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Purified compounds from marine organism sea pen induce apoptosis in human breast cancer cell MDA-MB-231 and cervical cancer cell Hela

https://doi.org/10.1016/j.ejphar.2020.173075Get rights and content

Highlights

  • Three compounds 3beta-Cholest, 5 en, 3ol, Hexadecanoic acid, 2-Hexadecanol were purified from marine organism Sea pen.

  • Three compounds reduced viability of MDA-MB-231 and Hela human cancer cells concentration-dependently.

  • Compounds 1, 2 and 3 induced apoptosis in cancer cells. Purified compounds decreased significantly the relative Bcl-2/Bax ratio in comparison to the control group.

Abstract

Marine organisms are an important source of chemical compounds which are appropriate for use as therapeutic agents. Among them, Sea pens produce valuable chemical compounds being used as anti-cancer drugs. The aim of this study was to investigate anti-cancer property of extracted and purified compounds from marine organism Sea pen and evaluate their effects on inducing of apoptosis. The extracts were prepared from dried colony of Virgularia gustaviana. The compounds (3β)-Cholest,5en,3ol (cholesterol) (15 mg), Hexadecanoic acid (2.5 mg) and 2-Hexadecanol (10.7 mg) were identified by GC-MS and NMR. The cytotoxic effects of the compounds were evaluated on Hela and MDA-Mb-231 human cancer cell lines with MTT assay. Immunocytochemistry and Western Blot analyses were used to evaluate the expression of apoptosis related markers Caspase 3, Caspase 8, Bax and BCL2 in cancer cells after treating with three compounds. The purified compounds reduced viability of human breast cancer cell line MDA-MB-231 and human cervical cancer cell line Hela concentration-dependently. 2-Hexadecanol reduced significantly the viability of both cancer cell lines in comparison to the other purified compounds. Treatment of cancer cells with the three purified compounds increased the expression of caspase-3, caspase-8 and Bax proteins and decreased the relative Bcl-2/Bax ratio, demonstrating induction of apoptosis as possible mechanism of action. According to the results, three purified compounds inhibit the growth of cancer cells by inducing of apoptosis pathway; an effect which needs to be further investigated in the future studies.

Introduction

Marine situations (ecological, chemical and biological diversity) possess productive sources of macroscopic or microscopic organisms with bioactive compounds which have a great potential for treatment of variety of diseases (Barkia et al., 2019). This diversity produces many unique chemical compounds (Ercolano et al., 2019). A lot of compounds were recently approved as drug with unique mechanism of action from marine organism and some of them are employed for chemotherapy of cancer (Jimenez et al., 2018) (Rocha et al., 2011).

Cancer is one of the major causes of morbidity and mortality in the world despite the increasing advances in prevention and treatment (Stonik and Fedorov, 2014). Breast cancer and cervical cancer are two common cancers in women (Pacheco et al., 2018). Since the current treatments for cancer like chemotherapy and radio-therapy are not always effective and there is a possibility of recurrence and also due to side effects of current anti-cancer agents, the scientists have focused on natural products as anti-cancer drugs (Branco et al., 2019), (Garcia et al., 2016) (Rotchell and Ostrander, 2011). Use of medicinal marine organisma as source of anticancer drugs in the recent years and identifying their mechanism of actions have attracted the attention of many scientists. Due to their attachment to beds life styles, sessile marine invertebrates such as corals, sponges, tunicates and ascidians do not have any physical defense mechanism(van de Water et al., 2018). Therefore, sessile marine organisms are adapted to their environment with producing chemicals called secondary metabolite including terpenes, steroids, polyketides, peptides, alkaloids, and porphyrins which give them the ability to defense against predator and competing species (Montaser and Luesch, 2011; Raimundo et al., 2018). The mentioned secondary metabolites demonstrate potent anti-cancer activities (Li, 2016). Among the marine compounds with remarkable activity, fatty acids provide several health benefits for resistant diseases such as cancer (Pacheco et al., 2018). It has been shown that fatty acid based compounds may reduce the growth of cancer cells (Gerber, 2012). About 41% of last decade studies about Cnidarians (a sessile invertebrate) bioactive molecules were focused on the potential treating of neoplasm (Rocha et al., 2011). Cnidarians are classified as sessile invertebrates, sub class octocorallina (or Alcyonaica), order Pennatulacea which contains sea pens (Koeberle and Werz, 2014). There are several studies on the extraction and fractionation of sea pen-derived compounds and investigation of their biological effects (Thomas et al., 2019; van de Water et al., 2018). Sesquiterpene Junceol A and two known Diterpenoids, Sclerophytin A and Cladiellisin which were extracted from sea pen Virgularia juncea demonstrated anticancer property (Chen et al., 2001). We recently showed that the crude extract of sea pen Virgularia gustaviana has cytotoxic effect on cancer cells which was comparable to cembrane diterpene Sarcophine (Sharifi et al., 2016), (Sharifi et al., 2018). Due to previous research, the aim of this study was to purified, identify and evaluate anti-cancer effects of compounds responsible for anticancer property of sea pen Virgularia gustaviana. To evaluate anti-cancer effects of chemical compounds, their cytotoxic effects were measured on Hela and MDA-MB-231 cancer cell lines. Moreover, the expressions of caspase 3 and caspase 8, and Bax (as apoptosis markers) and the expression of Bcl-2 were assessed following treatment of cancer cells with purified compounds.

Section snippets

Animal material

According to protocol for sampling and preservation of marine organisms established by V. Haussernann (Haussernann V. 2009), marine sea pen Virgularia gustaviana was collected by patrolling on the intertidal zone of the estuary Sura in Bandar Abbas city, south of Iran (Persian Gulf). The sea pens were carefully detached from the substratum and transferred to lab at 4 °C. The collected samples were kept at -20 °C.

Nuclear magnetic resonance spectroscopy

NMR spectra were recorded at a target temperature of 18 °C on a Bruker Avance III

Chemical extraction and purification of compounds 1-3

The compounds were separated and purified by flash column chromatography (FCC), recrystallization and HPLC system. Then, the structure of the purified compounds was determined based on NMR spectra, MS analysis and comparison of their spectral data with those of the literature. The isolated compounds were identified as known compounds (3β)-Cholest-5en-3ol (1), hexadecanoic acid (2), 2-Hexadecanol (3).

Compound 1 with molecular formula C27H46O was obtained as colorless needle-like structures with

Discussion

In this study, the chemistry and biology of the bioactive metabolites of marine sea pen Virgularia gustaviana was assessed by separation and purification techniques. Further biological and toxicological property of purified ingredients were examined by immunocytochemistry and Western blot techniques. Based on the results, a sterol compound (3beta-Cholest, 5en, 3ol) and two fatty acid based compounds hexadecanoic acid and 2-Hexadecanol were isolated from sea pen Virgularia gustaviana which all

Conclusion

In conclusion, the treatment of human cervical cancer cell line Hela and breast cancer cell line MDA-Mb-231 with a sterol compound (3beta-Cholest, 5en, 3ol) and two fatty acid based compounds hexadecanoic acid and 2-Hexadecanol isolated from sea pen Virgularia gustaviana concentration-dependently decreased the viability of both cancer cell lines and induced apoptosis in cancer cells by increase of caspoase-3, caspase-8 and Bax pro-apoptotic proteins and decrease of anti-apoptotic protein Bcl-2.

Author's contributions

SS and HN conceived and designed the research. All authors conducted the experiment. SS and HN analyzed the data. SS wrote the manuscript. All authors read and approved the final manuscript.

Ethical approval and consent to participate

The study was approved by Research Ethics Committee of School of Medicine, Shahid Beheshti University of Medical sciences.

CRediT authorship contribution statement

Sharareh Sharifi: Conceptualization, Methodology, Formal analysis, Investigation, Writing - original draft, Writing - review & editing. Pargol Ghavam Mostafavi: Conceptualization, Validation, Formal analysis, Writing - review & editing, Supervision. Roghayeh Tarasi: Methodology, Writing - original draft, Writing - review & editing. Ali Mashinchian Moradi: Validation, Formal analysis, Writing - review & editing. Mohammad Hadi Givianrad: Validation, Writing - review & editing. Mahdi Moridi

Declaration of competing interest

Authors declare no conflict of interest in this study.

Acknowledgement

The authors would like to thank Prof. H. Peirovi for his comments. The results of this paper were extracted from a part of PhD thesis of S. Sharifi. Research reported in this publication was supported by Researcher Grant Committee under award number [963951] from the National Institutes for Medical Research Development (NIMAD), Tehran, Iran.

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