherva,
Mehsana (N.G.), India
______________________________________________________________________________

ABSTRACT

Biological in-vitro cell-based cytotoxicity assay is an easy and cost effective tool for hit ranking
and lead optimization at the early stage of drug discovery, drug design and drug optimization. In
the present research investigation, spiro[azetidine-2, 3’-indole]-2’, 4(1’H)-dione derivatives
were evaluated for cytotoxicity against HEK-293T (Human erythrocyte kidney cell line), MDA-
MB453 (Human Breast carcinoma cell line), MDA-MB468 (Human Breast carcinoma cell line),
NCI-H522 (Human Lung cancer cell line) and NCI-H23 (Human Lung cancer cell line) with use
of short term cytotoxicity MTT and XTT assay protocol. The IC50 was determined by dose
response curve analysis and statistical analysis using GraphPad Prism application, by plotting
the graph of log concentration vs % growth Inhibition. Compound 7g displayed significant
cytotoxicity (IC50) in breast cancer cell lines after 48 hrs comparable with the standard control
drug doxorubicin and are good candidate for development of novel drugs based on these
derivatives.

Keywords: Spiro[azetidine-2, 3’-indole]-2’, 4(1’H)-dione derivatives, cytotoxicity, lung and

breast cancer cell line, MTT, XTT.
______________________________________________________________________________

INTRODUCTION

Cancer is a class of diseases in which a group of cells display uncontrolled growth (division
beyond the normal limits), invasion (intrusion on and destruction of adjacent tissues), and
sometimes metastasis (spread to other locations in the body via lymph or blood). These three
malignant properties of cancers differentiate them from benign tumours, which are self-limited,
and do not invade or metastasize. [1] 2−Azetidinones, commonly known as β − lactams, are well-
known heterocyclic compounds among the organic and medicinal chemists. The activity of the

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famous antibiotics such as penicillins, cephalosporins and carbapenems are attributed to the
presence of 2−azetidinone ring in them. Recently, some other types of biological activity besides
the antibacterial activity have been reported in compounds containing 2−azetidinone ring. Such
biological activities include anti-fungal, anti-tubercular, anti-tumour, cholesterol absorption
inhibition and enzyme inhibition activity. The β − lactams also serve as synthons for many
biologically important classes of organic compounds. [2,3,4]

Common basic steps of in-vitro cytotoxic screening include: (a) isolation of cells, (b) incubation
of cells with drugs, (c) assessment of cell survival and (d) interpretation of the result. The trypan
blue dye exclusion assay is the most commonly accepted method for the measurement of cell
viability. It relies on the alteration in membrane integrity as determined by the uptake of dye by
dead cells, thereby giving a direct measure of cell viability. It is now well-documented that
apoptosis or programmed cell death is the key mechanism by which Chemotherapeutic agents
exert their cytotoxicity. These colorimetric assays (MTT) are mainly useful in determination of
cellular proliferation, viability and activation. The need for sensitive, quantitative, reliable and
automated methods led to the development of standard assays. Cell proliferation and viability
assays are of particular importance for routine applications. Tetrazolium salts MTT are
especially useful for assaying the quantification of viable cells. Both, MTT and XTT dye work
by being converted to a formazan dye only by metabolic active cells. These formazan dyes were
solubilized and is directly quantified using an ELISA reader with respective reference
wavelengths. [1]

The development of in-vitro cytotoxicity assays has been driven by the need to rapidly evaluate
the potential toxicity of large numbers of compounds, to limit animal experimentation whenever
possible, and to carry out tests with small quantities of compound. Evidence for the utility of in-
vitro cytotoxicity tests has led many pharmaceutical companies to screen compound libraries to
remove potentially toxic compounds early in the drug discovery process. XTT assay and the
MTT assay are the most common employed for the detection of cytotoxicity or cell viability
following exposure to toxic substances. [1]

MATERIALS AND METHODS

Compounds:
A series of ten compounds (7a-7j) were procured from Pharmaceutical Chemistry Department,
S. K. Patel College of Pharmaceutical Education & Research, Ganpat University, N. Gujarat,
India as shown in Table 1. These synthesized compounds were screened for anti-lung and anti-
breast cancer activity.
Table 1: Spiro[azetidine-2, 3’-indole]-2’, 4(1’H)-dione derivatives used in the present investigation

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Mol. Formula /
Entry Compound Code R1 R2 R3 R4
Mol. Wt.
1 7a Br Br CH3 H C17H11Br2ClN2O2470
C17H11Br2ClN2O2
2 7b Br Br H CH3
470
C16H8Br2ClN3O4
3 7c Br Br NO2 H
502
C16H8Br2ClN3O4
4 7d Br Br H NO2
502
C16H9Br2ClN2O2
5 7e Br Br H H
456
C17H13ClN2O2
6 7f H H CH3 H
312
C17H13ClN2O2
7 7g H H H CH3
312
C16H10ClN3O4
8 7h H H NO2 H
344
C16H10ClN3O4
9 7i H H H NO2
344
C16H11ClN2O2
10 7j H H H H
299

Media [1,5,6]
Leibovitz L-15 Medium with L-Glutamine (Biological Industries, Lot No: 928726), FBS (Fetal
Bovine Serum, South American origin) (Quaditive, Lot No: 103128), SFM HEK-293 (Serum
Free Media, Hyclone, Lot no: ARF26635), Thioglycollate medium (TGM) (Himedia, Lot No:
YHI25), Tryptone soya broth (TSB) (Himedia, Lot No: YH031), Cell proliferation kit (MTT)
1000 tests (Biotium, Inc., Cat. No: 30006), Cell proliferation kit (XTT) 1000 tests (Biological
Industries, Lot No: 910395).

Cell lines [1,5,6]

HEK-293T (Human embryonic kidney normal cell line), NCI-H23 (Human Non-Small Cell
Lung cancer cell line), NCI-H522 (Human Non-Small Cell Lung cancer cell line), MDA-MB453
(Human breast adenocarcinoma cell line), and MDA-MB468 (Human breast adenocarcinoma
cell line) were procured from NCCS, Pune.

Microbial and fungal culture

Candida albicans, Bacillus subtilis, Candida sporogenes, Microbial Type Culture Collection
(MTCC), Institute of Microbial Technology, Chandigarh.

Subculture of adherent cell lines (HEK-293T, MDA-MB453 and NCI-H23) [1,5,6]

Cultures were observed using an inverted microscope to assess the degree of confluency and the
absence of bacterial and fungal contaminants was confirmed. Cell monolayer was washed with
PBS without Ca2+/Mg2+ using a volume equivalent to half the volume of culture medium.
Trypsin/EDTA was added on to the washed cell monolayer using 1 ml per 25 cm2 of surface
area.

Flask was rotated to cover monolayer with trypsin. Flask was returned to the incubator and left
for 2-10 mins. The cells were examined using an inverted microscope to ensure that all the cells
were detached and floated. The cells were resuspended in a small volume of fresh serum
containing HEK-293 medium. 100-200 µl was removed to perform a cell count. The required
number of cells were transferred to a new labeled flask containing pre-warmed HEK-293
medium and incubated as appropriate for the cell line.

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Determination of bacteria and fungi in normal and carcinoma cell lines [1,5,6]
Cell line was cultured in the absence of antibiotics at NCCS, Pune. Cell suspension was prepared
by scrapping attached cells with the use of a cell scraper and maintained the pH 7.5-8.0. In 1.5
mL cell suspension, 2 mL thioglycollate medium (TGM) and 2 mL tryptone soya broth (TSB)
were added and inoculated with two different strains; Candida albicans (0.1 mL) Bacillus
subtilis (0.1 mL). Then in 1.5 mL cell suspension, 1 mL TGM was added and inoculated with 0.1
mL Candida sporogenes and 2 mL (TGM), 2 mL (TSB) were left uninoculated as negative
controls. Broths were incubated at 32 ºC. Test and Control broths were examined for turbidity
after 14 days.

Anti-cancer Activity [1,5,6]

XTT assay:
XTT assay was employed to assess cell proliferation. Viable cells were seeded into 96-well
microtitre plates at 5 × 104 cells/well in L-15 media supplemented with FBS (fetal bovine
serum), 100 units/ml penicillin, 100 µg/ml streptomycin and cultured in a humidified atmosphere
of 5 % CO2 at 37 0C. 180 µl of cell suspension was cultured with 20 µl of various concentrations
of synthesized compounds (0.005-100 µg/ml) dissolved in 2 % DMSO solution and Doxorubicin
as standard. Control cells were incubated in culture medium only. Wells containing only media
were considered as a blank. All aliquots dilution doses were tested in duplicates.

Figure 1: Principle of XTT assay

The cell proliferation is based on the ability of the mitochondrial succinate-terazolium reductase
system to convert yellow tetrazolium salt XTT (sodium 3´-[1- (phenylaminocarbonyl)- 3,4-
tetrazolium]-bis (4 methoxy- 6-nitro) benzene sulfonic acid hydrate) to orange formazan dye.
The test denotes the survival cells after toxic exposure. 50 µl of XTT mixture was added to each
well. After 48 hrs incubation at 37 0C temperature and 5 % CO2, the absorbance of soluble
formazan product produced by viable cells was measured at 450 nm using ELISA plate reader
(Thermo, USA). Reference wavelength used was 650 nm.

MTT Assay:
The cells were preincubated at a concentration of 1 × 106 cells/ml in culture medium for 3 hrs at
37 °C and 6.5 % CO2. Then, the cells were seeded at a concentration of 5 × 104 cells/well in 100
µl culture medium and at various concentrations (0.005-100 µg/ml) of standard doxorubicin and
synthesized compounds (dissolved in 2 % DMSO (dimethylsulphoxide) solution) into
microplates (tissue culture grade, 96 wells, flat bottom) and incubated for 24 hrs at 37 °C and 6.5
% CO2. The cell proliferation is based on the ability of the mitochondrial succinate-terazolium
reductase system to convert 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

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(MTT) to a blue colored formazan. The test denotes the survival cells after toxic exposure. Then,
10 µl MTT labelling mixture was added and incubated for 4 hrs at 37 °C and 6.5 % CO2. Each
experiment was done in triplicates. Then 100 µl of solubilization solution was added into each
well and incubated for overnight. The spectrophotometric absorbance of the samples was
measured using a microplate (ELISA) reader. The wavelength to measure absorbance of the
formazan product in between 550 and 600 nm according to the filters available for the ELISA
reader (Thermo, USA) was used. The reference wavelength should be more than 650 nm.

Figure 2: Principle of MTT assay

IC50, the concentration of compound required to inhibit 50 % cell growth, was determined by
plotting a graph of Log (concentration of compound) vs % cell inhibition. A line drawn from 50
% value on the Y axis meets the curve and interpolate to the X axis. The X axis value gives the
Log (concentration of compound). The antilog of that value gives the IC50 value. Percentage
inhibition of novel compounds against all cell lines was calculated using the following formula:

(At − Ab)
% cell survival = ------------ × 100
(Ac − Ab)

Whereas,
At = Absorbance of Test,
Ab= Absorbance of Blank (Media),
Ac= Absorbance of control (cells)

% cell inhibition = 100 − % cell survival

RESULTS AND DISCUSSION

Total bacterial and fungal count

The examination of the test and control broths after 14 days incubation confirmed the absence of
turbidity. Absence of turbidity in the test broth means that there was no evidence of bacterial,
fungal and cross contamination.

Cytotoxicity Assay
The effect of novel compound aliquots (test) and doxorubicin (control) on the growth of MDA-
MB468, MDA-MB453, NCI-H522 and NCI-H23 cell lines were examined by the MTT assay.

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Figure 3: Activity of Compound 7g against various cell lines in MTT assay (48 hrs)

Figure 4: Activity of Compound 7g against various cell lines in XTT assay (48 hrs)

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Figure 5: Activity of Doxorubicin (control) on various cell lines in MTT assay (48 hrs)

Figure 6: Activity of Doxorubicin (control) on various cell lines in XTT assay (48 hrs)

Table 2: IC50 values of Compound 7g and Doxorubicin on various cell lines by MTT and XTT Assay

Lung cancer cell Normal

Breast cancer cell line
line cell line
Comp. No. Assay
R1 R2 R3 R4 MDA- MDA- NCI- NCI- HEK-293T
MB453 MB468 H522 H23
MTT 71.23 76.72 >100 >100 90.21
7g H H H CH3
XTT 71.99 78.82 >100 >100 87.98
Doxorubicin MTT 23.98 20.58 25.92 26.76 70.23
* * * *
(Standard) XTT 22.17 20.18 25.43 26.61 74.09
* NA = Not applicable

Dose response curves constructed between the range 0.005 – 100 µg/ml and 0.005 – 100 µM for
compound aliquots and doxorubicin (control) respectively, express decreasing number of viable

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cells with increasing concentration of compounds aliquots as well as doxorubicin. Calculation of
IC50 value was done using GraphPad Prism Software (Ver. 5.01). The susceptibility of cells to
the compound aliquots and doxorubicin was characterized by IC50 values (Table 2). Results
indicate that the cytotoxic effect linearly and steadily strengthens with increase in the
concentration.

In XTT method, the cells were treated with various compound dilutions followed by staining
with XTT dye and % cell growth inhibition was calculated. Results of XTT assay have been
tabulated in Table 2 and graphically presented in Figure 4. The data from graph revealed that as
the concentration of test compounds increases as the % growth inhibition of cell per well
increasing. IC50 of Compound 7g was 71.99 µg/ml on MDA-MB453 cell line, 78.82 µg/ml on
MDA-MB468 cell line respectively; but no activity found in lung carcinoma cell lines.

In MTT assay, comparable cytotoxicity of Compound 7g was found against breast cancer cell
line with the IC50 of 71.23 µg/ml on MDA-MB453 cell line and 76.72 µg/ml on MDA-MB468
cell line but no activity found in lung carcinoma cell lines. Graphical representation of the MTT
results is shown in Figure 3. However, both of the compounds were found to be devoid of any
activity against HEK 293T (normal) cell line but doxorubicin was found active against same lung
cancer and breast cancer cell lines.

CONCLUSION

A series of new Spiro[azetidine-2, 3’-indole]-2’, 4(1’H)-dione derivatives were screened for anti-
cancer activity at various concentrations (0.005-100 µM/ml) using Doxorubicin as standard by
MTT and XTT assay. Data indicates that among the synthesized compounds, 7g Compound
displayed greater cytotoxicity with comparable IC50. The results described indicate that these
compounds could serve as the basis for the development of a new group of cancer
chemotherapeutics and certainly holds great promise towards good active leads.

Acknowledgement
We wish to thank Dr. L. J. Patel (Dean and Head of Department, Pharmaceutical Chemistry, S.
K. Patel College of Pharmaceutical Education & Research, Ganpat University), Miss Neha R.
Modi and Mr. Ravi J. Shah for providing the synthesized compounds with proper guidance of
QSAR and Dr. N. J. Patel (Principal, S. K. Patel College of Pharmaceutical Education &
Research, Ganpat University) for providing excellent facilities and cooperation for this present
research work.

REFERENCES

[1] P. S. Sukhramani; S. A. Desai; M. P. Suthar. Journal of Pharmacy Research 2011, 4, 1, 124-

127.
[2] G. S. Singh; E. Mbukwa, T Pheko. ARKIVOC 2007, (ix), 80-90.
[3] D. P. Maia; D. V. Wilke; J. Mafezoli; J. N. da Silva; M. O. de Moraes; C. Pessoa; L. V.
Costa-Lotufo. Chemico-Biological Interactions 2009, 180, 2, 220-225.
[4] M. J. Meega; M. Carr; A. J. S. Knox; D. M. Zisterer; D. G. Lloyd. Journal of Enzyme
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[6] R. M. Patel; N. J. Patel. International Journal of Pharmaceutical Research, 2010, 2, 1, 88-
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