Structure-activity relationship studies of Longicalcynin A analogues, as anticancer cyclopeptides

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Highlights

  • In this study, analogues of Longicalcynin A, an anticancer cyclopeptide, were synthesized by SPPS method.

  • The cytotoxicity analyses of the peptides were evaluated against HepG2, HT-29 and fibroblast (as control) cell lines.

  • By these peptides, apoptosis was most probably occurred as flow cytometry analysis demonstrated.

  • Apoptosis might be happened through lysosomal membrane damage for some peptides.

  • Considering the amino acid sequences of the peptides, cyclopeptides 11 and 17 can be good anticancer candidates.

Abstract

Cancer has emerged as the main cause of the highest rate of mortality in the world. Drugs used in cancer, although, show some beneficial effects on cancerous organs, demonstrate side effects on other normal tissues. On the other hand, anticancer peptides, being effective on target tissues, should be safe and less harmful on healthy organs, since peptides have several advantages, i.e., high activity, specificity, affinity, being less immunogenic and not accumulate in the body. In the present work, analogues of Longicalcynin A, a naturally occurring anticancer cyclopeptide, were synthesized and evaluated their cytotoxicity in order to gain information from structure-activity relationships of the such cyclopeptides which may lead to find novel and safer anticancer peptide compound(s) to be used in clinic. Peptides were prepared by the solid-phase peptide synthesis method using trityl-resin. Peptide cyclization was performed in liquid phase. To study anticancer activity of the peptide analogues of Longicalycinin A, several methods including MTT, flow cytometry analysis and Lysosomal membrane integrity assay were employed using two cell lines HepG2 and HT-29. Fibroblast cells were used to control the safety of the synthesized cyclopeptides on normal cells. Two cyclopeptides 11 and 17 with the sequences of cyclo-(Thr-Val-Pro-Phe-Ala) and cyclo-(Phe-Ser-Pro-Phe-Ala), respectively were cytotoxic against the colon as well as hepatic cancer cells with safety profile against fibroblast cells, probably with the mechanism of apoptosis as lysosomal membrane integrity damaged. These cyclopeptides showed to be more favorable compounds better than Longicalycinin A and good candidates to develop cyclopeptides as anticancer agents.

Introduction

For the years, peptides have been considered as promising agents for the treatment of various diseases; i.e., diabetes, cardiovascular disorders, infection and cancer [1]. That is because peptides have several advantages, i.e., high activity, specificity, affinity, being less immunogenic and not accumulates in the body [2,3]. The best example of using peptides in cancer, are LHRH agonists in the treatment of prostatic cancer [4,5]. Anticancer activities of peptides are related to many different mechanisms which inhibit tumour growth. These mechanisms include; angiogenesis inhibitions, gene expression, signal transduction and enzyme activity prohibition [6,7]. Peptides, useful in many diseases, however, have some limited applications due to their low stability, short half-life, and susceptibility to degradation by protease enzymes [8]. To tackle with some of these pitfalls, various techniques have been employed to change the chemical backbone structure of the peptides to raise their stability while maintain their activities in biological systems. These techniques include; change and rearrangement the sequence of the peptide residues [9], replacement of l-amino acids with D-counterparts [10,11], cyclization of the linear peptides [12], hybridization with other compounds, fragmentation of the peptide chain and N-terminal/C-terminal modification of the linear peptides [13]. It was shown that amino acid substitution strategy in the peptide sequence can also vary the mode of action of the peptides [14]. On the other hand, cyclization of peptides, in addition to increasing biological stability, can fix peptides conformation and improve their interaction with biological active site, and therefore, give better pharmacological activity [12]. Anticancer activity of naturally originated cyclopeptides has attracted many researchers to look for various analogues in order to increase activity while reduce some of the side effects of these peptides on biological application [[15], [16], [17]]. From a Chinese medicinal plant, Dianthus superbus, initially two and then four cyclohexapeptides were isolated called as Dianthin A-F [16,17]. Among them, cytotoxic activity of Dianthin E on HepG2 hepatic cell line was profound, while reduced or no activity was resulted against other cancerous cell lines [17]. In another research, from Dianthus superbus var.longicalycinin, a cyclopentapeptide named Longicalycinin A was isolated [18]. The activity of Longicalcynin A, studied on cancerous hepatic, breast and lung cell lines, showed that this peptide was considerably toxic against HepG-2 [18], although this effect was less than that of Dianthin E. The study on the synthetic Longicalycinin A revealed that this peptide had a high anthelmintic activity, while low or no activity against gram positive and gram negative bacteria as well as fungi was achieved [19]. Also, researchers showed that the cytotoxic activity of Longicalycinin A on two cell lines of Dalton's lymphoma ascites (DLA) and Ehrlich's ascites carcinoma (EAC) was high, even more than 5-FU, a cytotoxic agent used as the positive control [19]. Meanwhile, Dianthin A was reported to show antifungal action on Candida albicans, moderate activity on worms and low or no effect on dermatophytes and bacteria. Dianthin A also demonstrated anticancer effects on DLA and EAC cell lines, comparable with 5-FU [20]. In a late study from Dianthus superbus two cyclopeptides, Dianthins G and H along with Dianthin E were isolated. All these three cyclopeptides showed proliferative activity on MC3T3-E1 osteoblast cells as MTT experiment demonstrated and so, the authors suggested that these cyclopeptides be considered for the treatment of osteoporosis [21]. Longicalycinin A, being reconsidered, was synthesized by solid phase peptide synthesis strategy and meanwhile its activity as anticancer agent was reevaluated on EAC and DLA cell lines [22]. In order to expand our knowledge on Longicalycinin A, as an anticancer cyclopeptide, the present study was focused on the synthesis of Longicalycinin A and its synthetic several analogues followed by examining and comparing their activities against hepatic and colon cancerous cell lines. This study should give useful information about the structure-activity relationship data of Longicalycinin A and its analogues as anticancer agents. Examining the effect of these peptides on normal fibroblast cells may also give a degree of safety profile which would be desirable to introduce new anticancer cyclopeptides for clinical application.

Section snippets

Designing the sequence structures of Longicalycinin A peptide analogues

A strategy to make various peptide analogues of Longicalycinin A was chosen as the replacement of one, two and three of amino acids in the Longicalycinin A sequence, and thus three classes of peptide analogues were made (Table 1). For each class, six peptides were designed and thus, eighteen peptide (linear and cyclic) analogues along with longicalycinin A were synthesized (Table 2).

Designing the synthesis of Longicalycinin A linear analogues

Two-step synthesis of peptides on 2-CTC resin was chosen as the main strategy in this study due to several

Materials and methods

Trifluoroacetic acid (TFA), Fmoc amino acids and coupling reagents O-(7-Azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (HATU), (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBop), Solvents like acetonitrile (MeCN), Piperazine, N,N-diisopropylethylamine (DIPEA), Diethylether, Dichloromethane (DCM), N,N-dimethylformamide (DMF), and methanol (MeOH) were purchased from Merck Co. Germany. 2-chlorotritylchloride resin (1% DVB, 200–400 mesh, 1 mmol/g) was

Conclusions

The synthesized cyclic analogues of Longicalycinin A showed toxic effects on chosen cancerous cells of liver and colon in a meaningful way, compared with the linear peptide analogues. Meanwhile, among the cyclopeptides, compounds 11 and 17 were cytotoxic against HT-29 as well as HepG2 cancer cells probably with the mechanism of lysosomal membrane integrity damage. The favorable safety profiles resulted for the compounds 11 and 17 against fibroblast cells promote us to choose these cyclopeptides

Author contributions

Conceptualization, M.H.H.T.; methodology, M.H.H.T. and M.G. investigation, M.G., resources, M.H.H.T., A.B., B.M.M. and H.R.B., supervision, M.H.H.T. and A.B., data curation, M.G., writing, M.G. and M.H.H.T.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

The authors are grateful to the Shahid Beheshti University of Medical Sciences by grant number 1338 and the University of Kashan by grant number 159148/51 for providing support to this work.

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