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Anticancer activities and cell death mechanisms of 1H-indole-2,3-dione 3-[N-(4 sulfamoylphenyl) thiosemicarbazone] derivatives.

INTRODUCTION

Cancer is caused by abnormal cell division and growth of certain body cells and the invasion of surrounding tissues (WHO 2018). Even though many influential chemotherapeutic agents have been approved to treat cancer, their uses are limited due to serious side effects and toxicity. Therefore, researchers have been trying to develop effective but also less deleterious new anticancer agents (Sarkar and Li 2006; Remesh 2012).

1H-indole-2,3-dione (isatin) is a versatile moiety and compounds bearing the isatin chemical scaffold demonstrate diverse pharmacological or biological properties (Patel et al. 2006; Zhou et al. 2006; Karali et al. 2007; Pakravan et al. 2013; Liu et al. 2014). Furthermore, various structure activity relationship (SAR) studies have demonstrated that the presence of electron-withdrawing groups (fluor, chlor, trifluoromethoxy, nitro etc.) at the position 5 of the isatin ring scaffold enhances anticancer activity (Hall et al. 2009; Vine et al. 2009; Lv et al. 2011; Gabr et al. 2017). After approval of the 2-indolinone derivatives (unitinib, as a multi-targeted receptor, tyrosine kinase inhibitor for the treatment of renal cell carcinoma and imatinib-resistant gastrointestinal stromal tumor by FDA), 2-indolinone analogs have aroused interest because of their anticancer activities (Gan et al. 2009; Vine et al. 2009; Eldehna et al. 2015).

A series of 1H-indole-2,3-dione 3-thiosemicarbazones obtained from the condensation of substituted isatin with thiosemicarbazides has been reported as active against ovarian carcinoma, cervix carcinoma and uterine sarcomacell lines (Pape et al. 2016; Singh et al. 2017). The inhibitory effects of 1H-indole-2,3-dione 3-thiosemicarbazones bearing a 4-sulfamoyl phenyl moiety were investigated using human carbonic anhydrase (hCA) I, II, IX and XII isoenzymes. The tested compounds displayed selectivity against hCA IX and XII. [K.sub.l]values of the compounds were found to be at low nanomolar levels (Karali et al. 2017).

In the present study, 1H-indole-2,3-dione 3-[N-(4-sulfamoylphenyl)thiosemicarbazone] derivatives were evaluated for their anticancer activities and different cell death mechanisms using cell kinetic parameters including the cell index, mitotic index, labelling index and apoptotic index.

MATERIALS AND METHODS

Cytotoxicity

Cell Culture

The HeLa cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco) supplemented with FBS (Foetal Bovine Serum), streptomycin (100 [micro]g/mL), and penicillin (100 IU/mL; Gibco).

Compound Concentrations

Stock solutions of each of 4a-d were freshly prepared in DMSO. Seven different working solutions of each of 4a-d (5, 10, 20, 40, 80, 100 and 160 [micro]M) were prepared.

Real Time Cell Analyzer (RTCA): Cell Index

For this purpose, the real time cell analyzer (RTCA, xCELLigence, Roche) is used for label-free and real-time monitoring of cell properties. This system is impedance-based technology and uses specially designed microtiter plates containing interdigitated gold microelectrodes to non-invasively monitor the viability of cultured cells using electrical impedance as the measure.

In the experimental process, 100 [micro]L of cell culture medium was added to each well for the impedance background measurement. After adding 6000 cells for each well, the final volume was 200 [micro]L. The E-Plates (16 E-Plate) were incubated at 37[degrees]C with 5% C[O.sub.2] and monitored on the RTCA system at 15-minute time intervals for up to 24 hours without treatment of compounds and following 72 hours with treatment of compounds (Cetin and Topcul 2017).

Mitotic Index

HeLa cells were plated on coverslips and treated with either the control or experimental group for 0-72 h. The cells were then fixed using Carnoy fixative (ethanol:acetic acid, 3:1) and stained using the Feulgen method. The number of cells in the mitotic phases (including the late prophase, metaphase, anaphase and telophase; n) per total cells (3,000-3,500; C) was determined under light microscope. The MI (%) was scorred using the following formula: MI=(n/C)x100 (Topcul et al. 2013).

[.sup.3]H-Thymidine Labelling Index

For [.sup.3]H-thymidine labelling index analysis (which determines cells in the S phase), HeLa cells were seeded into round coverslips which were in 24-well plates at a density of 2x104 cells per well and incubated for 24 hrs. Then the cells were treated with the 4a-d experimental concentrations. At the end of the experimental period, cells were treated with medium containing 1 [micro]Ci/mL [.sup.3]H-thymidine for 20 min to evaluate the labelling index (Cetin and Topcul 2017).

Autoradiography

After exposure for 3 days at 4[degrees]C, autoradiograms were developed with a D-19 developer solution (Kodak, New York, USA) and fixed with Fixaj B (Kodak, New York, USA). The coverslips were evaluated after being stained with Giemsa for 3 min. The labeling index was determined by counting at least 3000 cells/coverslip. The index is expressed as percentage labeled nuclei (Cetin and Topcul 2017).

Apoptotic index (AI)

HeLa cells were collected and then fixed with methanol:Phosphate Buffered Saline (PBS) (1:1) and methanol. Fixed cells mounted on slides, stained with 0.5 mg/mL4',6-diamidino-2-phenylindole (DAPI) for 30 min and washed with PBS. Nuclear morphology of the cells was visualized using an Olympus fluorescence microscope. For evaluation of the AI, at least 250 cells were counted for control and each of the experimental groups (Cetin and Topcul 2017).

RESULTS AND DISCUSSION

Chemistry

(4-Sulfamoylphenyl)isothiocyanate 1 was prepared by reacting of sulfanilamide with thiophosgene in water containing concentrated hydrochloric acid. Hydrazine hydrate was reacted with 1 in ethanol to give N-(4-sulfamoylphenyl)thiosemicarbazide 2 1H-indole-2,3-dione 3-[N-(4-sulfamoylphenyl)thiosemicarbazones] 4a-d were synthesized by reacting 2 with 5-substituted 1H-indole-2,3-dione 3a-d in ethanol containing a catalytic amount of sulphuric acid (Karali et al. 2017) (Scheme 1).

Anticancer Activity

Cell Index

The cell index values obtained from the real-time cell analysis system were examined following the application of 4a-d to HeLa cells. 5, 10, 20, 40, 80, 100 and 160 [micro]M concentrations were used for all compounds. Figures 1-4 show the curves of the most effective concentrations of these compounds.

When the cell index values obtained following application of 4a-d to HeLa cells were compared with the standard curve, it was shown that there were different effects for different concentrations for all compounds. The bromine substituted 4c was tested at 5, 80 and 100 [micro]M concentrations using the control group. 4c showed significant DNA damage on HeLa cells at 80 and 100 [micro]M concentrations. The trifluoromethoxy substituted 4a and the nitro substituted 4d were examined using the control group on HeLa cells. Both compounds had cytostatic effects as a distinct cell death type at 160 [micro]M. The fluorine substituted 4b showed a cytostatic effect with uncertainty at 100[micro]M concentration to HeLa cells.

Mitotic Index

After administration of 4a at 160 [micro]M, 4b at 100 [micro]M, 4c at 80 [micro]M and 4d at 60 [micro]M for 0-72 h on HeLa cells, 3000 cells were counted for both the control and experimental groups. The mitotic index values belonging to 4a-d are shown in Table 1.

The mitotic index is a scale for the proliferation case of a cell population. When the mitotic index values were examined, it was seen that while the bromine substituted 4c had the most antiproliferative activity at 80 [micro]M, the fluorine substituted 4b had no significant effect compared with the other substances. The trifluoromethoxy substituted 4a and the nitro substituted 4d showed effects at 160 [micro]M. The difference was significant between the control and experimental groups (p<0.01). In addition, a statistically significant difference was noted among all experimental groups (p<0.01).

Labelling Index

Labelling index parameters were applied at 80, 100 and 160 [micro]M concentrations on HeLa cells for 4a-d. 3000 cells were counted for both the control and experimental groups. Labelling index values of 4a-d are shown in Table 2.

The effects of 4a-d on the S phase of HeLa cells are similar to those of mitotic index values. 4c saw the most greatly reduced DNA synthesis of HeLa cells among 4a-d. The bromine substituted 4c produced significant inhibition at 80 [micro]M, while the trifluoromethoxy substituted 4a and the nitro substituted 4d showed an inhibitory effect at 160 [micro]M for DNA synthesis. The difference was significant between the control and experimental groups (p<0.01). In addition, a statistically significant difference was noted among all experimental groups (p<0.01).

Apoptotic Index

In the apoptotic index parameter, 80, 100, and 160 [micro]M concentrations were applied to 4a-d respectively. 250 cells were counted for both the control and experimental groups. Apoptotic index values of 4a-d are shown in Table 3.

As in other parameters, the apoptotic index values of the bromine substituted 4c were shown to significantly increase apoptotic cell ratio at 80 [micro]M. The trifluoromethoxy substituted 4a and the nitro substituted 4d were found to increase the apoptotic index values at 160 [micro]M. The fluorine substituted 4b had no significant apoptotic effect compared with the other substances. The difference was significant between the control and experimental groups (p<0.01). In addition, a statistically significant difference was noted among all experimental groups (p<0.01).

Peer-review: Externally peer-reviewed.

Author Contributions: Concept--I.C., N.K.; Design--I.C., P.E.E., M.T., N.K.; Supervision--M.T., N.K.; Resource--M.T., N.K.; Materials--I.C., M.T., N.K.; Data Collection and/or Processing--I.C., M. T., N.K.; Analysis and/or Interpretation--I.C., M.T., N.K.; Literature Search--I.C., P.E.E.; Writing--I. C., P.E.E., N.K.; Critical Reviews--N.K., M.T.

Conflict of Interest: The authors have no conflict of interest to declare.

Financial Disclosure: This work was supported by Istanbul University Scientific Research Projects (Project Number: 4074).

REFERENCES

* (2018) World Health Organization, http://www.who.int/media-centre/factsheets/fs297/en/. Accessed 01.02.2018.

* Cetin I, Topcul MR (2017). In vitro antiproliferative effects of nabpaclitaxel with liposomal cisplatin on MDA-MB-231 and MCF-7 breast cancer cell lines. J BUON 22: 347-354.

* Eldehna WM, Altoukhy A, Mahrous H, Abdel-Aziz HA (2015). Design, synthesis and QSAR study of certain isatin-pyridine hybrids as potential anti-proliferative agents. Eur J Med Chem 90: 684-694. [CrossRef]

* Gabr MT, El-Gohary NS, El-Bendary ER, El-Kerdawy MM, Ni N (2017). Isatin-[beta]-thiocarbohydrazones: Microwave-assisted synthesis, antitumor activity and structure-activity relationship. Eur J Med Chem 128: 36-44. [CrossRef]

* Gan HK, Seruga B, Knox JJ (2009). Sunitinib in solid tumors. Expert Opin Investig Drugs 18: 821-834. [CrossRef]

* Hall MD, Salam NK, Hellawell JL, Fales HM, Kensler CB, Ludwig JA, Szakacs G, Hibbs DE, Gottesman MM (2009). Synthesis, activity, and pharmacophore development for isatin-[beta]-thiosemicarbazones with selective activity toward multidrug-resistant cells. J Med Chem 52: 3191-3204. [CrossRef]

* Karali N, Gursoy A, Kandemirli F, Shvets N, Kaynak FB, Ozbey S, Kovalishyn V, Dimoglo A (2007). Synthesis and structure antituberculosis activity relationship of 1H-indole-2,3-dione derivatives. Bioorg Med Chem 15: 5888-5904. [CrossRef]

* Karali N, Akdemir A, Goktas F, Eraslan Elma P Angeli A, Kizilirmak M, Supuran CT (2017). Novel sulfonamide-containing 2-indolinones that selectively inhibit tumor-associated alpha carbonic anhydrases. Bioorg Med Chem 25: 3714-3718. [CrossRef]

* Liu W, Zhu HM, Niu GJ, Shi, EZ, Chen J, Sun B, Chen WQ, Zhou HG, Yang C (2014). Synthesis, modification and docking studies of 5-sulfonylisatin derivatives as SARS-CoV 3C-like protease inhibitors. Bioorg Med Chem 22: 292-302. [CrossRef]

* Lv K, Wang LL, Liu ML, Zhou XB, Fan SY, Liu HY, Zheng ZB, Li S (2011). Synthesis and antitumor activity of 5-[1-(3-(dimethylami-no)propyl)-5-halogenated-2-oxoindolin-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxamides. Bioorg Med Chem Lett 21: 3062-3065. [CrossRef]

* Pakravan P Kashanian S, Khodaei MM, Harding FJ (2013). Biochemical and pharmacological characterization of isatin and its derivatives: from structure to activity. Pharmacol Rep 65: 313-335. [CrossRef]

* Pape VFS, Toth S, Furedi A, Szebenyi K, Lovrics A, Szabo P, Wiese M, Szakacs G (2016). Design, synthesis and biological evaluation of thiosemicarbazones, hydrazinobenzothiazoles and arylhydrazones as anticancer agents with a potential to overcome multidrug resistance. Eur J Med Chem 117: 335-354. [CrossRef]

* Patel A, Bari S, Talele G, Patel J, Sarangapani M (2006). Synthesis and antimicrobial activity of some new isatin derivatives. Iran J Pharm Res 4: 249-254.

* Remesh A (2012). Toxicities of anticancer drugs and its management. Int J Basic Clin Pharmacol 1: 2-12. [CrossRef]

* Sarkar FH, Li Y (2006). Using chemopreventive agents to enhance the efficacy of cancer therapy. Cancer Res 66: 3347-3350. [CrossRef]

* Singh A, Raghuwanshi K, Patel VK, Jain DK, Veerasamy R, Dixit A, Rajak H (2017). Assessment of 5-substituted isatin as surface recognition group: Design, synthesis, and antiproliferative evaluation of hydroxamates as novel histone deacetylase inhibitors. Pharm. Chem. J 51: 366-374. [CrossRef]

* Topcul MR, Cetin I, Kolusayin Ozar MO (2013). The effects of anastrozole on the proliferation of Fm3a cells. J BUON 18: 874-878.

* Vine KL, Matesic L, Locke JM, Ranson M, Skropeta D (2009). Cytotoxic and Anticancer Activities of Isatin and Its Derivatives: A Comprehensive Review from 2000-2008. Anticancer Agents Med Chem 9: 397-414. [CrossRef]

* Zhou L, Liu Y, Zhang W, Wei P Huang C, Pei J, Yuan Y, Lai L (2006). Isatin compounds as noncovalent SARS coronavirus 3C-like protease inhibitors. J Med Chem 49: 3440-3443. [CrossRef]

Idil Cetin (1)[iD], Pinar Eraslan Elma (2)[iD], Mehmet Topcul (1)[iD], Nilgun Karali (2*)[iD]

(1) Department of Biology, Faculty of Science, Istanbul University, 34116, Istanbul, Turkey

(2) Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Istanbul University, 34116, Istanbul, Turkey

Cite this article as: Cetin I, Eraslan Elma P, Topcul M, Karali N (2018). Anticancer activities and cell death mechanisms of 1H-indole-2,3-dione 3-[N-(4 sulfamoylphenyl)thiosemicarbazone] derivatives. Istanbul J Pharm 48 (3): 63-67.

Address for Correspondence :

Nilgun Karali, e-mail: karalin@istanbul.edu.tr

Received: 12.04.2018

Accepted: 04.10.2018

DOI: 10.26650/IstanbulJPharm.2018.414805
Table 1. Mitotic index (%) values of 4a-d on HeLa cells

                                     Mitotic Index (%)
                                     4a                4b
Time (Hours)  Control                160 [micro]M (*)  100 [micro]M (*)

24            6.24[+ or -]0.11 (SD)  3.56[+ or -]0.03  4.15[+ or -]0.07
48            8.27[+ or -]0.07       3.18[+ or -]0.01  4.72[+ or -]0.05
72            8.96[+ or -]0.05       2.96[+ or -]0.03  5.14[+ or -]0.02

              Mitotic Index (%)
              4c                4d
Time (Hours)  80 [micro]M (*)   160 [micro]M (*)

24            2.94[+ or -]0.04  5.18[+ or -]0.06
48            1.18[+ or -]0.01  4.15[+ or -]0.04
72            0.38[+ or -]0.02  4.13[+ or -]0.05

(*) Significantly different p<0.01
(SD): Standard deviation

Table 2. Labelling index (%) values of 4a-d on HeLa cells

                                     Labelling Index (%)
                                     4a                4b

Time (Hours)  Control                160 [micro]M (*)  100 [micro]M (*)
24            5.13[+ or -]0.06 (SD)  2.24[+ or -]0.02  3.21[+ or -]0.03
48            6.17[+ or -]0.03       2.11[+ or -]0.03  3.12[+ or -]0.02
72            6.96[+ or -]0.07       1.03[+ or -]0.01  4.14[+ or -]0.03

              Labelling Index (%)
              4c                4d

Time (Hours)  80 [micro]M (*)   160 [micro]M (*)
24            1.97[+ or -]0.03  3.95[+ or -]0.04
48            1.16[+ or -]0.01  3.17[+ or -]0.04
72            0.21[+ or -]0.02  2.96[+ or -]0.02

(*) Significantly different p<0.01
(SD): Standard deviation

Table 3. Apoptotic index (%) values of 4a-d on HeLa cells

                                     Apoptotic Index (%)
                                     4a
Time (Hours)  Control                160 [micro]M (*)

24            6.17[+ or -]0.06 (SD)  12.15[+ or -]0.02
48            7.21[+ or -]0.05       12.19[+ or -]0.03
72            7.96 [+ or -]0.09      14.33[+ or -]0.01

              Apoptotic Index (%)
              4b                 4c                 4d
Time (Hours)  100 [micro]M (*)   80 [micro]M (*)    160 [micro]M (*)

24             8.16[+ or -]0.03  18.19[+ or -]0.11  10.23[+ or -]0.06
48             9.18[+ or -]0.04  19.01[+ or -]0.08  14.06[+ or -]0.03
72            10.12[+ or -]0.03  22.15[+ or -]0.03  15.04[+ or -]0.07

(*) Significantly different p<0.01
(SD): Standard deviation
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Title Annotation:Original Article
Author:Cetin, Idil; Elma, Pinar Eraslan; Topcul, Mehmet; Karali, Nilgun
Publication:Journal of the Faculty of Pharmacy of Istanbul University
Article Type:Report
Date:Dec 1, 2018
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