INTRODUCTION:

International Agency for Research on Cancer (2018), reported that global cancer cases have risen to 18.1 million new cases and 9.6 million deaths in 2018¹. Up to the year of 2016, 1, 308,061 cancer patients in Indonesia is undergoing treatment². The etiology of cancer is still unknown, but according to Ling et al. (2018) cause of cancer is related to smoking, alcohol consumption, and viral infection (Human Papilloma Virus)³. Other cancer etiologies include accumulating genetic alterations, including mutations, amplification, or DNA deletions⁴.

Nowadays, chemotherapy is a standard modality in cancer management.

Most chemotherapy drugs have unwanted side effects such as nausea, vomiting, anemia, mucositis, diarrhea, thrombocytopenia, bleeding, and hair loss. These drugs are also not selective against cancer cells and lead to toxicity and drug resistance^5,6. This issue has led to the development of alternative technologies that are safer and more effective in curing cancer, one of which is nanoparticle technology⁷.

Nanoparticles are biological and synthetic materials with dimensions <1μm (International Institute Cancer). Nanoparticles have functioned as a carrier for drugs or anti-cancer substances. The small size of the nanoparticles facilitates drug administration and can penetrate directly into the target cell^8,9. Nanoparticles can enter and accumulate into the cancer cells through endocytosis. Among the various types of nanoparticles, biopolymer-based nanoparticles are the most biocompatible and inert with active substances. Thus, biopolymers are widely developed in matrix carrier systems¹⁰. Chitosan is a biodegradable polysaccharide widely used to synthesize nanoparticles and comes from crustacean shells such as crab, shrimp, and lobster¹¹.

Herbal medicines have fewer side effects compared with synthetic medicines. Moringa oleifera plant has been used as a drug in various types of diseases¹². Moringa leaves are part of the Moringa plant containing phytochemicals such as isothiocyanates, flavonoids, polyphenols, terpenoids, alkaloids, and saponins¹³. These phytochemicals show anti-cancer activity through induction of caspase-3 activity, decrease in anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) and increase in pro-apoptotic proteins such as Bcl2-Associated X protein (BAX)^14–16.

With the high incidence of cancer and the great benefits of Moringa oleifera as an anti-cancer agent, as well as the many advantages of the nanosystem in carrying drugs to cancer cells, the authors aim to prove the potential of chitosan-based Moringa oleifera leaves extract nanoparticles (Nano-MOLE) as an anti-cancer agent in cancer cells (in vitro).

MATERIALS AND METHODS:

This research was a true laboratory experimental study with a post-test-only controlled group design. The study has been approved by the Health Research Ethics Committee, Faculty of Dental Medicine, Universitas Airlangga (approval number 467/ HRECC.FODM/ VII/ 2019) (466/HERCC.FODM/VII/2019).

Preparation of Moringa oleifera leaves extract.

Moringa oleifera leaves were harvested from the Lawang sub-district in Malang, Indonesia. Harvested leaves (300g) were sterilized and dried, then softened using a blender. Then maceration was done by adding 96% ethanol solvent with a ratio of 1:2 to obtain filtrate for three days. The whole filtrate is then rotary evaporated until a thick extract is obtained.

Preparation of chitosan-based Moringa oleifera leaves extract nanoparticles (Nano-MOLE).

Chitosan-based Moringa leaves extract nanoparticles were prepared using the ionic gelation method¹⁷. Chitosan (0.2 g) was dissolved in 100mL of 1% glacial acetic acid. Moringa leaves extract (20mg) was dissolved in 700μL ethanol in a glass bottle. The solution of Moringa leaves extract and ethanol was added to the 50mL of chitosan and 1% glacial acetic acid solution. Glass bottles that contain a mixture of the solution are placed on a magnetic stirrer. The solution mixture formula was then added with 0.5mL of Tween 80. Then added 0.1% NaTPP by 10mL to the formula. The formula is mixed on a magnetic stirrer for 16-18 hours at a speed of 1000rpm.

Particle size analysis:

The laser diffraction method is used to measure the size of the moringa leaf extract nanoparticles using Mastersizer (Malvern Mastersizer 3000 Instrument Ltd., UK).

Phytochemistry Screening:

Phytochemical tests used Thin Layer Chromatography (TLC) methods to screen alkaloid, flavonoid, polyphenol, and terpenoid or steroid compounds. Froth test was used to screen saponin compounds and glycoside by using anhydrous acetic acid and sulfuric acid.

Cell culture:

HeLa cells line were obtained from the Department of Stem Cell (Institute of Tropical Disease, Universitas Airlangga, Indonesia) were cultured on Roswell Park Memorial Institute (RPMI) media (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA), penicillin-streptomycin 2% (Gibco, USA). Cells were incubated in a 5% CO₂ incubator at 37°C.

Experimental procedure:

The study began with the preparation of Nano-MOLE. Characterization of Nano-MOLE determines the particle size and distribution. It was then divided into five concentrations (20, 40, 60, 80, 100µg/mL in DMSO) for the MTT test to determine the IC50 value. Based on IC50 values, chitosan-based Moringa leaves extract nanoparticles were divided into three concentrations (250, 500, 750µg/mL). The samples (HeLa cells line) were divided into four groups, namely treatment group 1, treatment group 2, treatment group 3, and control group, then treated with Nano-MOLE with 250, 500, 750, and 0µg/mL, respectively. The caspase-3 expression of each groups were observed by immunofluorescence test.

Determination of IC50 value:

IC50 values were obtained through the MTT assay18. HeLa cells line (5×103 cells/well) were planted on 96-well plates and incubated 24 hours (37°C, 5% CO₂). Then induced Nano-MOLE with different concentrations (20, 40, 60, 80, 100µg/mL) into each well and incubated for 24 hours. 25µL of MTT solution (414.32g/mol, Sigma- Aldrich, USA) was added to all wells and re-incubated for 4 hours. If formazan was clearly formed, 50µL of 10% SDS (288.38g/mol, Sigma-Aldrich, USA) was added. The absorbance of the formazan formed was calculated using a Benchmark Microplate Reader (Bio-Rad, USA) at a wavelength of 595nm. The percentage inhibition results are used to calculate the IC50 value using linear regression analysis with SPSS.

Immunofluorescence staining:

HeLa cells suspension (4x104) was transferred over coverslip to 24-well plates, then incubated for 5 minutes in a 5% CO₂ incubator at 37°C. Before being induced with Nano-MOLE with series of concentrations (250, 500, dan 750μg/mL) for 24 hours, fixation using 500μL methanol (Merck, Germany) for 10 minutes, then rinsed with PBS (Sigma-Aldrich, USA).

Provision of blocking solution with (H₂O₂: methanol 1:10) then incubated for 10 minutes. It was then rinsed with distilled water followed by PBS. Coverslip taken with the tip of the needle and then placed in 24 well plates. 20μL of Blocking serum (Merck, Germany) was dropped on coverslip dan incubated for 10 minutes in a dark room at room temperature. The caspase-3 polyclonal antibody (Rabbit polyclonal AG1227703, BIOSS, USA) was dripped 50µL, then washed with PBS for 2 minutes. Then, 50μL of biotin (Sigma-Aldrich, USA) was added and incubated in a dark room at room temperature. 50μL of streptavidine peroxidase (Sigma-Aldrich, USA) was dripped and re-incubated in a dark room at room temperature, then washed with distilled water two times. 50μL of fluorochrome substrate (Bioss, USA) was dropped and set aside for 10 minutes, then washed with distilled water. The slide is immersed in alcohol and dried at room temperature mounting and reading of an immunofluorescence microscope (Olympus CKX53). Caspase-3 protein expression was evaluated using ImageJ software.

Statistical analysis:

Linear regression test by SPPS used to determine IC50 values. The normality test was conducted with the Shapiro-Wilk test. Then the homogeneity was assessed with the Levene test, followed by the Kruskal-Wallis and Post Hoc tests to analyze differences between groups of variables. If the p-value was lower than 0.05, the differences were considered to be significant.

RESULTS:

Particle Size Analysis:

The particle size distribution ranges from 0.01 – 0.5µm (10 – 500nm), with the smallest size of 18.7nm.

Phytochemical Screening:

The Nano-MOLE contains alkaloids, terpenoids or steroids, and saponins but does not contain glycosides, polyphenols, and flavonoids.

The IC50 value of Nano-MOLE on HeLa cells:

MTT assay was used to determine the ability of Nano-MOLE to inhibit the HeLa cells growth and to determine IC50 values. MTT assay was performed on treated HeLa cells with different concentrations (20, 40, 60, 80, and 100μg/mL). Nano-MOLE showed an increased ability to inhibit cell growth, dose dependent (figure 1), with an IC50 value of 287.13μg/mL after 24 hours of incubation.

Figure 1. Comparison graph of the mean number of cells expressing caspase-3 on HeLa cells in each study group.

Caspase-3 expression of HeLa cells induced by Nano-MOLE:

Caspase-3 expression in HeLa cells after induced Nano-MOLE was tested using immunofluorescence and evaluated using ImageJ software. In figure 2, the expression of caspase-3 treated with Nano-MOLE with concentrations of 250, 500, and 750μg/mL was compared with the control group. The average number of cells expressing caspase-3 in the control group, treatment group 1 (250μg/mL), treatment group 2(500 μg/mL), and treatment group 3(750μg/mL) are 18525.50; 9599.67; 4618.83; and 576.17, respectively. A decrease in the number of cells expressing caspase-3 was found along a higher Nano-MOLE concentration.

Statistical analysis:

Normality test showed normal distribution in the data of all groups. The homogeneity test using Levene test showed the data is inhomogeneous, indicated by the significance value is lower than 0.05. Thus, the statistical analysis was proceed by non-parametric test using Kruskal-Wallis test. The Kruskal-Wallis test showed significant differences in all of the groups, showed by significance value of 0.001 (p<0.05). Further analysis using Post Hoc Tukey HSD multiple comparison test revealed significant differences between all of the treatment groups and each treatment group and control group.

Figure 2. Microscopic image of Hela cells with a magnification of 200x. (A) Control Group (K) indicates the presence of a bond between antigen and polyclonal caspase-3 antibody with green color. (red arrow); (B, C, D) Treatment group after induction of Nano-MOLE with concentrations of 250, 500, and 750 μg/mL, for 24 hours. Yellow color indicates a bond between antigens in the form of caspase-3 with antibodies polyclonal caspase-3 (blue arrow).

DISCUSSION:

Moringa leaves extract nanoparticles were made by ionic gelation using chitosan as a biocompatible and biodegradable biopolymer. The nanoparticle formula was measured using a particle size analyzer (Malvern., UK) with the measurement results of moringa leaf extract nanoparticles is 100-500nm. Then phytochemical screening was carried out, and it was found that Moringa leaves extract nanoparticles contained alkaloids, terpenoids, and saponins. This research about the potential of Nano-MOLE as an anti-cancer agent was tested in vitro on human cervical cancer in the form of HeLa cells line. MTT assay was performed to determine the cytotoxicity and IC50 values of Nano-MOLE. Treated cells were analyzed using the Immunofluorescence test to evaluate the expression of caspase-3 induced by Nano-MOLE on HeLa cells.

Previous studies contradict ethanolic Moringa oleifera leaf extract as a cancer-killing agent, so this study suggested that further research is needed to understand these new findings. Nanoparticles can be used to answer this case. The results of our study showed that Nano-MOLE induces anti-cancer effects on cervical cancer cells through MTT assay. Other previous studies have reported that Moringa leaves extract can reduce the proliferation of HeLa cells and several other cancer cells such as B16F10 melanoma cancer cells and astrocytoma cancer cells⁸. IC50 Nano-MOLE was obtained at 287.13 μg/mL. An agent is said to be strong anti-cancer if the IC50 value is < 100μg/mL and moderate anti-cancer if IC50 value is more than 100μg/mL. Thus, Nano-MOLE has moderate anti-cancer properties¹⁸.

Cancer is an abnormal cell growth that can be induced by the p53 gene mutation. The p53 gene that has a mutation causes dysregulation in the process of apoptosis¹⁹. Apoptosis is an effective approach in cancer therapy by eliminating tumor cells that have mutations or grows abnormally²⁰. The primary key of the apoptosis process is the proteolytic enzyme cysteine protease-3 (caspase-3)²¹. The results showed the expression of caspase-3 in cells treated with Nano-MOLE. This phenomenon indicated the induction of apoptotic processes in HeLa cells.

Nano-MOLE enters HeLa cells through endocytosis. Based on the particle size, nanoparticles enter the HeLa cell through the mechanism of macropinosis endocytosis, while based on particle load, nanoparticles enter the HeLa cell through a clathrin-dependent endocytosis mechanism. Both of these processes involve forming vesicles or endosomes, which envelop the Nano-MOLE⁶. Nano-MOLE endosomes fuse with lysosomes in the cytoplasm. Hydrolytic enzymes derived from lysosomes degrade chitosan that carrying Moringa oleifera leaves extract. The release of Moringa oleifera leaves extract involves the proton-sponge effect, i.e., Nano-MOLE with secondary or tertiary amine groups when interacting with acids, protonation, or the addition of protons or H⁺ ions to vesicles, so that the fusion between endosomes and lysosomes experiences osmotic swelling²². This can cause the fusion of endosome and lysosome lysis, and release of Moringa oleifera leaves extract into the cytoplasm of HeLa cells^23–25.

Many phytochemical contents in Moringa oleifera leaves can be used as anti-cancer agents such as isothiocyanates, flavonoids, alkaloids, terpenoids, and saponins¹³. In the phytochemical screening test, the Moringa oleifera leaves extract used in this study obtained alkaloids, terpenoids, and saponins. Isothiocyanates and flavonoids are not detected in phytochemical screening due to their high solubility towards Nano-MOLE using 1% glacial acid^26,27. Phytochemical release of Moringa oleifera leaves extracts such as alkaloids, terpenoids, and saponins in increasing apoptosis can go through the same pathway, namely increasing pro-apoptotic protein (BAX) regulation and decreasing anti-apoptotic protein (Bcl-2) regulation^14,16,28,29. BAX initiates the intrinsic mitochondrial pathway and integrates with the outer membrane mitochondria so that the pores in outer membrane mitochondria open and the mitochondria outer membrane permeabilization (MOMP) occurs^30,31. This condition results in cytochrome c (Cyt c) present in the intermembrane mitochondrial space released into the cytoplasm. Cyt c binds to the apoptosis factor-1 (Apaf-1), activating protease and activates caspase-9^19,32–34. Caspase-9 activates caspase-3 and causes the division of poly ADP-ribose polymerase (PARP) in the cell nucleus^20,35. Cleavage of PARP causes DNA fragmentation which induces the HeLA's cell death marked by the initiation of cytoplasmic shrinkage and chromatin condensation^19,36–38.

The highest expression of caspase-3 is found in 250 μg/mL concentrations. Meanwhile, a minimum caspase-3 expression was observed in 500μg/mL concentration and almost unexisting in 750μg/mL concentration. This condition may be explained by the cell death mechanism, mainly apoptosis. Further research is needed on the causes of cell death. The limitation of this study is that phytochemical screening can only determine six kinds of phytochemical and Nano-MOLE load should be known to understand the cellular uptake mechanism of nanoparticles in cancer cells. Further research on Nano-MOLE cytotoxicity to normal cells is needed to support this research, and further research needs to utilize Zetasizer to determine the nanoparticle load of Moringa oleifera leaf extract.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGEMENT:

The authors express our gratitude to Unit Pelayanan Penelitian, Faculty of Pharmacy, Universitas Airlangga for the extraction process of Moringa oleifera leaves 100% concentration and phytochemical screening, Professor Nidom Foundation for manufacturing chitosan-based moringa leaf extract nanoparticles, Department of Stem Cell, Institute of Tropical Disease, Universitas Airlangga for grow HeLa cells and to conduct MTT assay and immunofluorescence testing, and the Faculty of Dental Medicine, Universitas Airlangga that has approved ethics and provides facilities to support this research.

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Received on 28.08.2021 Modified on 17.05.2022

Research J. Pharm. and Tech 2023; 16(1):35-40.

DOI: 10.52711/0974-360X.2023.00007