Open Veterinary Journal, (2024), Vol. 14(10): 2628–2633

Research Article

10.5455/OVJ.2024.v14.i10.12

Stem bark ethanolic extract of Pinus merkusii induces caspase 9-mediated apoptosis in HeLa cells

Agung Budianto Achmad^1*, Annise Proboningrat², Arif Nur Muhammad Ansori³, Amaq Fadholly⁴, Siti Eliana Rochmi¹, Rinza Rahmawati Samsudin⁵, Nanik Hidayatik⁶, Gracia Angelina Hendarti⁷ and Shara Jayanti⁸

¹Department of Health, Faculty of Vocational Studies, Universitas Airlangga, Surabaya, Indonesia

²Division of Veterinary Pathology, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia

³Postgraduate School, Universitas Airlangga, Surabaya, Indonesia

⁴Division of Pharmacology and Toxicology, School of Veterinary and Biomedical Medicine, IPB University, Bogor, Indonesia

⁵Department of Chemistry, Faculty of Health Sciences, Muhammadiyah University of Surabaya, Surabaya, Indonesia

⁶Division of Basic Veterinary Medicine, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia

⁷Division of Veterinary Anatomy, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia

⁸Marine and Fisheries Polytechnic, Surabaya, Indonesia

*Corresponding Author: Agung Budianto Achmad. Department of Health, Faculty of Vocational Studies, Universitas Airlangga, Surabaya, Indonesia. Email: ab.achmad [at] vokasi.unair.ac.id

Submitted: 07/07/2024 Accepted: 13/09/2024 Published: 31/10/2024

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Abstract

Background: Cervical cancer is a severe concern for women throughout the world. This percentage of cancer incidence causes sufferers to die at a high rate. It is believed that the bark of the Pinus merkusii tree contains anti-cancer compounds that inhibit cervical cancer cell growth.

Aim: This present study aims to examine the cytotoxic ability of P. merkusii tree bark ethanol extract (PMBE) by inducing apoptosis in HeLa cells.

Methods: We administered the PMBE at concentrations of 50, 100, 200, and 400 µg/ml to HeLa cell cultures. We then conducted the MTT cytotoxicity assay, detected apoptosis via Annexin V binding, and observed caspase 9 expression via immunocytochemistry.

Results: PMBE showed cytotoxic activity on HeLa cells with an IC₅₀ of 226.6 µg/ml for 24 hours of treatment. PMBE caused early apoptosis in up to 81.31% of HeLa cells, as well as increased caspase-9 expression.

Conclusion: Based on this study, PMBE is predicted to have dose-dependent antiproliferative or cytotoxicity effects on the HeLa cell line through the intrinsic pathway apoptosis mechanism.

Keywords: Anticancer, Cervical cancer, Apoptosis, Pinaceae.

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Introduction

Cervical cancer ranks fourth worldwide in terms of cancer-related mortality among women; over 85% of cases and deaths from the disease occur in developing countries (Proboningrat et al., 2019). Cervical cancer develops as a result of aberrant cell proliferation in the cervix (the lowest part of the uterus that terminates in the vagina) (Sharma et al., 2017). The cancer treatment process still uses many surgical procedures, which still have the potential to leave behind cancer cells that can grow again and metastasize to other body organs (Wijaya and Muchtaridi, 2017). Treatment with conventional chemotherapy methods often has negative impacts on the patient’s health due to systemic toxicity and drug resistance (Proboningrat et al., 2021b). Based on this problem, looking for anti cancer agents based on natural ingredients with low side effects and a better ability to kill cells is necessary. There are many side effects in cancer therapy with conventional models of treatment; it is essential to explore new candidates that are effective with lower side effects against cervical cancer.

Several compounds in plants have anti-cancer properties (Zarrinnahad et al., 2018), one of which is Pinus merkusii (Proboningrat et al., 2021a). Pinus merkusii is a member of the Pinaceae family, which is indigenous to Southeast Asia and extensively dispersed throughout Indonesia, Philippines, Laos, Vietnam, Cambodia, Thailand, and Burma (Proboningrat et al., 2021a). Pine trees are utilized as a folk cure in Asia for gastrointestinal issues, skin and topical conditions, inflammation, ulcers, itching, and snakebites (Sajid et al., 2018). The phytochemicals found in the pine plants, including polyphenols, flavonoids, alkaloids, triterpenes, triterpenoids, sterols, glycosides, lignans, and saponins, have been demonstrated to be responsible for the medicinal properties of the plant (Sudjarwo et al., 2018).

However, its complete anticancer potential against HeLa cells is still unknown. Previous research (Proboningrat et al., 2021a) used the WIDR cell line showed that P. merkusii crude extract was potently to killing cancer cells. Apoptosis is one of how cancer cells die through programmed cell death. It works by a cascade series involving increased activity of pro-apoptotic proteins (Bax) (Li et al., 2011), decreased expression of the anti-apoptotic protein (Bcl-2), and activation of Caspase 9 and 3 (Chu et al., 2012; Liu et al., 2015). Thus, in the current study, we assessed the cytotoxicity of the stem bark extract of P. merkusii (PMBE) against cervix cancer, HeLa cell line, and the possible mechanism of action by inducing apoptosis via caspase-9 activation.

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Materials and Methods

Reagents

Ethanol 96%, Dulbecco’s modified eagle medium (Gibco, USA), 2% penicillin-streptomycin (Gibco, USA), amphotericin B (Sigma-Aldrich, USA), HEPES (Sigma-Aldrich, USA), 10% fetal bovine serum (Rocky Mountain Biologicals, Inc., USA), 1 × phosphate buffer saline (PBS) (Sigma-Aldrich, USA), dimethyl sulfoxide (SigmaAldrich, USA), Trypsin/EDTA solution (Sigma-Aldrich, USA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, USA), HCl 0.1 N (Sigma-Aldrich, USA), sodium dodecyl sulfate (SDS) (Sigma-Aldrich, USA), Annexin V apoptosis detection kit with PI (BioLegend Inc., UK), anti-caspase-9 antibody (ab52298, Abcam), and Mayer’s hematoxylin solution (Sigma-Aldrich, USA).

Plant extract preparation

The stem barks of P. merkusii were gathered from Malang Regency, East Java Province, Indonesia. After being cleansed, the dried stem barks were chopped into tiny pieces. After that, they were ground into a powder. 350 g of powdered bark were steeped for 3 days in 1.75 l of 96% ethanol. A rotary evaporator running at 250 rpm and 60°C separated and concentrated the macerate (Proboningrat et al., 2021a).

Cell culture

The Department of Parasitology of Universitas Gadjah Mada’s School of Medicine contributed to the HeLa cell lines. The cells were cultured at 37°C with 5% CO2 in Dulbecco’s modified eagle medium supplemented with 10% fetal bovine serum, HEPES, 0.5% amphotericin B, and 2% penicillin-streptomycin.

MTT cytotoxicity assay

HeLa cells were seeded at a density of 104 cells/ml, and after being incubated overnight, they were exposed to a range of concentrations of PMBE starting with the highest concentration of 400 µg/ml (four folded dilution) for 24 hours. After incubation, 100 µl of MTT solution was added and left in each well for 4 hours. The absorbance was measured at a wavelength of 595 nm using a Benchmark Microplate Reader (Bio-Rad, USA) following the addition of 100 µl of SDS 10%. Graphs were plotted to find the percentage of live cells in a particular value.

Apoptosis detection by annexin V binding

Detection of the cells’ apoptosis was performed using the FITC Annexin V apoptosis detection kit with PI. HeLa cells were plated in 6-well plates at a density of 106 cells/well. After being incubated overnight (37°C, 5% CO2), they were treated with IC₅₀, 2IC₅₀, and 4IC₅₀ of PMBE for 24 hours. Following harvesting, the cells were rinsed with PBS, re-suspended in Annexin V binding buffer, and stained for 15 minutes at room temperature in the dark with 5 µl of Annexin V and 10 µl of PI. The distribution of cell populations in various quadrants was identified using the BD FACSCalibur flow cytometer (BD Biosciences, USA).

Caspase-9 detection by immunocytochemistry

HeLa cells (2 × 104 cells/well) were plated and incubated overnight on coverslips within 24-well plates at 37°C and 5% CO2 prior to being treated with IC₅₀, 2IC₅₀, and 4IC₅₀ of PMBE for 24 hours. After 10 minutes of fixation using absolute methanol, the cells were washed with PBS and treated with a 1% hydrogen peroxide solution for 10 minutes. They were then blocked with a background sniper and incubated with a 1:50 anti-caspase-9 antibody in a dark room for 1 hour. After washing, the Star Trek Universal HRP Detection System was added, and Mayer’s hematoxylin was used for counterstaining. The cells were dipped in ethanol, dried, and mounted. The stained cells were observed using a Nikon Eclipse Ci microscope (Nikon Corp., Japan) and evaluated semi-quantitatively by the H-score method (Mazières et al., 2013).

Statistical analysis

Results were analyzed using GraphPad Prism by one-way analysis of variance and Kruskal-Wallis, and differences were deemed statistically significant at the level of p-values ≤ 0.05.

Ethical approval

Not needed for this study.

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Results

Cytotoxicity effects of PMBE on HeLa cells

We examined the ethanolic bark extract of PMBE to determine its capacity to inhibit the cervical cancer HeLa cell’s growth or viability to verify the antiproliferative effect of the plant. Initially, an MTT assay was conducted using a range of dosages applied to the cells, starting from 400 µg/ml to 200, 100, 50, and 25 µg/ml. As shown in Figure 1, PMBE demonstrated a dose-dependent ability to inhibit cell growth, with an IC₅₀ of 226.6 µg/ml after 24 hours.

Apoptosis induction of HeLa cells by PMBE

To determine whether PMBE is involved in programmed cell death, we assessed the proportion of apoptotic cells in treated HeLa cells. The proportion of early (Annexin V+/PI) and late (Annexin V+/PI+) apoptotic cells shown in Figure 2 indicates the apoptotic cells. The HeLa cells treated with IC₅₀ PMBE exhibited the highest proportion of early apoptosis of any group, reaching up to 81.31%. Meanwhile, there was a dose-dependent rise in the percentage of late apoptosis, with the 4IC₅₀ PMBE group exhibiting the most significant proportion at 3.48%. It was indicated that treating HeLa cells with PMBE for 24 hours incubation tended to cause early apoptosis.

Caspase-9 activation of HeLa cells by PMBE

The dose-course effect of PMBE extract after 24 hours on the initiator caspase-9 activity is depicted in Figure 3. The results indicate that there is an increase in caspase-9 expression in all treated cells compared to untreated cells. Moreover, the results revealed that PMBE induced the potentially highest activity of caspase-9 in HeLa cells exposed to the 2IC₅₀ concentration. The findings show that PMBE causes HeLa cells to undergo apoptosis by upregulating the expression of caspase-9.

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Discussion

In recent decades, scientists have been striving to find and isolate novel anticancer medicines from natural resources. Cytotoxic substances are among the most efficacious anticancer medications (Sarvmeili et al., 2016). Some plant active components, such as alkaloids, flavonoids, and phenolic compounds, have been proven to possess cytotoxic activity against cancer cells (Ganadhal et al., 2021). Multiple investigations have demonstrated the existence of cytotoxic substances in plants of the Pinaceae family (Shi et al., 2016; Yi et al., 2016).

Pinus merkusii is a member of the Pinaceae family and is extensively found in Indonesia, particularly in Sumatera and Java Island (Imanuddin et al., 2020). Although prior research has conducted cytotoxicity screening of chitosan-based encapsulation P. merkusii (Proboningrat et al., 2019), there is still a significant lack of scientific data addressing the potential therapeutic effectiveness of the plant’s bark’s crude extract for cervical cancer and its mechanism of action. According to this study, PMBE extracted from P. merkusii decreased the growth of proliferative cells, which was associated with the induction of apoptosis, as indicated by the increase in caspase activity.

Prior studies have already documented the inhibitory activity of various pine plant species on HeLa cell proliferation (Amalinei et al., 2014; Li et al., 2016; Sarvmeili et al., 2016). In the present study, PMBE exhibited a dose-dependent growth inhibitory effect, which triggers cellular processes that suppress proliferation and induce cell death. Our MTT assay confirmed that PMBE inhibited the growth of 50% of HeLa cells at a concentration of 226.6 μg/ml, which was slightly lower than what was reported before (235.60 μg/ml, WiDR cells) (Proboningrat et al., 2021a). In addition, it has been documented that bark extracts derived from different species of pine trees are rich in proanthocyanidins, which possess promising therapeutic properties (Li et al., 2015). The potential of proanthocyanidins to inhibit tumor growth has been established on account of their capacity to regulate the activity of multiple targets implicated in carcinogenesis (Rauf et al., 2019).

Fig. 1. Cytotoxicity of Pinus merkusii stem bark ethanolic extract on HeLa cells.

Fig. 2. The flow cytometric histogram shows the activation of apoptosis in HeLa cells after a 24-hour treatment with PMBE. (a) control, (b) IC₅₀, (c) 2IC₅₀, and (d) 4IC₅₀. The lower left, lower right, upper right, and upper left, respectively, represent viable, early apoptotic, late apoptotic, and necrotic cells.

Fig. 3. Caspase-9 expression in (a-1) untreated HeLa cells and HeLa cells treated with (a-2) IC₅₀, (a-3) 2IC₅₀, and (a-4) 4IC₅₀ of PMBE for 24 hours (Nikon Eclipse Ci; 400×). (b) The bar displays the mean ± standard deviation of the caspase-9 expression score in treated HeLa cells.

We further investigated the mechanisms by which PMBE inhibits the growth of cervical cancer cells. Apoptosis may be responsible for the inhibitory effect on cancer cell proliferation. Apoptosis plays a significant part in eliminating mutated or rapidly proliferating tumor cells (Millimouno et al., 2014). Some studies have revealed that bark extracts from P. massoniana and P. maritima induce apoptosis in human ovarian cancer A2780 cells and human malignant melanoma A375 cells, respectively (Liu et al., 2015; Thaichinda et al., 2020). According to flow cytometry results from the current investigation, the proportion of apoptotic cells increased substantially after PMBE treatment. The findings suggest that PMBE triggers apoptosis in cervical cancer cells.

Apoptosis induction is associated with upregulation of pro-apoptotic proteins and downregulation of anti-apoptotic proteins (Singh et al., 2015). The Bcl-2 protein is crucial for modulating the mitochondria-mediated caspase activation pathway (Akl et al., 2014). When Bcl-2 is down-regulated, it leads to the decline of mitochondrial membrane potential and the release of cytochrome c from mitochondria into the cytosol, which in turn activates caspase-9 (Proboningrat et al., 2019). Cleaved caspase-9 additionally stimulates caspase-3, a pivotal enzyme in the intrinsic apoptotic pathway, thereby propelling subsequent apoptotic processes (Moghadamtousi et al., 2014). Herein, we observed an increase in caspase-9 expression in HeLa cells treated with PMBE. It could be suggested that PMBE may potentially trigger apoptosis via the mitochondria-associated apoptotic pathway. The proanthocyanidin content commonly present in pine bark extract may be responsible for the PMBE’s ability to induce apoptosis in cervical cancer cells. Consistently, Shi et al. (2019) documented that proanthocyanidin from grape seeds could trigger apoptosis of human colon cancer SW480 and SW620 cells by facilitating the expression of the pro-apoptotic proteins Bax and Bax, suppressing the expression of the anti-apoptotic protein Bcl-2, and activating caspase-9 and caspase-3.

The results of this study reported that PMBE was predicted to have cytotoxic activities by targeting the mitochondrial-apoptosis pathway in cervical cancer HeLa cell lines. However, further in vitro and in vivo research is required to improve the findings of this study.

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Conclusion

Our study has demonstrated that crude extracts from the bark of PMBE exhibited dose-dependent antiproliferative effects on the HeLa cell line. Furthermore, HeLa cells were induced to undergo apoptosis via a caspase-dependent pathway by the PMBE. Additional research in the field of anti-cancer drug discovery should be conducted, as demonstrated by the current study’s findings regarding the potency of PMBE as an anti-cancer agent.

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Acknowledgment

The authors thank Gadjah Mada University for providing facilities for this research.

Conflict of interest

All authors declare that there are no conflicts of interest in the present study.

Funding

This work was supported by Airlangga Research Fund, Universitas Airlangga.

Author’s contributions

Conceived, designed, and coordinated the study: ABA, AP, and SER. Designed data collections tools, supervised the field sample and data collection, and laboratory work as well as data entry: ANMA, AF, and SJ. Validation, supervision, and formal analysis: RRS and NH. Carried out the statistical analysis and interpretation and participated in the preparation of the manuscript: AP, ABA, and GAH. All authors have read, reviewed, and approved the final manuscript.

Data availability

All data supporting the findings of this study are available within the manuscript and no additional data sources are required.

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