Biological Cytotoxicity Evaluation of Sulfonamide Derivatives as Anti-Lung and Anti-Breast Cancer Activity

 

Sarav A. Desai ¹*, Prakash S. Sukhramani², Maulik P. Suthar³ and Vipul P. Patel³

¹Pioneer Pharmacy College, Ajwa Road, Nr. N.H. 8, Sayajipura, Vadodara, Gujarat, India

² Veerayatan Institute of Pharmacy, Bhuj-Mandvi Road, Jakhania, Mandvi, Dist: Kutch – 370460, Gujarat, India

³ Shree S. K. Patel College of Pharmaceutical Education and Research, Ganpat University, Mehsana – Gozaria Highway, Kherva – 382711, Ta and Dist: Mehsana (N.G.), India

*Corresponding Author E-mail: sonu.sukhramani@gmail.com, desaisarav@gmail.com

ABSTRACT:

The purpose of this research work was to evaluate the cytotoxic activity of Sulfonamide derivatives against the panel of lung cancer and breast cancer cell lines, which were MDAMB-453, MCF-7, MDAMB-468, NCI-H23, and NCI-H522 with one normal cell lines, which was HEK-293T (Human embryonic kidney cell line); by comparing the results with the standard drug Doxorubicin. For primary screening, sulfonamide derivatives were screened at 100 µM concentration against all cell lines with the use of XTT based cytotoxicity assay and the compounds which were found to be active as a anti-cancer and those with lower IC₅₀ value below 60 μM were again subjected to MTT assay for confirmation. From the results, Compound No. 1 having substitution with chlorine group showed cytotoxicity against panel of breast cancer cell lines (IC₅₀ = 48.39 µM in MCF-7, IC₅₀ = 36.86 µM in MDA-MB453 and IC₅₀ = 38.52 µM in MDA-MB468) but it is found to be inactive against lung cancer cell lines with no activity in normal cell line.

 

 

 

INTRODUCTION:

Most of the commonly used cytotoxic anticancer drugs were discovered through random high-throughput screening of synthetic compounds and natural products in cell-based cytotoxicity assays. In-vitro cytotoxicity assays have been used 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. Objective of the research work was to evaluate the synthetic Sulfonamide derivatives as anti-cancer against a breast cancer and lung cancer cell lines, which were MDAMB-453, HEK-293T, MCF-7, MDAMB-281, MDAMB-468, NCI-H23, NCI-H522 using a quantitative bioassay MTT and XTT. ¹ With this screening approach, mechanism of action is not a primary determinant in selecting agents for further development, and, as a result, none of the current drug directly targets the molecular lesions responsible for malignant transformation.

 

Specific cells that can grow indefinitely given the appropriate medium and conditions  i.e. living cells that are maintained in-vitro in artificial media of serum and nutrients for the study and growth of certain strains, experiments in controlling diseases, or study of the reaction to certain drugs or agents. ² Human tumor cell line panels combined with rapid high-throughput cytotoxicity testing have proven to be valuable tools for drug screening and early drug evaluation and investigation of drug resistance mechanisms. ¹⁰

 

Sulfonamides are derived from sulfanilamide. These chemicals are structural analogues of p-aminobenzoic acid (PABA). All sulfonamides are characterized by the same chemical nucleus. Functional groups are added to the amino group or substitutions made on the amino group to facilitate varying chemical, physical and pharmacologic properties and antibacterial spectra. Synthesized some novel sulfonamide derivatives and reported to show substantial protease inhibitor properties. Of particular interest are some metalloproteases inhibitors belonging to this class, which by inhibiting several matrix metalloproteases (MMPs) show interesting anti tumour properties. Some of these compounds are currently being evaluated in clinical trials. ³ E7070, novel sulfonamide antitumor agent exhibited potent anti-tumor activity in-vitro and in-vivo. This compound affects cell cycle progression in human tumour cells. ⁴

Cancer is a class of diseases in which a group of cells display the traits of uncontrolled growth (growth and 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 tumors, which are self-limited, do not invade or metastasize. Most cancers form a tumor but some, like leukemia, do not. ^{5, 6}

 

Worldwide, breast cancer is the second most common type of cancer after lung cancer and the fifth most common cause of cancer death. The most common pathologic types of breast cancer are invasive ductal carcinoma (malignant cancer in the breast's ducts) and invasive lobular carcinoma (malignant cancer in the breast's lobules). No etiology is known for 95% of breast cancer cases, while approximately 5% of new breast cancers are attributable to hereditary syndromes. BRCA1 (breast cancer Type 1, early onset) and BRCA2 (Breast Cancer Type 2 are human genes that belongs to a class of genes known as tumor suppressors, which maintains genomic integrity to prevent uncontrolled proliferation. Variations in these genes have been implicated in a number of hereditary cancers, namely breast, ovarian and prostate. ^{7, 8}

 

Lung cancer is a disease of uncontrolled cell growth in tissues of the lung. This growth may lead to metastasis, which is the invasion of adjacent tissue and infiltration beyond the lungs. The main types of lung cancer are small cell lung carcinoma and non-small cell lung carcinoma. This distinction is important, because the treatment varies; non-small cell lung carcinoma (NSCLC) is sometimes treated with surgery, while small cell lung carcinoma (SCLC) usually responds better to chemotherapy and radiation. ⁹

 

Doxorubicin is a drug used in cancer chemotherapy. It is an anthracycline antibiotic, closely related to the natural product daunomycin. It is commonly used in the treatment of a wide range of cancers, including haematological malignancies, many types of carcinomas, and soft tissue sarcomas. Due to low cost and relative efficacy against many common bacterial infections, sulfonamides and sulfonamide combinations with diaminopyrimidines are some of the most common antibacterial agents utilized in veterinary medicine.

 

MATERIALS AND METHODS:

The following materials were procured for the research work and were maintained at appropriate temperature as recommended.

 

Compounds:

Two Compounds of sulfonamide moiety (Fig 1, Table 1) were procured from Pharmaceutical Chemistry department, S. K. Patel College of Pharmaceutical Education & Research, Ganpat University, N. Gujarat, India under the supervision of Dr. L. J. Patel, Dean and Head of Department, Pharmaceutical Chemistry, S. K. Patel College of Pharmaceutical Education & Research, Ganpat University. These procured synthesized compounds were screened for anti-lung and anti-breast cancer activity.

 

Fig 1: Basic Sulfonamide moiety used in the present research study

 

Table 1: List of Compounds with substitutions at R on basic sulfonamide moiety

Compound No.	R
Compound 1	-Cl
Compound 2	-NO2

 

Media:

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:

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), MCF-7 (Human breast adenocarcinoma cell line), MDA-MB453 (Human breast adenocarcinoma cell line), and MDA-MB468 (Human breast adenocarcinoma cell line) were procured from NCCS, Pune and morphological data are listed in Table 2.

 

Table 2: List of Cell Lines (Normal, Lung and Breast carcinoma cell line) Used In Experiments with their morphological characterization

Cell line	Morphology	Origin	Species
HEK 293T	Epithelial	Kidney	Human
NCI – H23	Epithelial	Lung	Human
NCI – H522	Epithelial	Lung	Human
MDA-MB453	Epithelial	Breast	Human
MDA-MB468	Epithelial	Breast	Human
MCF-7	Epithelial	Breast	Human

 

Methods:

Characterization includes the checking of microbial growth contamination, Viability, and PDT (population doubling time). Microbial contamination was checked by using Fluid thioglycolate media (TGM) and Tryptone Soya broth (TSB) and direct observation using Grams stain. Contamination by bacteria, yeast or fungi was detected by an increase in turbidity of the medium and/or a decrease in pH (yellow in media containing phenol red as a pH indicator). It takes advantage of the ability of healthy cells with uncompromised cytoplasmic membrane integrity to exclude dyes such as Trypan blue. ¹¹ Population doubling time is the time expressed in hours, taken for cell No. to double and is reciprocal of the multiplication rate (1/r) and was calculated by using following equation.

n = 3.32 (logN^H ― logN^I)

Where,

N^H= No. of cells harvested at the end of growth period that is t₂

N^I = No. of cells inoculated at time t₁= 0

And multiplication rate was calculated by using equation.

r = 3.32 (logN^H ― logN^I)/t₂-t₁.

Test substances were stored as stock solutions prepared in 2% DMSO at concentration of 100 µM. The final concentration of DMSO (2%) used does not interfere cell viability. As a reference substance, Doxorubicin was prepared at 100 µM concentration using 2% DMSO and used. MCF-7, HEK – 293T, NCI – H23, NCI – H522, MDA – MB453 and MDA – MB468 cell cultures were used in these experiments were procured from National Centre for Cell Science (NCCS), Pune. Stock cells of these cell lines were cultured in L – 15, supplemented with 10% FBS (fetal bovine serum) (but in case of HEK cell line serum free media HEK^TM was used). Along with media cells were also supplemented with 5 % HBSS, penicillin, streptomycin and Amphotericin – B, in a humidified atmosphere (RH 75%) of 5 % CO₂ at 37 °C until confluence reached. The cells were dissociated with 0.2 % trypsin, 0.02 % EDTA in Phosphate Buffer Saline solution. The stock cultures were grown initially in 25 cm² tissue culture flasks, than in 75 cm² and finally in 150 cm² tissue culture flask and all cytotoxicity experiments were carried out in 96 microtitre well-plates. 2 × 10⁴ cells/well was added in to each well of 96 well-plates. Finally, Calculated for no. of cells required for 100 wells ≈ 96 well,

No. of cells/well × 100

= 2 × 10⁴ × 100

= 2 × 10⁶ cells/plate

Total volume of media for 100 wells

= volume of media/well × 100

= 100 µl × 100

= 10 ml

 

Therefore, we need a total of 2 × 10⁶ cells in 10 ml of medium, then aliquot the required volume of cell suspension in to each wells. Cell lines in exponential growth phase were washed, trypsinized and re-suspended in complete culture media. Cells were seeded at 2 × 10⁴ cells/well in 96 well microtitre plate and incubated for 24 hrs during which a partial monolayer forms. The cells were then exposed to various concentrations of the test compounds (as indicated in plate assignment) and standard doxorubicin. The plates were incubated at 37 °C in a humidified incubator with 5% CO₂ for a period of 24 hrs. Morphological changes of drug treated cells were examined using an inverted microscope at different time intervals and compared with the cells serving as control. At the end of 24 hrs, cellular viability was determined using MTT and XTT assay.

MTT assay:^{10, 11}

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 (MTT) to a blue colored formazan (Fig 2). 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.

 

Fig 2: Reaction Principle of MTT bioassay: Conversion of yellow MTT dye to Purple color formazan

 

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

 

 

Fig 3: Reaction Principle of XTT bioassay: Conversion of yellow tetrazolium salt (XTT) to orange formazan dye

 

XTT ASSAY:^{10, 11}

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.

 

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 (Fig 3). 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.

 

RESULTS:

Characterization of cell lines was performed for detection of microbial and cross contamination (Table 3). Cell lines used in our experiments were free from any kind of microbial or fungal contamination which is essential in order to continue our screening experiments.

 

According to the FDA, IC₅₀ represents the concentration of a drug that is required for 50% inhibition of cells in-vitro. For primary screening, we used a threshold of 50% cell growth inhibition as a cut off for compound toxicity against cell lines. IC₅₀ determined from plot of Dose Response curve between log of compound concentration and percentage growth inhibition. IC₅₀ value has been derived using curve fitting methods with Graph Pad Prism (V.5.01) (Vanicha vichai et al; 2006). IC₅₀ values were calculated using the nonlinear regression program Origin The average of two (duplicates manner) were taken in determination. Graph was plotted by keeping log concentration of drug on X axis and % cell growth inhibition or % cytotoxicity Y axis. IC₅₀ was estimated as a concentration of drug at 50% position on Y axis.

 

Table 3:  Viability, pH, Contamination and PDT data for cell lines used

 

Table 4: IC₅₀ value (in µM) of sulfonamide derivatives by MTT Assay

 

 

 

Fig 4. A. DRC of Compound 1 (MDA-MB453) 

 

Fig 4. B. DRC of Compound 1 (MDAB468)

 

Fig 4. C. DRC of Compound 1 (MCF-7) 

                       

Fig 4. D. DRC of Compound 2 (NCI-H23)

 

Fig 4. E. DRC of Compound 2 (NCI-H522)

 

Fig 5. A. DRC of Compound 1 (MCF-7)                 

 

Fig 5. B. DRC of Compound 1 (MDA-MB453)

 

Fig 5. C. DRC of Compound 1 (MDA-MB468)

 

 

Table 5: IC₅₀ Value (µM) of sulfonamide derivatives against following cell lines by XTT Assay

Sr. no	Comp.  No.	MCF-7	MDAMB453	MDAMB468	NCIH23	NCIH522	HEK293T
1	Comp. 1	51.05	40.01	42.20	>100	>100	>100
2	Comp. 2	>100	>100	>100	56.07	54.09	>100

 

Fig 5. D.  DRC of Compound 2 (NCI-H522)

 

Fig 5. E. DRC of Compound2 (NCI-H23)

 

Table 6: IC₅₀ Value (µM) of doxorubicin against following cell lines by XTT AND MTT Assay

 

 

Fig 6. C. DRC of Doxorubicin (MDA-MB468)

 

Fig 6. D. DRC of Doxorubicin (NCI-H23)   

 

Fig 6. E. DRC of Doxorubicin (MCF-7)

 

Fig 6. F. DRC of Doxorubicin (MDA-MB453)

 

Fig 6. G. DRC of Doxorubicin (MDA-MB468)

 

Fig 6. H. DRC of Doxorubicin (NCI-H23)

 

Fig 6. I. DRC of Doxorubicin (NCI-H522)  

 

Fig 6. J. DRC of Doxorubicin (NCI-H522)

Doxorubicin shown good IC₅₀ value against both cancer cell lines where as in case of HEK 293T, Doxorubicin was found to be inactive. In case of XTT assay, Doxorubicin was again found active against both cancer cell lines while found to be inactive against HEK 293T cell line, which confirm the cytotoxicity and inactiveness of Doxorubicin against cancer cell lines and HEK 293T cell line respectively. Synthetic derivatives under investigation were found to be Cytotoxic against cancer cell line but found inactive against normal cell line HEK 293T. Data from MTT assay reveals that sulfonamide derivatives have significant cytotoxicity against breast cancer cell lines and lung cancer cell lines respectively with IC₅₀ below 100 µM. These compounds were further evaluated by XTT assay against same breast cancer cell line and lung cancer lines and HEK 293T cell line for confirmation of cytotoxicity as well as for comparison purpose between MTT and XTT assay. As the result concluded that, Compound 1 and 2 with positive IC₅₀ results in case of MTT assay and XTT assay particularly against lung and breast carcinoma cell lines reveals good basis for the development of a new group of cancer chemotherapeutics.

 

ACKNOWLEDGEMENT:

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

 

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Received on 08.12.2010        Modified on 30.12.2010

Accepted on 27.01.2011        © AJRC All right reserved