INTRODUCTION:

Cancer is one of the most threatening diseases in the world of health. World Health Organization (2012) reported that the two types of cancer with the most deaths in the world are lung cancer (1.69 million victims) and liver cancer (788,000 victims)¹. Programmed cell death (apoptosis) in cancer cells occurs through 2 pathways, namely, the extrinsic pathway or the death receptor (DR) pathway which involves the immune system and the intrinsic pathway or mitochondrial pathway².

In the mitochondrial pathway, apoptosis is caused by the release of cytochrome-c through channel formation by the mitochondrial permeability transition pore (PTP) and Bax protein. Cytochrome-c released into the cytosol will form an apoptosome complex with Apaf-1, ATP, and caspase 9³. Extrinsic apoptosis is played by the coordination of the immune cells, both innate immune cells such as natural killer cells (NK) and macrophages, as well as adaptive immune cells like B cells and T cells. These immune cells are activated by the presence of cytokines, such as Interferon-gamma (IFN-γ) and Tumor necrosis factor-α (TNF-α).

Interferon-gamma is the main cytokine for MAC and plays a major role in cellular nonspecific and specific immunity. Interferon gammas are also called type II interferons and are produced by Th1 cells and natural killer cells (NK cells)⁴. These interferons are the main activator of macrophages⁵. Tumor necrosis factor-α is mainly produced by activated macrophages, T lymphocytes, and NK cells. Other cells also express lower levels of TNF-α, such as fibroblasts, smooth muscle cells, and tumor cells. The local effects of TNF release include killing target cells, regulating adhesion of molecules for cell migration, activating neutrophils and macrophages to kill microbes and other pathogens, stimulating the release of other cytokines (IL-1 and IL-6), and increasing MHC class I molecules to increase the presentation of pathogenic peptides⁶. Therefore, the systemic release of TNF-α plays an important role in the inflammatory process.

Indonesia has many types of plants that can be used for medicinal purposes. Okra (Abelmoschus esculentus L.), also known as slime beans in Riau and chickpeas in West Kalimantan, is a plant rich in polysaccharides. The fruits and leaves also contain vitamin C and secondary metabolite compounds¹. The fruit is fiber rich, low calorie, and has a slimy texture. Okra is often used as food ingredients in daily basis. It can easily grow in soil that is rich with humus and has a full sun exposure. It also needs a pH level of 6 to 6.7. The fruits can be harvested one week after the flowers bloomed⁷.

Okra fruit contains flavonoid compound in the form of catechin and quercetin oligomers and vitamin C^8,9. These components play role as exogenous antioxidant against free radicals^9,10. Okra pods also known to have a relatively high scavenger activity against superoxide compared to other vegetables^11,12. Besides, okra also has a phenolic component in the form of tannins. Tannin compounds can be antibacterial by damaging the membrane and antiviral by inhibiting the activity of viral enzymes. Wahyuningsih et al. have also proven that the polysaccharide okra (Abelmoschus esculentus L.) plays role as immunomodulator in mice (Mus musculus) infected with Staphylococcus aureus bacteria¹³. The administration of okra polyssacharide could increase spleen weight and B-lymphocytes proliferation. This role is due to the ability of okra polysaccharides to activate the nuclear factor kappa-light-chain-enhancer transcription factor of activated B cells (NF-кB) from normal cells^13,14. Activation of NF-кB increases the transcription of genes related to the production of cytokines which are needed to activate immune cells, both innate and adaptive immune cells.

In our previous studies, administering okra raw polysaccharide extract (ORPE) at a dose of 600 µg / mL was shown to be able to cause 9.13% of Huh7it cells to experience apoptosis and 88.85% of activated NK cells¹. We also proved that the administration of ORPE effectively modulated the immune system by suppressing the T-regulator level and stimulating the production of interleukin-2, in mice with hepatocarcinogenic conditions¹⁴. Therefore, we examined the effect of ORPE on cytokine production and apoptosis to determine its effectiveness as an immunomodulatory agent, especially for a liver cancer patient.

MATERIAL AND METHODS:

Preparation of okra raw polysaccharide extract:

The okra fruits were grown in Surabaya, Indonesia. After being cleaned, Fresh pods (500g) were cut, smoothen, and macerated with 500ml of distilled water for 24 hour. The maceration was done three times. Supernatants were collected. Then, it was centrifuged at 4300rpm for 5 min, precipitated in absolute ethanol 1X sample volume. After that, the mixture was incubated for 24 h at 4^oC and centrifuged. The pellet was dissolved in distilled water. Then, it was centrifuged and the supernatant was collected^1,7. The supernatant was lyophilized and the powder obtained was labelled as Okra Raw Polysaccharide Extract (ORPE).

Animal ethical clearance:

All animals were treated based on protocol and acclimated in a pathogen-free chamber. All procedures has been approved by the Animal Care and Use Committee (ACUC) of Veterinary Faculty, Universitas Airlangga, Surabaya, Indonesia (No.701-KE).

Experimental design:

The used samples consisted of thirty six mice. Mice were adult male mice (BALB/C), 3-4 months old, with total weight of 30 to 40g. Diethylnitrosamine (DEN) injection was performed to cause severe liver damage and promote the growth of the tumor^14,15. Mice were acclimatized for 14 days and then they were randomly divided into six groups: the normal control group without any treatment (CN), negative control induced by DEN with no ORPE administration (C-), positive control induced by DEN with 10mg/kg BB doxorubicin administration (C+), and ORPE treatment of dose 50 mg/kg BW (P1), 100mg/kg BW (P2) and 200mg/kg BW (P3) which previously have been injected with DEN. The 100mg/kg BB DEN was injected intraperitoneally for three days. Okra raw polysaccharides extracts administration was performed for four week by gavage. Blood samples wand liver samples were collected in the end of the experiments.

Serum isolation and cytokine assay:

The upper layer contained the serum was collected by centrifuging the whole blood at 2000-3000RPM for 20 minutes. Cytokine analysis of IFN-γ and TNF-α was performed using an ELISA kit (Bioassay Technology, Shanghai, China) according to the manufacturer’s protocol. Around 40μl blood serum was placed to wells and 50μl streptavidin-HRP and 10μl antibody (anti- IFN-γ; anti-TNF-α) were added to each wells. The mixtures were then incubated for 60 minutes at 37°C. After that, the plate was washed 5 times with wash buffer and each wells were added with 50μl substrate solutions, A and B. After 10 minutes incubation at 37°C in the dark, 50μl stop solution was added to each well. Within 10 minutes, the optical density was measured using a microplate reader at 450nm wavelength.

Hepatocytes apoptosis and necrosis analysis:

The liver was smashed and the supernatant was removed using a micropipette and transferred to a microtube. The supernatant containing hepatocytes was washed with PBS then centrifuged at 2500rpm for 5 minutes. The supernatant was then discarded and the pellets on each microtube were washed twice with Cell Staining Buffer BioLegend and centrifuged at 1500rpm at 4°C. Cells were re-suspended in Annexin V Binding Buffer with a concentration of 0.25-1.0 x 10cells/mL. A total of 100 μL of cell suspension was transferred to a 5mL test tube. A total of 5µL of FITC Annexin V and 10µL of propidium iodide solution were added, then vortexed. Cells were incubated for 15 minutes at room temperature (25°C) in dark conditions. A total of 400μL of Annexin V binding buffer was added to each test tube. The cells were then analyzed using the BD FACS Verse flow cytometer (Becton Dickinson BioScience).

Statistical analysis:

The statistical analysis was performed using the GraphPad Prism version 8 (GraphPad Software, La Jolla, CA, USA). The differences between the experimental and control groups were compared by the One-Way ANOVA test followed by the Dunnett test. All results were written as mean ± standard deviation (SD) of five independent experiments. P-values less than 0.05 was considered significantly different.

RESULT AND DISCUSSION:

The effectiveness of ORPE as an immunomodulator in this study was observed through its effect on cytokine levels. The immunosurveillance capability resulting from ORPE administration was expected to cause the death in cancer cells itself. Cancer cell death occurs through two pathways, namely, the extrinsic pathway or the death receptor (DR) pathway and the intrinsic pathway or mitochondrial pathway. The extrinsic pathway is played by the performance of the body's immune cells, both adaptive immune cells and innate immune cells. Meanwhile, the mitochondrial pathway involves various protein groups, such as Bax, Bcl, p53, and caspase groups, which can trigger apoptosis in cancer cells^3,16.

In our previous research, it has been proven that giving okra polysaccharide extract was able to activate the nuclear factor kappa-light-chain-enhancer of activated B cells or what we know as NF-κB transcription factor^1,13,14. The activation of transcription factor NF-κB has an impact on stimulating the production of cytokines in the body. In this study, the cytokines tested were TNF-α and IFN-γ.

Table 1. Data of TNF- α and IFN-γ levels on DEN-induced mice blood serum.

Groups	TNF-α level (µg/mL)	IFN-γ level (µg/mL)
CN	1.67 ± 0.103	2.20 ± 0.260
C-	1.69 ± 0.038	2.04 ± 0.160
C+	1.99 ± 0.079^**,##	2.83 ± 0.548^*,#
P1	1.99 ± 0.048^**,##	2.04 ± 0.218
P2	2.13 ± 0.192^***,###	2.09 ± 0.229
P3	2.08 ± 0.162^***,###	1.99 ± 0.358

Data are expressed as mean ± SD (n=5). The differences between the experimental and control groups were compared by One-Way ANOVA test followed by Dunnett test. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. control. #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. negative control.

The immunostimulant effect of ORPE was seen in the TNF-α cytokine. Tumor necrosis factor-alpha (TNF-α) is a type of proinflammatory cytokine that plays a role in inflammation in cancer. TNF-α levels showed a significant increase in the ORPE group at doses of 50, 100, and 200mg/kg BW, compared to normal controls and negative controls (Table 1). At doses of 100 and 200 mg/kg BW, TNF-α levels were successfully increased up to two times compared to normal controls and negative controls (Table 1). Kang et al. have previously revealed that administering Citrus unshiu polysaccharide extract can increase the production of pro-inflammatory cytokines TNF-α and IL-6 as well as anti-inflammatory cytokines IL-12 in RAW264.7 macrophages¹⁷. Another study by Zhou et al. has also proven that giving Lonicera japonica (LJP) polysaccharide extract can restore serum levels of IL-2 and TNF-α in mice whose immune system is suppressed by induction of cyclophosphamide¹⁸.

However, giving ORPE to carcinogenic mice did not affect the IFN-γ levels. The levels of IFN-γ had no significant differences compared to control groups (Table 1). Interferon-gamma is also called type II interferons and is produced by Th1 cells and NK cells. These interferons are the main activator of macrophages. Immune cells work collectively, integrated and have a hierarchy¹⁹. Studies using transgenic mice show that macrophages of mice that are not sensitive to IFN-γ are not efficient in the formation of Th1 subsets because these mice are unable to produce IL-12¹³. Previous research proved that giving ORPE did not affect increasing Th CD-4¹⁴. Th1 cell activation is characterized by secretion of IFN-γ and IL-12. The findings by Gazzinelli et al. and Hayaza et al. are also in line with the results of the present study, where administering ORPE to carcinogenic mice did not have an effect on IFN-γ levels therefore the percentage of activated Th1 cells on the previous study was not significantly different from the control group^14,20.

Figure 1. The percentage of apoptosis and necrosis cells on mice hepatocytes. Data were obtained by flow cytometry analysis. Data are expressed as mean ± SD (n=5). The differences between the experimental and control groups were compared by One-Way ANOVA test followed by Dunnett test. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. control. #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. negative control.

A normal cell undergoes regulated cell division, differentiation and apoptosis. When normal cell has lost the usual control over their division, differentiation and apoptosis they become tumor cells²¹. Apoptosis is the programmed cell death of various cells in an organism. In apoptosis, cell shrinkage occurs where there will be changes in biochemistry and cell morphology, where intracellular material is not dispersed out of the cell²². This is different from cell necrosis where intracellular material will be dispersed outside the cell². In healthy tissues, apoptosis prevents the propagation of faulty DNA unlike the cancer cells which evade apoptosis²³. The administration of ORPE did not affect the percentage of hepatocytes apoptosis (Fig.1). On the other hand, the administration of doxorubicin (C+ groups) significantly increased hepatocytes apoptosis compared to normal control groups (Fig.1). Therefore, the administration of ORPE at all doses is considered safe for healthy cells.

CONCLUSION:

In summary, the administration of ORPE can increase the levels of TNF- α. It did not cause a significant number of hepatocytes apoptosis in DEN-induced liver cancer mice. This study demonstrates that polysaccharide contents in crude okra pods extract may be a promising and safe immunomodulatory agent in nutraceutical and pharmaceutical therapies of a liver cancer patient in the future.

ACKNOWLEDGEMENT:

The authors thank the Ministry of Education of the Republic of Indonesia for giving the PMDSU doctoral scholarship for SH and RJKS. The authors also wish to thank Dr. Junairiah from the department of biology for providing taxonomic identification of okra. This work was funded by the DRPM research grant of Universitas Airlangga and the Ministry of Education of the Republic of Indonesia [893/UN3/2019].

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 27.04.2021 Modified on 20.06.2021

Research J. Pharm.and Tech 2022; 15(2):546-550.

DOI: 10.52711/0974-360X.2022.00088