Polyalthia longifolia Methanolic Leaf Extracts (PLME) induce apoptosis, cell cycle arrest and mitochondrial potential depolarization by possibly modulating the redox status in hela cells

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Abstract

Medicinal plants have been accepted as a gold mine, with respect to the diversity of their phytochemicals. Many medicinal plants extracts are potential anticancer agents. Polyalthia longifolia var. angustifolia Thw. (Annonaceae) is one of the most significant native medicinal plants and is found throughout Malaysia. Hence, the present study was intended to assess the anticancer properties of P. longifolia leaf methanolic extract (PLME) and its underlying mechanisms. The Annexin V/PI flow cytometry analysis showed that PLME induces apoptosis in HeLa cells in dose-dependent manner whereas the PI flow cytometric analysis for cell cycle demonstrated the accumulation of cells at sub G0/G1, G0/G1 and G2/M phases. Investigation with JC-1 flow cytometry analysis indicated increase in mitochondria membrane potential depolarisation corresponding to increase in PLME concentrations. PLME was also shown to influence intracellular reactive oxygen species (ROS) by exerting anti-oxidant (half IC50) and pro-oxidant (IC50 and double IC50) affect against HeLa cells. PLME treatment also displayed DNA damage in HeLa cells in concentration depended fashion. The proteomic profiling array exposed the expression of pro-apoptotic and anti-apoptotic proteins upon PLME treatment at IC50 concentration in HeLa cells. Pro-apoptotic proteins; BAX, BAD, cytochrome c, caspase-3, p21, p27 and p53 were found to be significantly up-regulated while anti-apoptotic proteins; BCL-2 and BCL-w were found to be significantly down-regulated. This investigation postulated the role of p53 into mediating apoptosis, cell cycle arrest and mitochondrial potential depolarisation by modulating the redox status of HeLa cells.

Introduction

Medicinal plants rich with various phytochemicals have received great attention in the development of anticancer agents due to their numerous medicinal properties [1]. It is presently estimated that >50% of all patients diagnosed with cancer explore complementary and alternative medicine, especially herbal medicine [2] Although many medicinal plant’s curative properties were reported in the literature but there are still various medicinal plants need comprehensive scientific study. Accordingly, certifying the traditional use of these medicinal plants and studying the possible mechanism of bioactivities can offer a rich source of new pharmaceutical products particularly in the development of anticancer agents. Hence, the present study was intended to assess the Polyalthia longifolia var. angustifolia Thw. (Annonaceae) leaf methanolic extract (PLME) anticancer properties and the underlying mechanisms. P. longifolia is one of the most significant native medicinal plants and is found throughout Malaysia. Various phytochemicals such as diterpenes, alkaloids, steroids, and miscellaneous lactones have been isolated from its bark. The stem bark extracts and isolated compounds have been studied for various biological activities, such as cytotoxicity, and antibacterial and antifungal activity [3], [4]. Various bioactivities of this important medicinal plant was recently reported in the literature such as antioxidant activity, hepatoprotective activity [5] genoprotective activity, acute oral toxicity [6] and in vivo radioprotective activity [4] of leaf extract. Furthermore, P. longifolia is widely used in traditional medicine as a febrifuge and tonic [7]. Our preliminary study showed that the standardized methanolic leaf extract of P. longifolia (PLME) has exhibited cytotoxicity against HeLa cell line in vitro [8]. However, possible pathway and detailed mechanisms of action of P. longifolia leaves have not been investigated this far. Therefore this study was conducted to establish the possible apoptotic pathways induced in HeLa cells by P. longifolia leaves.

The increase search for competent anticancer agents now resides greatly upon the strategic to identify agents with selective ability in cancer cell annihilation. Cancer is an outcome to a loss of balance between cell proliferation and cell death during which a normal cell becomes malignant by evading cell death or apoptosis [9]. Due to the fact that apoptotic mechanism can be altered to influence activation of cell death in cancers, such strategic becomes feasible with explorations continuing to confine the cytotoxicity effect to only cancer cells and minimize the tendency of side effects in normal cells. Therefore, the important mechanisms in the avoidance and suppression of cancer are inhibition of cell cycle machinery and induction of apoptosis in the abnormal cells [1]. The induction of apoptosis is highly preferred pathway for therapeutic intervention even with the emergence of alternative paths leading to cell death due to its limited side effects to the adjacent normal cells [10]. Moreover, cell cycle arrest by therapeutic agents in the Sub G1, G0 and G1 phases can lead to apoptosis [11]. In this study, HeLa cells were exposed to varying PLME concentrations to investigate whether the growth inhibitory effects of the PLME are due to changes in cell cycle progression and/or apoptosis with their underlying molecular pathways involved in the regulation of cell death. Understanding of the underlying molecular pathways of cell cycle machinery and induction of apoptosis in cancer cells could provide new rational strategy and targets for effective cancer therapy.

Section snippets

Plant material collection and extraction

The leaves of P. longifolia were collected from various areas in Universiti Sains Malaysia and authenticated at the Herbarium of the School of Biological Sciences, Universiti Sains Malaysia, Pulau Pinang, Malaysia, where a sample was deposited (Voucher specimen: USM/HERBARIUM/11306). The leaves were cut, washed with distilled water and oven-dry at 30 °C for 7 days. The dried leaves are then grounded into fine powder. A sample of 100 g of plant powder was soaked in 400 mL of methanol at RT (23 °C ±2)

Gas chromatography profile of PLME using GC–MS analysis

The PLME extract was characterized by GC–MS. The chromatographic analysis revealed 14 major peaks with four major compound namely Phenol, 2,4-bis(1,1-dimethylethyl)-; Pentadecanoic acid, 14-methyl-, methyl ester; 9-Octadecenoic acid, methyl ester, (E)- and Octadecanoic acid, methyl ester (Fig. 1, Table 1).

PLME induced apoptosis in HeLa cells

The percentage of apoptotic cells in PLME treated HeLa cells were monitored using FITC-Annexin V and propium iodide (PI) double staining by FACS (Fig. 2). The vehicle control cells visibly

Discussion

Apoptosis is tracked continuously as the most scrutinised mechanism in the biological field ever since it was coined by Kerr and colleagues in 1970s [17]. The process is comprehensively studied as such that it was found role-playing in both physiological and pathological state [18], [19]. Every now and then, millions of cells enter into dying state as consequence to apoptosis regulation. This type of molecular mechanism seems to result from diversified pathways and has proved imperative in the

Conclusion

As shown in Fig. 8, the treatment of PLME with different concentrations of HeLa cells resulted in a concentration-dependent increase in PS exposure on the outer leaflet of the mitochondrial membrane, cell cycle arrest, loss of ΔΨm, generation of ROS and DNA fragmentation. The PLME may have caused oxidative stress by increasing the formation of free radicals and ROS generation. The effective cell killing by PLME may be postulated as leading to the induction of DNA damage and the loss of DNA

Funding

This project was funded by the Fundamental Research Grant Scheme (FRGS; Grant No.: 203/CIPPM/6711379) from the Ministry of Education Malaysia, Government of Malaysia, Malaysia.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgement

Soundararajan Vijayarathna was supported by the MyPhD fellowship from the Ministry of Higher Education, Government of Malaysia, Malaysia.

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