https://doi.org/10.3889/oamjms.2020.3289 eISSN: 1857-9655
Category: A - Basic Sciences Section: Pathology
Apoptotic with Double-Staining Test, P53, and Cyclooxygenase-2 to Proliferation Colon Cancer Cell (WiDr) of Dolichol in Three Mangrove Leaves
Meighina Atika Istiqomah1, Mohammad Basyuni2,3*, Poppy Anjelisa Zaitun Hasibuan1
1Department of Pharmacology, Faculty of Pharmacy, Universitas Sumatera Utara, Medan 20155, Indonesia; 2Department of Forestry, Faculty of Forestry, Universitas Sumatera Utara, Medan 20155, Indonesia; 3Center of Excellence for Mangrove, Universitas Sumatera Utara, Medan 20155, Indonesia
Abstract
BACKGROUND: Mangroves secondary metabolites are mostly consisting of sterols, ubiquinones, isoprenoids, and polyisoprenoids. Polyisoprenoid is divided into two types, namely, polyprenol and dolichol, which has been reported to have biological and pharmacological activities.
AIM: This research was aimed to analyze apoptosis 48 h with double staining and immunocytochemistry (ICC) 48 h of P53 and cyclooxygenase-2 (COX-2) gene expression from chemical constituents of dolichol in three mangrove leaves of Ceriops tagal, Nypa fruticans, and Rhizophora mucronata.
METHODS: Apoptosis with the double-staining method was employed to analyze the genes expression in growth and development of cancer cells, P53, and COX-2 with ICC and flow cytometry method. The data were statistically analyzed using one-way ANOVA parametric statistical analysis followed by Duncan’s test.
RESULTS: The result revealed that the increased apoptosis of samples C. tagal was 70% fluorescence orange, while N. fruticans and R. mucronata were 35% and 30% fluorescence orange, respectively. However, it was compared with the positive control; it produced orange fluorescent as much as 75%, suggesting that C. tagal have a position similar to 5-FU. Predominance dolichol in N. fruticans and C. tagal leaves led the expression gene of p53 to have 1.57% M1 phase, indicating the domination in G0-G1 phase (70–80%). Inhibit the expression for 48 h in p53 and COX-2 showing that n-hexane extract of C. tagal had the most percentage (80.733 ± 0.11%) to upregulate the p53 and less percentage (20.16 ± 1.19%) to downregulate the COX-2, indicating positive extract belong to N. fruticans and R. mucronata leaves.
CONCLUSION: The present study confirmed the pharmacological properties of dolichol from three mangrove leaves as an anticancer of tumor suppressor genes and significantly proliferated of cancer cell growth from mangrove leaves.
Edited by: Ksenija Bogoeva-Kostovska Citation: Istiqomah MA, Basyuni M, Hasibuan PAZ.
Apoptotic with Double-Staining Test, P53, and Cyclooxygenase-2 to Proliferation Colon Cancer Cell (WiDr) of Dolichol in Three Mangrove Leaves. Open Access Maced J Med Sci. 2020 Feb 05; 8(A):37-42.
https://doi.org/10.3889/oamjms.2020.3289.
Keywords: Polyisoprenoid; Dolichol; Anticancer; Ceriops tagal; Nypa fruticans; Rhizophora mucronata
*Correspondence: Mohammad Basyuni, Department of Forestry, Faculty of Forestry, Universitas Sumatera Utara, Medan 20155, Indonesia. E-mail: [email protected] Received: 26 June 2019 Revised: 07 February 2020 Accepted: 08 February 2020 Copyright: © 2020 Meighina Atika Istiqomah, Mohammad Basyuni, Poppy Anjelisa Zaitun Hasibuan Funding: Funding: This research was supported by Penelitian Tesis Magister 2019 scheme (11/E1/
KP.PTNBH/2019) from the Directorate for Research and Community Service, Ministry of Research, Technology and Higher Education, Republic of Indonesia.
Competing Interests: The authors have declared that no competing interests exist.
Open Access: This is an open-access article distributed under the terms of the Creative Commons Attribution- NonCommercial 4.0 International License (CC BY-NC 4.0)
Introduction
Mangroves plants are known to produce a varied range of secondary metabolites such as sterols, ubiquinones, isoprenoids, and polyisoprenoids.
Polyisoprenoid comprised polyprenol and dolichol, which is efficacious to have anticancer and antibacterial activities [1], [2], [3]. The composition and diversity of polyisoprenoids compounds in North Sumatran mangrove forests, Indonesia, have been described [4], [5]. Mangrove plants are, therefore, an ideal plant model to study the biological activity of polyisoprenoid at the cellular level [5].
Recently, the screening of cytotoxic activity of polyisoprenoids of 17 mangrove plants against colon cancer has been reported [1]. Polyisoprenoid extract from Nypa fruticans has been shown as potent cytotoxicity effect [1] as well as polyisoprenoids from
Rhizophora mucronata and Ceriops tagal leaves significantly induced apoptosis and suppressed the expression of Bcl-2 and cyclin D1 protein [1].
However, the cytotoxicity potential of polyisoprenoids in mangrove leaves remains unclear concerning the cytotoxicity effect in other genes and protein expression.
Despite the importance of new possible chemotherapy and chemopreventive agents from mangrove polyisoprenoids, a few studies have focused on the biological activity role of polyisoprenoids. Here, we extend our work and the present study was aimed to analyze anticancer against proteins that oppose the proliferation of colon cancer cells, namely, p53 (tumor suppressor gene) and cyclooxygenase-2 (COX-2) from chemical component of dolichol from three mangrove leaves, C. tagal, N. fruticans, and R. mucronata and to determine the apoptotic of colon cancer cells with a double-staining test.
Materials and Methods
The mangrove leaves were collected from Lubuk Kertang, Langkat mangrove forest, North Sumatra Province, Indonesia: N. fruticans, C. tagal, and R. mucronata. The species of plants are determined at the Bogor Research Center for Biology, the Indonesian Institute of Science (LIPI). Cell lines and cell culture conditions (WiDr) cells isolated human colon cancer cells from the large intestine of 78-year-old women obtained from the Laboratory of Parasitology collection, Faculty of Medicine, Gadjah Mada University, Yogyakarta, Indonesia. WiDr cell lines cultured in RPMI 1640 medium and supplement with 10% (v/v) fetal bovine serum, 1% penicillin and streptomycin, fungizone 0.5%, and in a 37°C incubator with 5% CO2 [6].
N. fruticans, C. tagal, and R. mucronata dry leaves are homogenized by blending, to obtain 500 g of powder, then maceration with chloroform:methanol (2: 1, v/v) for 48 h and this treatment procedure follows the procedure as stated in Kurisaki et al. and Sagami et al. [7], [8]. Lipid extract from leaves was saponified at a temperature of 65°C for 24 h in 86% ethanol comprising KOH 2 M. The non-saponified portion was then dissolved with n-hexane; then, the solvent was evaporated. Then, the extract was redissolved in n-hexane.
The procedure for apoptosis with double- staining method was carried out by taking the cell from the CO2 incubator, then observe the conditions and the cell calculated then prepared the 24-well plates and slipcover. A 200 µl of cell suspension was transferred just above the coverslip evenly and slowly. The cell kept for 3–30 min in the incubator, to attach to the coverslip, 800 μl of culture media was added for 48 h incubation.
Dispose of culture media slowly then washed cell with phosphate-buffered saline (PBS) of 500 µl each.
Sample and media were inserted into the well for cell control and incubated. Dimethyl sulfoxide (DMSO) solvent dissolved into the well for 10 h. All media from the well were removed with the Pasteur pipette slowly.
Cells in wells washed with PBS. The coverslip was taken using tweezers, placed the coverslip on the glass object, and labeled it. Ten microliters of the reagent mixture of ethidium bromide-acridine orange were added over the slipcover. The mixture was flattened gently rocking. The apoptosis was observed under a fluorescence microscope [9].
Inhibition expression of p53 and COX-2 was analyzed using immunocytochemistry (ICC) methods, which was performed as described previously by Galgano with slight modifications [10], [11]. WiDr cells were seeded on 24-well plate, first included coverslip on each well. Cells were seeded with a density of 5×104 cells/well, incubated for 48 h at 37°C with 5% CO2. Furthermore, the polyisoprenoids from N. fruticans, C. tagal, and R. mucronata leaves in various concentrations were added to the cells and incubated for
48 h with 5% CO2. The cells were washed with PBS.
Then, cells were placed in the glass object for 5 min and added hydrogen peroxidase to the glass object and incubated at room temperature for 10–15 min. The cells washed twice with PBS and onto the glass object then added p53 (Santa Cruz Biotechnology, Dallas, TX, USA) and COX-2 [10] and incubated 1 day at room temperature. The cells were washed 3 times with PBS, then added with secondary antibody, and incubated at room temperature for 15 min, and washed twice with PBS, added 3,3-diaminobenzidine, then incubated for 5 min. The cells were washed with distilled water and added hematoxylin solution and incubated for 10 min at room temperature. Inhibition expression of p53 and COX-2 was statistically analyzed using one-way ANOVA parametric statistical followed by Duncan’s test. Data were expressed as the mean ± standard deviation of triplicate experimental value (n = 3).
Expression p53 was analyzed with flow cytometry of C. tagal. First, cell WiDr was harvested are then counted, taken 2.5 ul cells inserted into the cone then adverted with culture media up to 12 ml. Cells were taken as much as 1 ml, put into the well (inside plate 6 wells). Cell distribution was observed and documented and created a series of sample concentrations (IC50 and 1/5 IC50). Take cells in each well and media, then discard media with micropipette, wash with PBS as much as 500 ul in each well, put 1 ml sample with concentration series and 1 ml culture media then incubate for 48 h, provide 6 cones for each cell well, take media from 1 ml well, add 500 ul PBS to in the well, add 200 EDTA 0.25% trypsin, incubate for 5 min 1 ml MK, resuspend until the cells are released one by one, then observe under the microscope, transfer it to the cone, add 500 ml PBS to the well/5 min, drops 500 ul alcohol 70%, centrifuge with 600 rpm/5 min, and add propidium iodide as reagent flow cytometry for 30 min, the results are read with flow cytometer [10], [11].
Results
Figure 1 shows the apoptosis double-staining test and is detected by staining acridine orange- ethidium bromide. This method based on differences in DNA fluorescence in cells that live and die due to the binding of acridine orange-ethidium bromide [11], [12].
Cell control after being observed in a fluorescence microscope, it appears that cells produce green as much as 99%. With the treatment of samples, N. fruticans and R. mucronata only occupied 35% and 30% fluorosis orange (dead cells) while C. tagal was 70% fluorosis orange (dead cells). Moreover, when compared with the positive control (5-FU), it produces orange fluorosis as much as 75% which means that C. tagal may have a position similar to the first-line therapy cancer colon (5-FU).
To test the COX-2 expression by ICC method for 48 h, it showed that COX-2 expression was decreased which means that COX-2 levels decreased in colon cancer (WiDr), to angiogenesis inhibition (new blood vessel formation) occurred [12], [13].
Figure 2 depicts the n-hexane extract from C. tagal, N. fruticans, and R. mucronata decreased the percentage of COX-2, 20.16, 27.93, and 44.24%, respectively. The positive control (6-FU) was reduced by 12.11% in the COX-2 expression.
Figure 2: Cyclooxygenase-2 relative expression from Ceriops tagal, Nypa fruticans, Rhizophora mucronata, and 5-FU
Based on Table 1, it is noteworthy that polyisoprenoid from C. tagal leaves of 120 µg/mL and 24 µg/mL concentration increases the COX-2 expression of 20.16 ± 1.19 % and 30.90 ± 0.16 %, respectively.
Similarly, polyisoprenoid content from N. fruticans leaves of 260 µg/mL and 52 µg/mL enhances the COX-2 percentage of 27.93 ± 1.24 % and 41.21 ± 0.12 %, respectively and polyisoprenoid from R. muconata leaves of 320 µg/mL and 64 µg/mL concentration increases the COX-2 expression of 44.24 ± 1.10 % and 48.41 ± 0.34 %, respectively. On the other hand, control positive (5-FU) with the treatment of 18 µg/ml and 3.6 µg/ml, the expressions were 12.11 ± 1.01% and 18.14 ± 1.71%. Table 1 shows that C. tagal leaves have the most potent apoptotic potential than N. fruticans and R. mucronata because C. tagal and N. fruticans have dolichol compound 100% [14].
Table 1: Percentage of COX-2 from 48 h of treatment
Treatment Expression of COX-2 (%)
Control 66.36±0.34
C. tagal 120 µg/mL 20.16±1.19*
C. tagal 24 µg/mL 30.90±0.16*
N. fruticans 260 µg/mL 27.93±1.24*
N. fruticans 52 µg/mL 41.21±0.12*
R. mucronata 320 µg/mL 44.24±1.10*
R. mucronata 64 µg/mL 48.41±0.34*
5-FU 18 µg/mL 12.11±1.01*
5-FU 3.6 µg/mL 18.14±1.71*
*Referred to significant difference to control (p<0.05), C. tagal: Ceriops tagal, N. fruticans: Nypa fruticans, R. mucronata: Rhizophora mucronata, COX-2: Cyclooxygenase-2.
Figure 3: Cyclooxygenase-2 expression 48 h in WiDr cell (10 × 40) (a) Cell control WiDr; (b) Nypa fruticans 260 µg/mL; (c) N. fruticans 52 µg/mL; (d) Ceriops tagal 120 µg/mL; (e) C. tagal 24 µg/mL;
Rhizophora mucronata 320 µg/mL; (g) R. mucronata 64 µg/mL; (h) 5-FU 18 µg/mL; (i) 5-FU 3.6 µg/mL
: Positive expression: : Negative expression It has been shown that dolichol from C. tagal leaves 120 μg/mL inhibits COX-2 protein expression.
Similarly, administration of dolichol from C. tagal leaves 120 μg/mL showed that purple cells are more precise than controls [Fgure 3]. Furthermore, the percentage of COX-2 expression suppression data in each treatment and control group was not significant from statistically analyzed using one-way ANOVA parametric statistical analysis followed by Duncan’s test. Statistical analysis showed COX-2 protein expression suppression in various treatment and control groups significant difference at p < 0.05.
Based on Table 1, the increase in the level of the extract was able to provide significantly increased percentage COX-2 expression when compared with the control.
Figure 4 shows that the increased percentage of p53 for the n-hexane extract C. tagal rather than N. fruticans and R. mucronata which each is 80.73%,74.74%, and 66.81% in this research using positive control (5-FU) is 86.11%.
Figure 4: p53 expression from Ceriops tagal, Nypa fruticans, Rhizophora mucronata, and 5-FU
Table 2 shows that C. tagal has the same potential as 5-FU in increasing value p53 (tumor suppressor genes) than cell control that has the potential to prevent the development of cancer cells.
Figure 1: Apoptotic with double-staining test. (a) Cell control, (b) 5-FU, (c) Ceriops tagal, (d) Nypa fruticans, (e) Rhizophora mucronata
d
b c a
e d
h i
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g
b
f a
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The p53 is a tumor suppressor protein that acts as a regulator cell cycle.
Table 2: Relative expression p53 from 48 h of treatment
Treatment Expression of p53 (%)
Control 66.36±0.34
C. tagal 120 µg/mL 80.73±0.11*
C. tagal 24 µg/mL 73.27±0.32*
N. fruticans 260 µg/mL 74.74±2.74*
N. fruticans 52 µg/mL 60.22±0.17*
R. mucronata 320 µg/mL 66.81±1.54*
R. mucronata 64 µg/mL 56.76±0.13*
5-FU 18 µg/mL 86.11±2.43*
5-FU 3.6 µg/mL 77.86±0.49*
*Referred to significant difference to control (p<0.05). C. tagal: Ceriops tagal, N. fruticans: Nypa fruticans, R. mucronata: Rhizophora mucronata.
Figure 5: p53 expression 48 h in WiDr cell (10 × 40) (a) Cell control WiDr; (b) Ceriops tagal 120 µg/mL; (c) C. tagal 24 µg/mL; (d) Nypa fruticans 260 µg/mL; (c) N. fruticans 52 µg/mL; (f) Rhizophora mucronata 320 µg/mL; (g) R. mucronata 64 µg/mL; (h) 5-FU 18 µg/
mL; (i) 5-FU 3.6 µg/mL
: Positive expression: : Negative expression It has been shown that dolichol from C. tagal leaves 120 μg/ml increased p53 expression (Figure 2).
Similarly, administration of dolichol from C. tagal leaves 120 µg/ml shows that purple cells are more transparent than controls. Furthermore, the percentage of p53 expression suppression data in each treatment and
control group was statistically analyzed using one-way ANOVA parametric statistical analysis followed by Duncan’s test. Statistical analysis showed that p53 expression in various treatment and control groups gave a significant difference (p < 0.05). The increase in the dose level of the given test extract was able to provide a significantly increased percentage p53 expression when compared with the control [Figure 5].
Dolichol content (100%) [14], as a significant compound in C. tagal leaves, has more dolichol concentration than R. mucronata (90.2%) [14] tas shown in Figure 6 to have higher p53 expression, were found to have in 1.57% M1 phase which indicated dominated in G0-G1 phase (70-80%).
Discussion
The present data enable us to analyze the apoptosis using double-staining test. The working principle of apoptosis testing is the double-staining method, orange acridine penetrated all parts of the cell, and the nucleus will appear green color, whereas ethidium bromide only intercalated with cell membranes already damaged and the nucleus will be red. The color caused by ethidium bromide in cells death is dominant when compared to orange, so the nucleus in the dead cell was orange color [15]. Living cells with intact membranes have a green nucleus uniform. As long as the cell undergoes an apoptosis process, and membrane begins to occurs, ethidium bromide can enter the cell and give an orange color [15].
COX-2 is overexpressed in many malignancies, such as in the colon, lung, mammary, prostate, bladder, abdomen, and esophagus. Therefore, COX-2 may play a role in promotion and progression of tumors [16]. In this regard, COX-2 plays a key role in the formation of prostaglandin as a trigger for vascular endothelial growth factor (VEGF) expression [17]. COX-2 is an enzyme that induces inflammatory and prostaglandin biosynthesis [18] and is expressed at very high levels in tumor cells. Emphasis on VEGF expression inhibits the angiogenesis which is the process of forming new blood vessels that play a role in cell transfer (metastasis) and ensure premature cancer cells to survive so that the inhibition of angiogenesis causes cancer cells died because lack of nutrients and oxygen from blood and cannot metastasize or spread to other body parts [19].
COX-2 is an enzyme that plays a role in turning arachidonic acid into prostaglandin. This final product of COX-2 contributes to various factors biological in triggering tumor growth. COX-2 will induce occurrence angiogenesis through three products of arachidonic metabolism, namely, TXA2, PGI2, and PGE2, which stimulates VEGF to form new blood vessels [20], [21], [22], [23]. Besides that, COX-2 too acts as an d
h i
c
g
b
f a
e
File: P53 WIDR KS.008 Sample ID: P53 WIDR KS Acquisition Date: 09-May-19 Total Events: 20000
Marker Events % Gated % Total Mean Median All 20000 100.00 100.00 3.48 2.53
M1 314 1.57 1.57 22.08 16.25
File: P53 WIDR CERIOPS.013 Sample ID: P53 WIDR CERIOPS Acquisition Date: 09-May-19 Total Events: 20000
Marker Events % Gated % Total Mean Median All 20000 100.00 100.00 5.62 4.61
M1 643 3.21 3.21 24.36 16.11
File: P53 WIDR 5FU.011 Sample ID: P53 WIDR 5 FU Acquisition Date: 09-May-19 Total Events: 20000
Marker Events % Gated % Total Mean Median All 20000 100.00 100.00 6.25 5.67
M1 830 4.15 4.15 19.68 15.40
Figure 6: p53 expression (a) Cell control, (b) Ceriops tagal, (c) 5-FU c
b a
immunosuppressant which causes a decrease in cytotoxic activity from NK cells, COX-2 also inhibits the occurrence of apoptosis and increases activity from MMPs so that the risk of tumorigenesis increases and invades metastasis tumor [24], [25]. COX-2 is an inducible form and is not detected in all normal networks. However, COX-2 is induced by various types of inflammation and mitogenic stimuli [26].
The p53 protein plays a key role in response to cellular stress, for example, exposure to carcinogens.
This protein inhibited the proliferation of abnormal cells that have been initiated carcinogens to prevent the development of neoplasms. Protein inactivity can cause malignancy until cancer is malignant [13]. Besides functioning regulating cell proliferation, p53 also regulates apoptosis, inhibits angiogenesis, and regulating DNA repairment. In cancer, p53 is mutation generally [27]. Most p53 mutations a lot happens missense mutation. The mutation can be in the form of degradation of p53, loss of the ability of p53 induces cell cycle arrest or apoptosis, and loss affinity of p53 to bind damaged DNA [28]. This circumstance can be opened with the increase of p53 and decrease COX-2 where p53 and COX-2 work on G0-G1 phase on cancer cell cycle, so the growth and development of colon cancer cells (WiDr) are inhibited.
Percentage dolichol of C. tagal and N.
fruticans is 100% [14] although the percentage dolichol of R. mucronata is 90.2% [14] which is in the same max range (90–100%). The three species belong to type-1 of polyisoprenoid categories, the predominance of dolichol over polyprenol. The difference between the three mangrove plants is the location and condition of the plant soil, where the factors that affect it are salinity. Most salinity R. mucronata is more tolerant than N. fruticans and C. tagal [29]. C. tagal leaves have 75, 80, and 85 carbon chain length of the polyisoprenoid alcohols, N. fruticans leaves have 75, 80, 85, and 90 carbon chain length of the polyisoprenoid alcohols, and R. mucronata leaves have 75, 80, 85, 90, and 95 carbon chain length of the polyisoprenoid alcohols [14].
The shorter the carbon chain, the higher the level of dolichol from the mangrove plant [30]. The results of apoptosis testing indicate this and upregulation of p53 and downregulation of COX-2 so that the growth and development of colon cancer cells to form new blood vessels and get the energy to continue the cell cycle of the colon cancer is blocked.
Conclusion
This study confirmed the pharmacological properties of dolichol from three mangrove leaves as an anticancer of tumor suppressor genes, significantly apoptotic and proliferated of cancer cell growth from mangrove leaves.
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