Anticancer Effects of Polyisoprenoid From Nypa fruticans Leaves by Controlling Expression of p53,EGFR,PI3K,AKT1,and mTOR Genes in Colon Cancer (WiDr) Cells

Abstract

The current research seeks to examine the anticancer effect of polyisoprenoids from mangrove palm, Nypa fruticans, leaves in WiDr cells by analyzing the cell cycles of cancer and regulating the expression of p53, epidermal growth factor receptor (EGFR), PI3K, AKT1, and mammalian target of rapamycin (mTOR) genes by using the reverse transcription-polymerase chain reaction (RT-PCR). An inhibited cell cycle analysis was conducted using the flow cytometry, and the upregulation or downregulation of the expression of p53, EGFR, PI3K, AKT1, and mTOR genes was obtained using RT-PCR. The data were then statistically analyzed using one-way analysis of variance by a post hoc test, a parametric statistical analysis using Tukey’s honest significant difference. Polyisoprenoids in N. fruticans extracts worked as chemotheraupetic in the G0-G1 cycle is 79.0%, however, with positive control 5-fluorouracil as 88.1% and are carried out by the specific upregulation of the expression of the p53 gene and the downregulation of the expression of the EGFR, PI3K, AKT1, and mTOR genes. This study can also explain the significant pharmacological properties of the leaves of the species N. fruticans that work specifically in the G0-G1 phase to upregulate the expression of the p53 gene and downregulate the expression of the EGFR, PI3K, AKT1, and mTOR genes. This study also revealed polyisoprenoid (100% dolichol), which blocked the growth and development of WiDr colon cancer cells.

Keywords

polyisoprenoid anticancer up and downregulation

Cancer is the leading cause of death in the world in which colorectal cancer rank third in 2018.¹ Furthermore, more than 70% of deaths due to cancer occur in low-income and middle-income countries, and the percentage of patients diagnosed with cancer is escalating.¹ Risk factors for cancer include tobacco use, alcohol, obesity, environmental hazard viruses, and ionizing radiation.² Cancer is a disease with a growth phase faster than that of normal cells. Here, uncontrolled cells form lumps known as tumors or occur in blood vessels (leukemia) or lymph nodes (lymphoma).³ 5-Fluorouracil (5-FU) is most commonly used to the treat colon cancer; however, in the long term, it can have toxic effects or resistance to effectiveness.⁴

Nypa fruticans, a member of mangrove palm, is known to produce a variety of secondary metabolites such as lignin, hemicellulose, phenolic, flavonoid, and polyisoprenoids.^5
-7 Polyisoprenoids are long-chain secondary metabolites (>C50) found in almost all living cells, including various plant tissues.^7
-9 Furthermore, polyisoprenoid comprise of polyprenol and dolichol, which are efficacious in anticancer and antibacterial activities.^10
-12 The polyisoprenoid composition in N. fruticans has been reported to have 100% dolichol.⁷ Moreover, our previous screening study on cytotoxic and antiproliferative activity of polyisoprenoids in 17 mangroves found N. fruticans to exhibit the highest anticancer activity, with half-maximal inhibitor concentration (IC₅₀) at 180.2 µg/mL, on WiDr cells through the inhibition of cyclooxygenase-2.¹⁰ This study is an extension of our previous work to analyze the anticancer effects against gene expressions that oppose the proliferation of colon cancer cells, namely, p53, epidermal growth factor receptor (EGFR), PI3K, AKT1, and mammalian target of rapamycin (mTOR) pathway in colon cancer cells (WiDr) using reverse transcription-polymerase chain reaction (RT-PCR) in various inhibitions of growth, development of cancer cells, and development of molecular biology for the treatment of toxic chemotherapy.

Materials and Methods

Plant and Chemical Materials

Nypa fruticans Wurmb (Arecaceae) leaves were collected from Lubuk Kertang mangrove forest, Langkat, North Sumatra, Indonesia in February 2019. The mangrove forest is situated at 04°07′39.71″ North latitudes and 98°30′97.87″ East longitudes. The plant material has been identified in the Indonesia Institute of Science, Research Center for Biology, Bogor, Indonesia. A voucher specimen has been deposited in the herbarium. Chloroform, methanol, hexane, ethanol, HCl and KOH were obtained from Merck (Germany). Prediluted serum blocking, streptavidin horse enzyme peroxidase, 3,3′-diaminobenzidine, Mayer solution-hematoxylin, xylol, Roswell Park Memorial Institute (RPMI) 1640 medium, dimethylsulfoxide (DMSO), [3-(4,5-dimethylimidazole-2-yl)−2,5-diphenyl tetrazolium bromide], phosphate buffer saline (PBS) and sodium dodecyl sulfate were purchased from Sigma-Aldrich (USA). PI3K, AKT1, mTOR, p53, and EGFR genes were used.¹³ Regarding the condition of the colon cancer cell culture (WiDr) cells, the human colon cancer cells were extracted from the large intestine of a 78-year-old woman and later isolated. WiDr was cultured in RPMI 1640 and supplements with 10% (v/v) fetal bovine serum, 1% penicillin and streptomycin, and 0.5% fungizone were inserted inside an incubator at 37°C with 5% CO₂.^13

-16

Sample Preparation

Nypa fruticans leaves powder (500 g) was homogenized and macerated with solvent reagent chloroform:methanol (2:1, v/v) for 48 hours, and this treatment procedure was borrowed from the previous work.⁷ The lipid extract of the N. fruticans leaves was saponified at 65°C for 24 hours in 86% ethanol comprising of 2 M KOH.⁵ The unsaponifiable lipids of mangrove palm leaves were extracted with hexane, and the organic solvent evaporated and redissolved in hexane.⁹ Leaf extracts (50 μg) were applied to the thin layer chromatography plate to identify the polysioprenoid composition.⁷ The polyisoprenoid compound in N. fruticans leaves contained the dolichol family 100% (C75-C90) with quantities of 10.7 mg/g dolichol.⁷

Preparation of Stock Solution

Respectively, Fifty mg of polyisoprenoids from N. fruticans was dissolved in 10 mL of RPMI 1640 medium with the aid of 1 mL of DMSO (Sigma-Aldrich, St. Louis, MO, USA) to obtain a concentration of 5000 μg/mL.

Inhibition Analysis of Cellcycles with Flow Cytometry

The WiDr cells (5 × 10³ cells/well) were inserted inside a 6-well plate and incubated. Subsequently, the cells were treated with various concentrations of polyisoprenoid in N. fruticans and then incubated again. The floated cells found attached were collected by using 0.025% trypsin and then transferred into the cone tube. Then, 1 mL of PBS was added, after which PBS was extracted using a micropipette and centrifuged at 2500 rpm for 5 min. The liquid was released, and the RNase/propidium iodide (PI) dye solution added for 10 min in a dark place (avoiding light) at 37°C and then analyzed using the FACScan flow cytometer so that the percentage of cells obtained in each cell cycle phase (G1/S and G2/M) could be calculated using Modfit Lt. 3.0s.⁸

Analysis of Gene Expressions In Vitro with RT-PCR

The total ribonucleic acid (RNA) used (3000 ng) was reverse transcribed with a 1 µg random primer and ReverTra Ace (Toyobo) to yield complementary deoxyribonucleic acid (cDNA) (20 µL) for 10 min at 30°C, 60 min at 42°C, and 5 min at 99°C as per the manufacturer’s protocol. The resulting cDNA mixture was diluted in a TE buffer solution and directly used for the subsequent PCRs. Semiquantitative RT-PCR for p53, EGFR, PI3K, AKT1, and mTOR genes ^17

-21 was determined using 1 μL cDNA added to the 25 μL PCR Master Mix (12.5 μL GoTaxGreen, 9.5 μL DNase/RNase free water, 1 μL primer forward and 1 μL primer reverse, as displayed in Table 1). Subsequently, 8 µL of the mixed solution (5× RT-buffer 4 µL, random primary 1 µL, deoxynucleoside triphosphate 2 µL and Revers Tra-Ace 1 µL) was added. PCR was performed at 30°C for 10 min, 42°C for 60 min, and 99°C for 5 min.²² The RT-PCR product was obtained in the form of cDNA and stored at −20 °C. The expression of the gene was examined using 1 μL cDNA added to 25 μL PCR Master Mix with 35-40 cycles carried out for 15-30 s at 94°C, 45 s at 94°C, and 10 s at 55-60°C with the final extension phase at 72°C for 5 min; the PCR products were then stored at 4°C.

Table 1

Forward and Reverse Primer Sequences Used in This Study Were Derived From Previous Reports.^{^11

-15}

Genes		Primer sequence (5′-3′)	Product length (bp)
p53	F	5′-ATT CAG CTC CTC CTC CAT GAA GAA TCG GCG-3′	390
p53	R	5′-GCT TTG CTG CTG AGG CCA CCA GTA TCC ACT-3′	390
EGFR	F	5′-CAA CAT CTC CGA AAG CCA-3′	495
EGFR	R	5′-CGG AAC TTT GGG CGA CTA-3′	495
PI3K	F	5′-GGA CAA TCG CCA ATT CAG-3′	195
PI3K	R	5′-TGG TGG TGC TTT GAT CTG-3′	195
AKT1	F	5’′-ATG AGC GAC GTG GCT ATT GTG ATT-3’	330
AKT1	R	5′-GAG GCC GTC AGC CAC AGT CTG GAT G-3′	330
mTOR	F	5′-CCA ATC ATT CGC ATT CAG TCC-3′	110
mTOR	R	5′-AAC AAA CTC ATG TCC GTT GCT G-3′	110
β-actin	F	5′-GCT CCT CCT AAG CGC GAG T-3′	100
β-actin	R	5′-TCA TAC TCG CTG TCC TGC AT TTG-3′	100

The bands were documented using the image scanner Dox XR (Bio-Rad) Gel.²³ Testing of activity avoidance from the gene p53, EGFR, PI3K/AKT1/mTOR was carried out by amplifying cDNA in the WiDr cells with a test solution, after which the amplified cDNA results were added using electrophoresis on agarose 2% with medium 10% TBE.²³

Statistical Analysis

The data comprised density base pairs of p53, EGFR, PI3K, AKT1, and mTOR genes that were first analyzed statistically using one-way analysis of variance and then with a post hoc test consisting of the Tukey’s honest significant difference (HSD) test, significant at P < 0.05 as the threshold.

Results

Inhibition Analysis of Cell Cycles with Flow Cytometry

The results of the cell cycle inhibition analysis using flow cytometry testing were carried out at 488 nm and medium speed (500 cells/s) light emission with PI reagents.²⁴ Figure 1 and Table 2 show that polyisoprenoids in the n-hexane extraction of N. fruticans worked as a chemotherapeutic in the G0/G1 phase of the cell cycle is 79.0%. Furthermore, cell accumulation in G0/G1 phase in N. fruticans was found to be greater with the control cell at 76.6% but higher in 5-FU (88.1%) as displayed in Table 2.²⁵ A higher percentage of positive controls indicated the presence of accelerated cells in the preparation of DNA material to be synthesized.

Figure 1

Cell cycle with flow cytometry. (a) Control cell, (b) Nypa fruticans, and (c) 5-fluorouracil (5-FU, positive control).

Table 2

Percentage Accumulated in Each Phase of the Cell Cycle Treated with of Mangrove Leaves Nypa fruticans

Sample	Concentration (µg/mL)	Phase of the cell cycle (%)
Sample	Concentration (µg/mL)	G0-G1	S	G2-M
Control cell	-	76.6	7.2	16.2
N. fruticans	1/5 IC₅₀	79.0	7.0	14.0
5-Fluorouracil	1/5 IC₅₀	88.1	9.1	2.8

IC₅₀, half-maximal inhibitory concentration.

The cell cycles were examined using flow cytometry on WiDr cells using the staining method.²⁶ PI was used to dye the DNA so that it could be detected by the fluorescence-activated cell sorting detector.

Gene Expression with RT-PCR

The measurement of expression of the genes p53, EGFR, PI3K/AKT1/mTOR was carried out by amplifying the cDNA WiDr cells given a test solution. Subsequently, the amplified cDNA results were separated using electrophoresis on agarose 2% with medium tris-borate-ethylenediaminetetraacetic acid (TBE) 10% and an electric current of 70 mv. The results of WiDr cell electrophoresis are displayed in Table 3. The bands of EGFR and PI3K are more expressed than the control group (Table 3).

Table 3

Gene Expression of P53, EGFR, PI3K, AKT1, mTOR, β-Actin on the Treatments of WiDr Cell Line (A) Control Cell, (B) 5-FU, and (C) Nypa fruticans.

Gene	Treatment			Amplicon (bp)
Gene	(A)	(B)	(C)	Amplicon (bp)
p53				390
EGFR				495
PI3K				195
AKT1				330
mTOR				110
β-actin				100

The electrophoresis results obtained were analyzed using the Quantity One software (Bio-Rad) to acquire the density of bands in the order in which they appeared, after which the average and standard deviations were calculated. The results of the thickness of the density of the gene expression p53, EGFR, PI3K/AKT1/mTOR are shown in Table 4, where N. fruticans polyisoprenoid upregulated the p53 expression and, in contrast, polyisoprenoid downregulated the expression of EGFR, PI3K, AKT1, and mTOR genes. It is notable that EGFR and PI3K gene expressions showed significant difference either with control cell or 5-FU as depicted in Table 4.²⁶

Table 4

Density Expression of Genes P53, EGFR, PI3K, AKT1, and mTOR in WiDr Cells.

No	Genes	Treatment	Density pair ± standard deviation
1.	p53	Control cell	1.0 ± 0.0^b
		N. fruticans 1/5 IC₅₀	1.2 ± 0.0^a
		5-FU 1/5 IC₅₀	1.3 ± 0.0^a
2.	EGFR	Control cell	1.0 ± 0.00^b
		N. fruticans 1/5 IC₅₀	0.4 ± 0.0^a ^b
		5-FU 1/5 IC₅₀	0.7 ± 0.0^a
3.	PI3K	Control cell	1.0 ± 0.0^b
		N. fruticans 1/5 IC₅₀	0.4 ± 0.0^a ^b
		5-FU 1/5 IC₅₀	0.1 ± 0.0^a
4.	Akt1	Control cell	1.0 ± 0.0^b
		N. fruticans 1/5 IC₅₀	0.3 ± 0.0^a
		5-FU 1/5 IC₅₀	0.2 ± 0.0^a
5.	mTOR	Control cell	1.0 ± 0.0^b
		N. fruticans 1/5 IC₅₀	0.3 ± 0.0^a
		5-FU 1/5 IC₅₀	0.2 ± 0.0^a
6.	β-actin	Control cell	1.0 ± 0.0^b
		N. fruticans 1/5 IC₅₀	1.1 ± 0.0^a
		5-FU 1/5 IC₅₀	1.1 ± 0.0^a

IC₅₀, half-maximal inhibitory concentration; 5-FU, 5-fluorouracil.

^aSig (P) < 0.05; there is a significant difference with control cell.

^bSig (P) < 0.05; there is a significant difference with 5-FU.

Figure 2 shows the ratio of p53, EGFR, PI3K, Akt1, and mTOR genes to the internal standard (β-actin) evaluated using a one-way post hoc test and Tukey’s HSD. EGFRis a dominant growth factor that shows a significant difference with the control cell and 5-FU as well as PI3K (Figure 2). Additionally, the downregulation of the EGFR of colon cancer cells (WiDr) takes place in the G0-G1 cycle in the life cycle of cancer cells. By contrast, p53, Akt1, and mTOR display significant differences with the control cell (Figure 2).

Figure 2

The density value of gene expression p53, EGFR, PI3K, Akt1, and mTOR ratio to internal standard (β-actin). (a) Sig (P) <0.05, a statistically significant difference with the control cell; (b) sig (P) < 0.05, a statistically significant difference with 5-fluorouracil (5-FU).

Discussion

This article expresses the ability of N. fruticans polyisoprenoids to inhibit the expression of EGFR, PI3K, AKT1, and mTOR genes in WiDr cells as a therapeutic option in the treatment of colon cancer.²⁷ Moreover, p53 can be utilized in the apoptosis process and cell cycle of WiDr. In this context, PI3K/AKT1/mTOR is a pathway in the process of developing cancer cells. This pathway facilitates the process of cell division and deactivates the apoptosis process.²⁸ Furthermore, the activity of the PI3K/AKT1/mTOR genes that decrease indirectly can cause cell cycle inhibition events and trigger the occurrence of apoptosis so that the development of cancer cells is inhibited. Our previous study showed that polyisoprenoids (from N. fruticans leaves) worked in the WiDr colon cancer cell line.¹⁰ It has been compared with more than 60% of apoptosis at 90 µg/mL; G0-G1 cell cycle arrest did not appear in this study. We used different normal cells (control cells) and positive control compared with the present study. In the present work, no difference percentage accumulated in the G0-G1 cell cycle arrest as shown in Table 2.

Additionally, EGFR has become an important target for cancer therapy. Some reports indicate that an increase in the number of copies of EGFR or gene mutations responsible for stream signaling is important to determine the response or resistance to anti-EGFR antibodies.²⁹ Furthermore, the excessive activation of the PI3K pathway causes an increase in PIP2 to PIP3. This results in the formation of phosphoinositide-dependent protein kinase-1 phosphorylation and protein kinase B (PKB) phosphorylation so that PKB can become active. This protein is a central mediator in the signal transduction from the PI3K pathway and can also phosphorylate other intracellular proteins that play a role in cell cycle regulation, cell proliferation, DNA repair systems, and apoptosis.³⁰ To avoid geroconversion, cancer cells need to lose the expression of cell inhibitor cycles, such as p53.³¹ Thus, between p53 and the mTOR signaling pathway, cells running into apoptosis can be determined. Further, inhibitors from mTOR can suppress geroconversion and protect stem cells from undergoing temporary premature cell aging while preventing oncogenic transformation.³²

Moreover, 5-FU can inhibit cell cycles and apoptotic triggering without involving p53; however, it entails the increased expression of p21 and pRb, both of which play an important role in the checkpoint system in the G1 phase.³³ Furthermore, the overexpression of pRb will inhibit E2F activity so that it causes the inhibition of cells to exceed R. The p21 expression will also inhibit the activity of cyclin E/CDK2 and cyclin A/CDK2 and can thus inhibit the cell cycle in the G1 and S phase.^34,35 Cells that are in the G1 phase will stop there, and cells in the S phase will be stopped at that phase.³⁶ Resistance caused by 5-FU can occur through the mediation of cell cycle inhibition. Cancer cells with mutant p21 cannot spur the cessation of cell cycle so that it can directly accelerate apoptosis; however, cells with normal p21 that spur the cessation of the cell cycle can trigger the emergence of resistant cells.³⁷ 5-FU activity in apoptotic tracking can be through the p53 pathway (dependent or independent p53). Additionally, it has also been proven that 5-FU can induce apoptosis in cancer cells with p53 deficiency or have mutant p53.³²

The messenger RNA (mRNA) can be used to describe the expression of a gene because the amount can vary according to the environmental conditions compared with DNA, which is relatively stable. RNA is a biological macromolecule with a number of different functions, and mRNA transcribed from DNA serves as a template for protein synthesis, which is carried out by ribosomes consisting of ribosomal RNA (rRNA) and protein.³⁸ Amino acids for protein synthesis are sent to ribosomes by transferring the RNA (tRNA) molecules. Most of these RNA molecules consist of tRNA (15%-20%) and rRNA (80%-85%). However, mRNA constitutes only 1%-5% of the total RNA despite the actual amount depending on the type and physiological condition of the cell.³⁸

The phase in the normal cycle in the number of chromosome sets, namely G1, and the number of sets of chromosomes is 2 n.³⁹ The S phase is 2 n and 4 n while the G2 and M phases are 4 n.⁴⁰ The number of the sets of chromosomes determines the intensity of the fluorescence measured in the flow cytometry tool.⁴¹ The more sets of chromosomes, the greater the intensity of fluorescence.^42,43 PI3K/AKT1/mTOR is the pathway process for developing cancer cells and facilitates the processes of cell division and apoptosis.⁴⁴ The activity of the PI3K/AKT1/mTOR gene, which decreases directly, does not facilitate cell cycle inhibition and improvement of apoptosis so that the development of cancer cells is hampered.⁴⁵ The inhibition of displaying the PI3K/AKT1/mTOR gene by N. fruticans is inseparable from the composition of the secondary metabolites (polyisoprenoid) in the extract. The mixture of sesquiterpene lactones, such as vernodalol, vernodalinol, and vernomygdin, has been shown to inhibit phosphorylation of PI3K/AKT1 in breast cancer cells.^46,47 To support these results, in vivo experiments on polysioprenoids compounds as anticancer agents are needed to gain a complete understanding of the colon cancer mechanism in mangrove plants.

Supplemental Material

All data analyzed in this study are included in this published article.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The authors are sincerely grateful to the Universitas Sumatera Utara in the Talenta scheme of International Collaboration (4167/UN5.1.R/PPM/2019) and the Magister Research Thesis (PTM) Program 2019 (No. 11/E1/KP.PTNBH/2019) from Directorate for Research and Community Service, Ministry of Research, Technology and Higher Education, Republic of Indonesia.

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