INTRODUCTION:

Cancer is a pathological state with a characteristic of failure in regulating tissue growth, leading to cell proliferation, which out of control and human death^1,2,3,4. Around 7.6 million people die each year from cancer, and it is estimated to increase to 13.1 million by 2030^4,5. Colon cancer is one of the most common malignant type of cancer in the world which cause high mortality in men and women^1,5. The number of case is expected to increase along with the population growth in both developed and developing countries⁶.

Chemotherapy is a commonly therapeutic method to treat colon cancer, although it has some adverse effects, such as toxic to normal cells and increasing drug resistance to the rest of untreated cancer cells. Therefore, new pharmacological strategies which more effective with minimal side effects are needed in cancer treatment^7,8. Plants are considered very promising in this aspect since they are an essential source of compounds with various therapeutic benefits. Nowadays, most anti-cancer drugs are obtained from plants^4,5.

Pinus merkusii is one of the species of Pinaceae family native to Southeast Asia. Its distribution areas include Burma, Thailand, Laos, Cambodia, Vietnam, Philippines, and Indonesia. Its bark has been investigated to have phytochemicals of flavonoids, alkaloids, tannins, saponins, and terpenoids⁹. Bark extracts from other species of pine plants showed anticancer activity in some cancer cells. These include the bark extract of P. massoniana, P. koraiensis, P. eldarica, and P. sylvestris¹⁰^-¹⁴.

Pine bark was previously regarded as waste product in the lumber industry. Now It is recognized as a natural ingredient that is rich in proanthocyanidin content with potential medicinal properties¹¹. Proanthocyanidin is a condensed tannin produced from polymerization of catechin and epicatechin from the flavonoid class¹⁵. Sudjarwo et al. reported that proanthocyanidin is a potent compound that has antioxidant, antibacterial, anti-inflammatory, immunostimulatory, anti-atherosclerotic, anti-diabetic, and anticancer activities¹⁶. Recent studies have shown the treatment of several cancer cells with proanthocyanidin, such as HSC-2 human oral carcinoma¹⁷, ECA109 human esophageal squamous cancer cells¹⁸, MCF-7 human breast cancer cells¹⁹, HepG2, HCC-LM3, Bel-7402, SMMC-7721 and Huh-7 human hepatocellular carcinoma cells^20,21, CaCo-2 HT-29, LoVo, SW480 and SW620 human colon cancer cells²²^-²⁴, BIU87 human bladder cancer cells²⁵, as well as SiHa and HeLa human cervical cancer cells²⁶, resulted in cell viability inhibition and apoptosis induction. Therefore, we aimed to investigate the cytotoxic effects of P. merkusii bark extract (PMBE) on WiDr cells.

MATERIAL AND METHODS:

Ethical Clearance and Plant Identification:

The treatment procedures have been examined by Medical and Health Research Ethics Committee, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia (Approval Reference Number: 3.1-007.2013.03). In addition, taxonomic identification of P. merkusii was carried out by the Purwodadi Botanical Garden, Indonesian Institute of Sciences, Purwodadi, Indonesia.

Chemicals and Reagents:

96% ethanol, dimethyl sulfoxide (DMSO) (Sigma-Aldrich, USA), Dulbecco's modified eagle medium (Gibco, USA), HEPES (Sigma-Aldrich, USA), 10% fetal bovine serum (Rocky Mountain Biologicals, Inc., USA), 2% penicillin-streptomycin (Gibco, USA), amphotericin B (Sigma-Aldrich, USA), 1× phosphate buffer saline (PBS) (Sigma-Aldrich, USA), trypsin-ethylenediaminetetraacetic acid solution (Sigma-Aldrich, USA), 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, USA), sodium dodecyl sulfate (SDS) (Sigma-Aldrich, USA), and HCl 0.1 N (Sigma-Aldrich, USA).

Preparation of Pinus merkusii Bark Extract:

Stem barks of P. merkusii were collected from Malang Regency, East Java, Indonesia. The dry stem barks were cleaned and cut into small pieces. Then they were mashed into powder. Three hundred fifty grams of bark powder soaked in 1.75 liters of 96% ethanol for three days. The macerate was separated and concentrated using a rotary evaporator at a speed of 250rpm at 60 °C²⁷.

Cell Culture:

WiDr cells were provided by the Department of Parasitology, Faculty of Medicine, Gadjah Mada University, Indonesia. Cells were grown in Dulbecco's modified eagle medium that had been supplemented with 10% fetal bovine serum, 0.5% fungizone, and 2% penicillin-streptomycin at 37°C in a 5% CO₂ incubator.

Cytotoxicity Test and Data Analysis:

The cytotoxicity of P. merkusii bark extract (PMBE) was evaluated using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) method. WiDr cells were seeded in 96-well plates at a density of 1×10⁴ cells/well overnight. Before the treatment, the medium of all wells was removed first. Cells were treated with four concentrations of PMBE (50, 100, 200, and 400 µg/mL) for 24 h. The medium was removed from all wells. Then the cells were incubated in 100µL of MTT solution for 4 h. After incubation, cell condition was examined using an inverted microscope to observe the formation of formazan crystals. Subsequently, 100 µL of 10% SDS in HCl 0.1 N solution was administered as the stopper and incubated in the dark at room temperature overnight. The absorbance was measured at 595nm using Benchmark Microplate Reader (Bio-Rad, USA). The percentage of inhibition was calculated, and the IC₅₀ determination employed linear regression analysis using Microsoft Excel 2016.

RESULTS AND DISCUSSION:

The cytotoxic activity test of PMBE was evaluated by administering 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagents to WiDr cells that have been treated with PMBE for 24 h. The yellow MTT absorbed by living cells were reduced by NAD(P)H-dependent cellular oxidoreductase to dark purple formazan ((E, Z)-5-(4,5-dimethylthiazol-2-yl)-1,3-diphenylformazan). The intensity of the dark purple indicates cell viability (Figure 1)^28,29.

MTT test results revealed that the cytotoxicity of treated WiDr cells increased gradually with increasing concentration of PMBE. The highest inhibition of WiDr cells was found at 400 µg/mL PMBE concentration, where the cytotoxicity was 96.57%. Meanwhile, the lowest cell inhibition was at 50µg/mL PMBE concentration, with cytotoxicity of 1.53%. Two other PMBE concentrations (100 and 200µg/mL), respectively, inhibited WiDr cells with cytotoxicity of 17.39% and 33.07%. PMBE showed an IC₅₀ value of 235.60µg/mL on WiDr cells with an incubation of 24 h. This study proves that PMBE has a cytotoxic effect on WiDr cells, which increases in a concentration-dependent manner. The cytotoxicity graph of PMBE on WiDr cells is shown in Figure 1.

Figure 1. The MTT test of PMBE on WiDr cells. A1-A4: represent the formation of formazan crystals at the administration of PMBE (50, 100, 200, and 400 µg/mL); B: cytotoxicity of PMBE against WiDr cells.

In the current study, we demonstrated the first report on the cytotoxic effects of PMBE in WiDr cells. We revealed that PMBE concentration-dependently increased cytotoxicity on WiDr cells, and an IC₅₀ value of 235.60µg/mL has been noted. According to Zulfafamy et al., the activity index of crude extract of natural products could be classified by IC₅₀ value into four categories, i.e. active (≤20µg/mL); moderate (>20-100µg/mL); weak (>100-1000µg/mL); and inactive (>1000µg/mL)³⁰. Thus, PMBE potentially inhibits colon carcinogenesis in spite of its weak cytotoxic effect against WiDr cells.

In the previous study, the cytotoxicity activity of another pine bark extract has already been observed in cancer cell lines. Li et al. reported that P.koraiensis bark inhibited the viability of HeLa cells (IC₅₀ of 196.38 µg/mL)¹². Another study conducted by Wu et al. and Li et al. showed that P. massoniana bark also could inhibit the viability of HeLa cells with IC₅₀ of 140µg/mL and 153.93µg/mL, respectively^10,31. In addition, Proboningrat et al. demonstrated the potency of chitosan-based Pinus merkusii bark extract nanoparticles as anti-cancer on HeLa cells²⁷.

As in other species of pine bark, P. merkusii bark is thought to contain proanthocyanidins, which are considered contribute to suppressing cell proliferation and inducing apoptosis in some cancer cell lines without causing any significant effects on normal cells^15,32. Proanthocyanidins from Uncaria tomentosa L. extract were found to be cytotoxic to AGS human gastric adenocarcinoma and SW620 human colon adenocarcinoma cells³³. Moreover, P. massoniana bark proanthocyanidins could trigger apoptosis and hinder cell migration of A2780 human ovarian cancer cells¹¹.

Apoptosis is a mechanism of cell self-destruction and a critical circumstance of cell death, which occurs in response to various agents, such as chemotherapy drugs and ionic radiation¹². Among various initiators of apoptosis, ROS generated by natural compounds has an important role, since its excessive accumulation in cancer cells causes mitochondrial membrane potential (MMP) disruption and oxidative damage to DNA³⁴. Liu et al. reported that P. massoniana bark proanthocyanidins induce MMP loss in A2780 ovarian cancer cells¹¹. Apoptosis also involves a balanced transcription of genes that prevent and promote apoptosis, such as Bcl-2 and Bax. A study has proven that the expression of Bcl-2 was found to decrease, while the Bax was overexpressed in HeLa cervical cancer cells treated with P. koraiensis bark proanthocyanidins¹². Similar mechanisms might contribute to the cytotoxicity of PMBE on WiDr cells. It might be difficult to explain the sensitivity of cells to anticancer agents by only observing single related major factor because it might involve multiple factors which have not been evaluated. Hence, further research is very needed to uncover the underlying mechanisms of the anticancer activity of PMBE.

CONCLUSION:

In sum, PMBE exerts anticancer properties on WiDr human colon carcinoma cell line through cytotoxic activity in a concentration-dependent manner with an IC₅₀ of 235.60µg/mL which can be categorized to weak cytotoxicity. However, the current study is the first report on the anticancer potential of PMBE in WiDr cells. On the other hand, further observation is needed to find the optimal dosage and mechanism of action of PMBE as a strategy in human colon carcinoma treatment.

ACKNOWLEDGEMENT:

This study was supported by PMDSU Grant Funds from the Ministry of Research, Technology, and Higher Education of the Republic of Indonesia (Number: 1341/UN3.14/LT/2018). We thank to EJA – Professional Translation Services for editing the manuscript.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 17.03.2020 Modified on 19.04.2020

Research J. Pharm. and Tech 2021; 14(3):1685-1688.

DOI: 10.5958/0974-360X.2021.00299.7