International Journal of Nutrition, Pharmacology, Neurological Diseases

ORIGINAL ARTICLE
Year
: 2022  |  Volume : 12  |  Issue : 2  |  Page : 72--78

Antiproliferative Effects of Olanzapine against MCF-7 Cells and Its Molecular Interactions with Survivin


V. A. Varahi Vedam1, Poojith Nuthalapati2, Mohan Krishna Ghanta3, Darling Chellathai David4, M. Vijayalakshmi5, Krishna Murthy Potla6, Y. Sheena Mary7,  
1 Department of Pharmacology, ESIC Medical College & PGIMSR, KK Nagar, Chennai, Tamil Nadu, India
2 PJ Biousys, Irving, TX, USA
3 Department of Pharmacology, MVJ Medical College and Research Hospital, Hoskote, Bangalore, Karnataka, India
4 Department of Pharmacology, SRMC & RI, Sri Ramachandra Institute of Higher Education and Research Institute, Porur, Chennai, Tamil Nadu, India
5 Department of Biotechnology, Dr MGR Educational and Research Institute, Maduravoyal, Chennai, Tamil Nadu, India
6 Department of Chemistry, Bapatla Engineering College (Autonomous), Acharya Nagarjuna University Post Graduate Research Centre, Bapatla, Andhra Pradesh, India
7 Department of Physics, University of Kerala, Thushara, Neethinagar, Kollam, Kerala, India

Correspondence Address:
Mohan Krishna Ghanta
MVJ Medical College and Research Hospital, Hoskote, Bangalore, Karnataka
India

Abstract

Background: Epidemiologic findings revealed approximately one-third of patients with breast cancer develop brain metastases. Recent research has found that schizophrenia patients who take antipsychotic medications on a long-term basis have a decreased risk of cancers than normal individuals. This serendipitous anticancer action of antipsychotic medications is now being investigated by many studies. The ability of these drugs to penetrate the blood–brain barrier may target brain metastases. We investigated antiproliferative activity of antipsychotic drug. The present study aimed to determine the antiproliferative effects of olanzapine against MCF-7 cells and also to examine its molecular interactions with survivin. Methods: The antiproliferative effects of olanzapine were demonstrated using MTT assay and molecular interactions were analyzed using AutoDock Vina ver4.0 between olanzapine (PubChem CID − 135398745) and survivin (PDB ID − 1E31). These molecular interactions were also compared with tamoxifen (PubChem CID: 2733526). Results: We found that olanzapine has extensive antiproliferative effects against MCF-7 human breast cancer cells, with an IC50 of 10.9 g/mL. We also discovered that olanzapine had possible interactions with the survivin protein at Lys15, Phe86, and Val89 amino acid residues, which could be related to effects of olanzapine on MCF-7 cell viability. Conclusion: Our research establishes that olanzapine has promising anticancer properties against breast tumors, with prospective application to target brain metastases in patients with breast cancer.



How to cite this article:
Varahi Vedam V, Nuthalapati P, Ghanta MK, David D, Vijayalakshmi M, Potla K, Mary Y. Antiproliferative Effects of Olanzapine against MCF-7 Cells and Its Molecular Interactions with Survivin.Int J Nutr Pharmacol Neurol Dis 2022;12:72-78


How to cite this URL:
Varahi Vedam V, Nuthalapati P, Ghanta MK, David D, Vijayalakshmi M, Potla K, Mary Y. Antiproliferative Effects of Olanzapine against MCF-7 Cells and Its Molecular Interactions with Survivin. Int J Nutr Pharmacol Neurol Dis [serial online] 2022 [cited 2022 May 26 ];12:72-78
Available from: https://www.ijnpnd.com/text.asp?2022/12/2/72/345019


Full Text



 Introduction



Breast cancer is one of the specific cancers that have a high incidence of metastasis.[1] Higher incidences of brain metastasis in patients with breast cancer were diagnosed in recent years.[2,3] In this scenario, there is more need for anticancer drugs that can reach the central nervous system.[1,4,5] Currently, antipsychotic drugs are being investigated for their anticancer potential against breast cancer, brain metastasis, and other cancer types as well. The pathways targeted by these drugs include inhibition of histone deacetylases, deubiquitination complex, and phosphoinositide 3-kinase pathway. These properties of antipsychotics make them suitable candidates for drug repurposing. The drug repurposing of antipsychotic drugs for treating breast cancer may provide new effective cancer therapeutics with a shorter time frame of the drug discovery process.[6],[7],[8],[9],[10],[11],[12],[13],[14],[15],[16],[17],[18],[19],[20],[21] Olanzapine, as an antipsychotic drug, has proven antagonistic actions on dopamine D2 receptors (DRD2) and serotonin receptors (5-HT2A).[21] Downregulation of DRD2 and 5-HT2A receptor activities were found beneficial in cancer chemotherapeutics. The DRD2 antagonism modulates the cyclic adenosine monophosphate (cAMP) pathway, epidermal growth factor receptor signaling, Wnt signaling, and calcium signaling pathways.[22] The 5-HT2A receptors modulate the beta-catenin pathway, cAMP pathway, and angiogenesis in tumor cells.[23],[24],[25] All these pathways influenced by DRD2 and 5-HT2A receptors affect survivin expression.[26],[27],[28],[29],[30]

Olanzapine treatment against lung, pancreatic cancer stem cells, and ovarian cancer cells made them more sensitive to anticancer drugs through survivin inhibition. Survivin is an antiapoptotic molecule and mitosis inducer as well. The accumulation of mitosis in a cell cycle was positively correlated to upregulated survivin expression and its inhibition could be a possible target for the treatment of breast cancer, lung cancer, and ovarian carcinoma.[31],[32],[33] Therefore, the aim of this study is to assess the in vitro antiproliferative property of olanzapine against MCF-7 cells, and also to predict the molecular interaction of olanzapine with survivin using the in silico molecular docking method.

 Materials and Methods



Chemicals and reagents

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT), cell culture reagents, and chemicals were obtained from Himedia Pvt Ltd (Mumbai, Maharashtra, India) and Tokyo Chemical Industry (Chennai, Tamil Nadu, India).

Cell culture

MCF-7 cells (NCCS, Pune, India) were cultured in minimum essential medium (MEM) containing 10% fetal bovine serum, 5% CO2, and incubated at 37°C with appropriate humidity. After observation for healthy cultured cells, the culture flask is split, then the media is discarded and the cells were subjected to 2× wash with MEM. Following this, the cells were incubated in trypsin phosphate versene glucose (TPVG) phosphate buffer saline for 1 minute, later TPVG was discarded, and the 10% MEM was added. The cells were passaged and shifted to 96 well-plates.

MTT assay

The MCF-7 cells were seeded in 96-well plates and incubated for 24 hours before being treated with olanzapine at various concentrations. The media was replaced every 2 days, and the cells were then washed and exposed to MTT salts using a standard procedure on the third day (72 hours).[34] The absorbance at 540 nm was used to determine the viability of the cells. The cell viability–concentration plot yielded the 50% cell viability (IC50) value. The % cell viability is given by[35]

[INLINE:1]

Molecular docking

To find candidate drugs for survivin inhibition property, we have carried out a computational study to screen for effective drugs, endoxifen (PubChem CID:10090750), tamoxifen (PubChem CID: 2733526), and olanzapine (PubChem CID: 135398745) which may work as an inhibitor for apoptosis.[36] The structure of survivin (PDB ID: 1E31) was obtained from the Protein Data Bank (PDB) database, and molecular docking was performed with AutoDock Vina ver4.0 and as in literature.[37],[38],[39]

 Results



Olanzapine inhibits MCF-7 cell proliferation

The antiproliferative property of olanzapine was screened using MTT assay. The percentage cell viability–concentration graph trendline showed a dose-dependent steep fall in MCF-7 cell viability with an increase in the dose of olanzapine. The IC50 concentration of olanzapine in breast cancer cell lines was 10.9 μg/mL, indicating sensitivity to the drug [Figure 1] and [Figure 2]].{Figure 1} {Figure 2}

Molecular interactions of ligands with survivin

In silico screening was performed to evaluate the inhibition of survivin by olanzapine. Tamoxifen and its metabolite endoxifen were also selected for comparison. The binding energy of the three compounds at different root-mean-square deviations (RMSD) is depicted in [Figure 3]. In this study, RMSD was set to 0 Å.{Figure 3}

Interactions of endoxifen with 1E31

At the ranges of 2.42, 2.66, and 2.78 Å, the amino acid ASP16 forms two salt-bridge H-bonds with the same NH2 group and a carbon–hydrogen (C–H) bond with the methylene group. GLN92, PHE93 show ‐-donor H-bond with RingI, RingIII centers at the distances of 4.18 Å and 3.77 Å, whereas GLU40 has an electrostatic ‐-anion interaction with RingI at the distance 3.57 Å. VAL89, PHE93, formulate hydrophobic ‐-sigma, hydrophobic ‐-‐ T-shaped, hydrophobic ‐-alkyl interactions, respectively, with RingII having the distances 3.83, 5.63, and 5.26 Å. The RingI and RingIII exist at distances of 4.90 and 5.13 Å with LYS15, exhibiting two hydrophobic-‐-alkyl interactions. At a distance of 4.32 Å, PHE86 exhibits a hydrophobic--alkyl linking to the methyl group [[Figure 4]].{Figue 4}

Interactions of tamoxifen with 1E31

At ranges of 2.18 and 4.90 Å, the amino acid VAL89 forms a typical H-bond with the NH group and hydrophobic- -alkyl linkages with the RingIII center. At 2.86 Å, PHE86 forms C–H linkages with the methyl ester, whereas Glu40 exhibits an electrostatic- -anion linking with the RingIII center at 4.03 Å. Lys15 formulates hydrophobic- -sigma interaction with RingI and hydrophobic-alkyl linkages to methyl esters at 3.66 and 5.28 Å. PHE93 exhibited t-shaped hydrophobic- - linkages with RingII at 5.76 Å [[Figure 4]].

Interactions of olanzapine with 1E31

The amino acids LYS15 and VAL89 forming hydrophobic alkyl interactions with RingIII and RingI center, respectively, are at the distances of 4.12 and 4.73 Å. Phe86 shows hydrophobic -alkyl interactions with RingI center at the distance of 4.64 Å [[Figure 4]].

 Discussion



Our study establishes the in vitro antiproliferative effects of olanzapine against breast cancer MCF-7 cells, which may be beneficial as an adjuvant in the treatment of brain metastasis. Olanzapine exhibited dose-dependent cytotoxicity in MTT assay. The apoptosis induced through mitochondrial complex I pathway[40] is well studied using MTT assay by previous studies[41] and relates antiproliferative actions of olanzapine in MCF-7 cells.

Molecular interactions of olanzapine against survivin were also demonstrated in our study. Survivin is a dimeric protein and its dimerization interface was the target site for inhibition of its activity. The dimerization sites of survivin constitute 6–10, 93–99, 101, and 102 amino acid residues. Another important site of survivin for regulating its activity is the linker segment of monomer which comprises valine 89 to threonine 97 codon. Tamoxifen and its metabolite endoxifen interacted with both dimerization interface and linker segment codons, whereas olanzapine interacted only with linker segment codons. Thus, these interactions signify the inhibition of survivin by the three compounds of the study.

The binding efficacy of olanzapine to survivin was comparable to endoxifen, a metabolite of tamoxifen, and approximate to tamoxifen. Olanzapine exhibited good binding affinities with Lys15, Val89, and Phe86. Similar interactions were also observed with tamoxifen and its metabolite endoxifen. Other ligands that were evaluated as potential survivin inhibitors showed molecular interactions with the same amino acid residues. With survivin protein, ZINC00689728 had a −9.3 kcal/mol binding energy and hydrophobic interactions with Phe93, Val89, and Phe86 residues.[42] The S12 enantiomer, which was anticipated as the dynamic isomer inhibiting survivin, showed interactions with amino acids Lys15, Phe86, Phe93, and Val89.[43] Similar interactions were found with LQZ-7, which triggered spontaneous apoptosis against several cancer cells without affecting the synthesis of survivin.[43] LQZ-7-induced survivin degradation was based on the concept that exposing a dimeric protein’s hydrophobic interface causes conformational changes[44,45] which promote protein instability and breakdown by autophagy or proteasome.[46] Andrographolide interacting with Thr34 and Glu36 of human survivin in silico was shown to possess in vitro cytotoxicity activity with cytotoxicity concentration-50 of 0.32 mM against breast tumor cells.[47]

 Conclusion



Thus, we demonstrated the anticancer property of olanzapine with its in silico molecular interactions against survivin and enhanced cytotoxicity to MCF-7 cells in vitro. We suggest that the binding of olanzapine to survivin at Phe86, Val89, and Lys15 amino acid residues may be the critical part of the effects of olanzapine. Therefore, olanzapine may be a potential adjuvant anticancer therapy in breast cancer which could be beneficial for targeting brain metastasis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1Eichler AF, Chung E, Kodack DP, Loeffler JS, Fukumura D, Jain RK. The biology of brain metastases-translation to new therapies. Nat Rev Clin Oncol 2011;8:344-56.
2Fontanella C, De Carlo E, Cinausero M, Pelizzari G, Venuti I, Puglisi F. Central nervous system involvement in breast cancer patients: is the therapeutic landscape changing too slowly? Cancer Treat Rev 2016;46:80-8.
3Hardesty DA, Nakaji P. The current and future treatment of brain metastases. Front Surg 2016;3:30.
4Weidle UH, Niewöhner J, Tiefenthaler G. The blood-brain barrier challenge for the treatment of brain cancer, secondary brain metastases, and neurological diseases. Cancer Genomics Proteomics 2015;12:167-77.
5Lin J, Jandial R, Nesbit A, Badie B, Chen M. Current and emerging treatments for brain metastases. Oncology (Williston Park) 2015;29:250-7.
6Lee J-K., Nam D-H., Lee J. Repurposing antipsychotics as glioblastoma therapeutics: potentials and challenges. Oncol Lett 2016;11:1281-6.
7Huang L, Zhao S, Frasor JM, Dai Y. An integrated bioinformatics approach identifies elevated cyclin E2 expression and E2F activity as distinct features of tamoxifen resistant breast tumors. PLoS One 2011;6:e22274.
8Ahern TP, Pedersen L, Cronin-Fenton DP, Sørensen HT, Lash TL. No increase in breast cancer recurrence with concurrent use of tamoxifen and some CYP2D6-inhibiting medications. Cancer Epidemiol Biomarkers Prev 2009;18:2562-4.
9Cheng HW, Liang YH, Kuo YLet al. Identification of thioridazine, an antipsychotic drug, as an antiglioblastoma and anticancer stem cell agent using public gene expression data. Cell Death Dis 2015;6:e1753.
10Ranjan A, Gupta P, Srivastava SK. Penfluridol: an antipsychotic agent suppresses metastatic tumor growth in triple-negative breast cancer by inhibiting integrin signaling axis. Cancer Res 2016;76:877-90.
11Yeh CT, Wu ATH, Chang PMHet al. Trifluoperazine, an antipsychotic agent, inhibits cancer stem cell growth and overcomes drug resistance of lung cancer. Am J Respir Crit Care Med 2012;186:1180-8.
12Wiklund ED, Catts VS, Catts SVet al. Cytotoxic effects of antipsychotic drugs implicate cholesterol homeostasis as a novel chemotherapeutic target. Int J Cancer 2010;126:28-40.
13Yin YC, Lin CC, Chen TTet al. Clozapine induces autophagic cell death in non-small cell lung cancer cells. Cell Physiol Biochem 2015;35:945-56.
14Suzuki S, Okada M, Kuramoto Ket al. Aripiprazole, an antipsychotic and partial dopamine agonist, inhibits cancer stem cells and reverses chemoresistance. Anticancer Res 2016;36:5153-61.
15Sanomachi T, Suzuki S, Kuramoto Ket al. Olanzapine, an atypical antipsychotic, inhibits survivin expression and sensitizes cancer cells to chemotherapeutic agents. Anticancer Res 2017;37:6177-88.
16Dilly SJ, Clark AJ, Marsh Aet al. A chemical genomics approach to drug reprofiling in oncology: antipsychotic drug risperidone as a potential adenocarcinoma treatment. Cancer Lett 2017;393:16-21.
17Huang X, He Y, Dubuc AMet al. EAG2 potassium channel with evolutionarily conserved function as a brain tumor target. Nat Neurosci 2015;18:1236-46.
18Azuine MA, Tokuda H, Takayasu Jet al. Cancer chemopreventive effect of phenothiazines and related tri-heterocyclic analogues in the 12-O-tetradecanoylphorbol-13-acetate promoted Epstein-Barr virus early antigen activation and the mouse skin two-stage carcinogenesis models. Pharmacol Res 2004;49:161-9.
19Naftalovich S, Yefenof E, Eilam Y. Antitumor effects of ketoconazole and trifluoperazine in murine T-cell lymphomas. Cancer Chemother Pharmacol 1991;28:384-90.
20Hait WN, Morris S, Lazo JSet al. Phase I trial of combined therapy with bleomycin and the calmodulin antagonist, trifluoperazine. Cancer Chemother Pharmacol 1989;23:358-62.
21Thomas K, Saadabadi A. Olanzapine. [Updated 2021 Aug 19]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022. Available from: https://www.ncbi.nlm.nih.gov/books/NBK532903/. [Accessed on 17 January 2022].
22Weissenrieder JS, Neighbors JD, Mailman RB, Hohl RJ. Cancer and the dopamine D 2 receptor: a pharmacological perspective. J Pharmacol Exp Ther 2019;370:111-26.
23Kline CLB, Ralff MD, Lulla ARet al. Role of dopamine receptors in the anticancer activity of ONC201. Neoplasia 2018;20:80-91.
24Sonier B, Arseneault M, Lavigne C, Ouellette RJ, Vaillancourt C. The 5-HT2A serotoninergic receptor is expressed in the MCF-7 human breast cancer cell line and reveals a mitogenic effect of serotonin. Biochem Biophys Res Commun 2006;343:1053-9.
25Ballou Y, Rivas A, Belmont Aet al. 5-HT serotonin receptors modulate mitogenic signaling and impact tumor cell viability. Mol Clin Oncol 2018;9:243-54.
26Zhao P, Meng Q, Liu LZ, You YP, Liu N, Jiang BH. Regulation of survivin by PI3K/Akt/p70S6K1 pathway. Biochem Biophys Res Commun 2010;395:219-24.
27Zhang L, Yan R, Zhang Qet al. Survivin, a key component of the Wnt/β -catenin signaling pathway, contributes to traumatic brain injury-induced adult neurogenesis in the mouse dentate gyrus. Int J Mol Med 2013;32:867-75.
28Wang Q, Greene MI. EGFR enhances survivin expression through the phosphoinositide 3 (PI-3) kinase signaling pathway. Exp Mol Pathol 2005;79:100-7.
29Cheung CHA, Huang CC, Tsai FYet al. Survivin − biology and potential as a therapeutic target in oncology. Onco Targets Ther 2013;6:1453-62.
30Ortiz J, Chou LL. Calcium upregulated survivin expression and associated osteogenesis of normal human osteoblasts. J Biomed Mater Res A 2012;100:1770-6.
31Ozbay T, Durden DL, Liu T, O’Regan RM, Nahta R. In vitro evaluation of pan-PI3-kinase inhibitor SF1126 in trastuzumab-sensitive and trastuzumab-resistant HER2-over-expressing breast cancer cells. Cancer Chemother Pharmacol 2010;65:697-706.
32Shimizu T, Nishio K, Sakai Ket al. Phase I safety and pharmacokinetic study of YM155, a potent selective survivin inhibitor, in combination with erlotinib in patients with EGFR TKI refractory advanced non-small cell lung cancer. Cancer Chemother Pharmacol 2020;86:211-9.
33Liu YK, Jia YJ, Liu SH, Ma J. FSTL1 increases cisplatin sensitivity in epithelial ovarian cancer cells by inhibition of NF-κB pathway. Cancer Chemother Pharmacol 2021;87:405-14.
34Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55-63.
35Kamiloglu S, Sari G, Ozdal T, Capanoglu E. Guidelines for cell viability assays. Food Front 2020;1:332-49.
36Chantalat L, Skoufias DA, Kleman JP, Jung B, Dideberg O, Margolis RL. Crystal structure of human survivin reveals a bow tie-shaped dimer with two unusual alpha-helical extensions. Mol Cell 2000;6:183-9.
37Aswathy VV, Alper-Hayta S, Yalcin Get al. Modification of benzoxazole derivative by bromine-spectroscopic, antibacterial and reactivity study using experimental and theoretical procedures. J Mol Struct 2017;1141:495-511.
38Kramer B, Rarey M, Lengauer T. Evaluation of the FLEXX incremental construction algorithm for protein-ligand docking. Proteins Struct Funct Bioinforma 1999;37:228-41.
39Trott O, Olson AJ. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 2010;31:455-61.
40Sharma LK, Lu J, Bai Y. Mitochondrial respiratory complex I: structure, function and implication in human diseases. Curr Med Chem 2009;16:1266-77.
41Surin AM, Sharipov RR, Krasil’nikova IAet al. Disruption of functional activity of mitochondria during MTT assay of viability of cultured neurons. Biochemistry (Mosc) 2017;82:737-49.
42Mishra S, Singh S. Identification of inhibitors against metastasis protein “survivin:„ in silico discovery using virtual screening and molecular docking studies. Pharmacogn Mag 2018;13:S742-8.
43Quispe PA, Lavecchia MJ, León IE. On the discovery of a potential survivin inhibitor combining computational tools and cytotoxicity studies. Heliyon 2019;5:e02238.
44Agashe VR, Shastry MCR, Udgaonkar JB. Initial hydrophobic collapse in the folding of barstar. Nature 1995;377:754-7.
45Lins L, Brasseur R. The hydrophobic effect in protein folding. FASEB J Off Publ Fed Am Soc Exp Biol 1995;9:535-40.
46Kubota H. Quality control against misfolded proteins in the cytosol: a network for cell survival. J Biochem 2009;146:609-16.
47Wanandi SI, Limanto A, Yunita Eet al. In silico and in vitro studies on the anti-cancer activity of andrographolide targeting survivin in human breast cancer stem cells. PLoS One 2020;15:e0240020.