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Table of Contents
ORIGINAL ARTICLE
Year : 2021  |  Volume : 5  |  Issue : 1  |  Page : 42-46

Cytotoxicity of red fruit ethyl acetate extract (Pandanus conoideus lam.) on squamous cell carcinoma cell line (HSC-3)


1 Department of Oral Biology, Faculty of Dentistry, Maranatha Christian University, Bandung, Indonesia
2 Department of Periodontics, Faculty of Dentistry, Trisakti University, West Jakarta, Indonesia
3 Department of Biochemistry and Molecular Biology, Faculty of Dentistry, Trisakti University, West Jakarta, Indonesia

Date of Submission10-Aug-2020
Date of Decision18-Oct-2020
Date of Acceptance09-Nov-2020
Date of Web Publication16-Feb-2021

Correspondence Address:
Melanie S Djamil
Department of Biochemistry and Molecular Biology, Faculty of Dentistry, Trisakti University, West Jakarta
Indonesia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/SDJ.SDJ_57_20

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  Abstract 


Background: Oral squamous carcinoma is a malignancy of the head-and-neck area that comprises 90% of all oral cancers. Research continues to look for therapies with low concentrations of cytotoxicity to reduce morbidity in patients with tongue carcinoma. The red fruit plant (Pandanus conoideus Lam.) is believed to have anticancer activity because of its antiproliferation activity. The high antioxidant content in red fruit is able to ward off and break free radicals that carry carcinogen compounds. Red fruit ethyl acetate extract has the highest antioxidant activity compared with other fractions, such as water, chloroform, methanol, and n-hexane. Objective: This study sought to evaluate whether red fruit ethyl acetate extract is able to inhibit the growth of the Human Squamous Carcinoma (HSC-3) cell line with varying concentration levels and exposure times. Method: The HSC-3 cell line was treated with extract concentrations of 10 μg/mL, 20 μg/mL, and 40 μg/mL and exposure times of 6 and 12 h. Doxorubicin was used as a positive control, and dimethyl sulfoxide was used as a negative control. The percentage of viable HSC-3 cells was calculated through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay cytotoxicity test. All groups were statistically compared, and P < 0.05 was considered to be a statistically significant difference. Result: A concentration of 20 μg/mL with an exposure time of 6 h and a concentration of 10 μg/mL with an exposure time of 12 h showed a significant difference compared with the positive control of doxorubicin (P < 0.05). Conclusion: The results showed that the higher the concentration of red fruit ethyl acetate extract, the lower the percentage of viable HSC-3 cells.

Keywords: Cytotoxicity, HSC-3, oral squamous cell carcinoma, Pandanus conoideus Lam., red fruit


How to cite this article:
Rahmawati DY, Anggraini W, Djamil MS. Cytotoxicity of red fruit ethyl acetate extract (Pandanus conoideus lam.) on squamous cell carcinoma cell line (HSC-3). Sci Dent J 2021;5:42-6

How to cite this URL:
Rahmawati DY, Anggraini W, Djamil MS. Cytotoxicity of red fruit ethyl acetate extract (Pandanus conoideus lam.) on squamous cell carcinoma cell line (HSC-3). Sci Dent J [serial online] 2021 [cited 2021 May 11];5:42-6. Available from: https://www.scidentj.com/text.asp?2021/5/1/42/309549




  Background Top


Oral cancer is a global issue and is one of the most common malignant cancers in Asia. About 274,300 new cases of oral cancer appear in Asia each year.[1],[2] Oral squamous cell carcinoma, especially of the tongue, is the most common malignancy of the head-and-neck area, comprising 90% of all oral cancers. The risk factors for this cancer are primarily exogenous, including tobacco and alcohol, which affect DNA mutation rates.[1],[2] High levels of cigarette and alcohol consumption in the world, especially in Indonesia, increase an individual's risk of developing oral cancer, especially tongue carcinoma. Failures that often occur in cancer therapy include a lower therapeutic effect compared with the massive stem cell ability of cancer cells, meaning that tumor cells remain alive and have a high ability to undergo mitosis. Therefore, researchers continue to seek therapies with low concentrations of cytotoxicity to reduce morbidity in cancer patients.[3]

There are many plants that people now believe to be anticancer; this has been supported by a large amount of research, and these herbal remedies can be relatively easy to find. Many studies in this field have shown that herbs have various specific anticancer mechanisms. The red fruit plant (Pandanus conoideus Lam.) is a source of provitamin A originating from Papua. The high content of carotenoids (beta-carotene) in red fruit makes it empirically viable for the treatment of cancer, arteriosclerosis, rheumatoid arthritis, and stroke.[4],[5],[6],[7] Carotenoids have several biological activities, such as the precursor of Vitamin A, antioxidant component, inhibiting free radicals, immune function settings, and cell division and proliferation settings.[5],[6],[7],[8] The high antioxidant content in red fruit is able to ward off and break free radicals of carcinogenic compounds. Red fruit ethyl acetate extract has the highest antioxidant activity compared with other fractions, such as water, chloroform, methanol, and n-hexane.[9],[10],[11],[12]

Many studies on the effects of red fruit extract on antiproliferation activity have been performed. Among other results, it has been reported that in vitro, red fruit extract can suppress the proliferation of lung cancer cell lines A549 and Lewis lung cancer, sarcoma 180, and cervical cancer cell lines HeLa, K562, and CaSki. However, its effects on human tongue squamous carcinoma (HSC-3) cell line have never been studied.[13],[14],[15],[16],[17] The current research related to oral cavity cancer cell therapy, including cell line HSC-3, is developing in the world of dentistry research. HSC-3 cell line has a high metastatic ability and high apoptosis induction capability through modulation of the transduction a signal that involves interleukin-6 as a result inhibition factors a transcription response cellular nuclear factor-kappa ß.[18] Red fruit ethyl acetate extract is also able to induce apoptosis through intrinsic and extrinsic pathways involving caspase-8, Bid, cytochrome C, and caspase-3. The main apoptotic pathway activates caspase dependent which, in turn, activates caspase-3. Taking this research gap into consideration, this study aims to evaluate whether red fruit ethyl acetate extract is able to inhibit the growth of the HSC-3 cell line with varying concentration levels and exposure times.


  Materials and Methods Top


The identification of red fruit plants was certified by the Ministry of Agriculture of the Agricultural Quarantine Agency of the Republic of Indonesia, Merauke Province, Papua, Indonesia. The taxonomy process was conducted at the Department of Chemistry FMIPA (Faculty of Math and Science) Laboratory at Padjadjaran University, Jatinangor. The extraction of red fruit ethyl acetate was carried out in the Laboratory of the Aretha Medika Utama Biomolecular and Biomedical Research Center, Bandung. Ethyl acetate extract of red fruit in the form of paste was obtained through the extraction of active compounds from red fruit (P. conoideus Lam.) with a 70% ethanol distillation, followed by two-stage maceration was performed using 70% ethanol and ethyl acetate. A preliminary study show the lowest concentration 10 μg/mL already reduce HSC-3 cell line viability.[6],[7] The concentrations of red fruit ethyl acetate extract were 10 μg/mL, 20 μg/mL, and 40 μg/mL, respectively. The HSC-3 cell line from ATCC (serial UPCI: SCC090 (ATCC® CRL-3239™) was cultured at the Biological Collaborative Research and Education Centre (BioCore) Faculty of Dentistry, Trisakti University. The HSC-3 cell line, a squamous cell epithelial human cancer of the oral cavity, was cultured in Dulbecco's modified Eagle's medium (Gamma Scientific Biolab, Malang, East Java) containing 10% fetal bovine serum and 1% penicillin-streptomycin. To remove cells attached to the flask or plate, 0.025% trypsin-ethylenediaminetetraacetic acid in phosphate-buffered saline (PBS) was used. Dimethyl sulfoxide (DMSO) was used as a negative control, while doxorubicin was used as a positive control.

Observation of cell viability was carried out with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay with a concentration of 5 mg/mL. The stopper reagent used was sodium dodecyl sulfate in 0.1 N HCl. The HSC-3 cell line with a concentration of 5 × 103 cells/100 L/well was distributed into the 96-well plate and incubated for 24 h at 37°C, 95% humidity atmosphere with 5% CO2 for cell adaptation and adherence to the wells. The next day, the media was removed, the adhered cells were washed with PBS, and then, 100 μL of culture media containing samples with concentrations of 10 μg/mL, 20 μg/mL, or 40 μg/mL of red fruit ethyl acetate extract was added to the wells containing washed cells and incubated for 6 or 12 h at 37°C with 95% humidity atmosphere and 5% CO2. After the media was discarded, MTT reagents at a concentration of 5 mg/mL were added and incubated for 3 h until formazan crystals formed. The reaction was stopped by the addition of a stopper reagent. The amount of formazan was determined by measuring the absorbance at 595 nm using the microplate reader (Bio-Rad, Hercules, California). In this study, each treatment was performed in triplicate. The percentage of cell viability was measured using the formula below:



Statistical analysis

Data from treatment results for 6 and 12 h were analyzed first using the Shapiro–Wilk normality test, and then, it was found that the data distribution was normal where P > 0.05. Differences between treatment groups were analyzed using two-way analysis of variance (ANOVA). The groups were compared, and P < 0.05 was considered statistically significant.


  Results Top


[Figure 1] shows that the DMSO-negative control group had the highest viability percentage compared with the doxorubicin-positive control and treatment groups. Regarding the treatment with 10 μg/mL, 20 μg/mL, or 40 μg/mL of red fruit ethyl acetate extract, the results showed at [Table 1] that as the concentration of red fruit ethyl acetate extract increased, the viability percentage of the HSC-3 cell line decreased.
Figure 1: Graph of the HSC-3 cell line viability after being treated for 6 and 12 h calculated via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. * P < 0.05 was considered statistically significant

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Table 1: The HSC-3 cell line viability after being treated for 6 h and 12 h calculated via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide technique

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Two-way ANOVA tests comparing the HSC-3 cell line viability results from the 10 μg/mL, 20 μg/mL, and 40 μg/mL treatment groups with the doxorubicin-positive control and DMSO-negative control groups at exposure times of 6 h and 12 h showed a significant difference between both 20 μg/mL concentration, exposure time of 6 h, and 10 μg/mL concentration, exposure time of 12 h, and the positive control of doxorubicin (P < 0.05). In the other data, there was no significant difference between the treatment groups and the control groups.


  Discussion Top


Research to find therapies with low concentrations of cytotoxicity continues to be conducted using cell line HSC-3, which is a model of tongue squamous cell carcinoma cells isolated from biopsy specimens. This study used cell line HSC-3 because it is easier to culture, has high metastatic ability and high metabolic ability, and has the ability to induce high apoptosis.[18]

Natural materials can yield safer and more biologically beneficial drugs compared with synthetic molecules.[19] There are variations in natural materials that have been shown to have better biocompatibility with biological systems and have low side effects. This suggests that natural ingredients can be options for alternative treatment.[20] Red fruit has antioxidant activity and antiproliferation activity that is close to exceeding even the activity of doxorubicin, a chemotherapy drug.[6] The high antioxidant content in red fruit is able to ward off and break free radicals of carcinogenic compounds.[11] Red fruit ethyl acetate contains secondary metabolites phenolics, tannins, saponins, triterpenoids, steroids, and alkaloids. The antioxidant properties of phenolic compounds in plants and fruits have strong activity and low toxicity compared to synthetic phenolic antioxidants such as butylated hydroxytoluene, butylated hydroxyanisole, and propyl gallate.[11],[12]

The red fruit ethyl acetate extraction process in this study was carried out with a maceration technique at room temperature because antioxidant compounds are sensitive to hot temperatures. Ethanol 70% was used to filter several compounds optimally. Ethyl acetate has semi-polar properties, so it was expected to filter a xanthone compound called α-mangostin, which is a highly effective antioxidant. α-mangostin as a highly effective antioxidant only can be extracted with semi-polar solvent. Rohman et al. showed that red fruit ethyl acetate extract has the highest antioxidant activity (10.35 ± 0.86 ppm) when compared with red fruit extract or oil itself, as well as compared with other red fruit fractions such as water, chloroform, methanol, and n-hexane.[9]

In the cell viability test for the HSC-3 cell line treatment group, there was a significant difference between the doxorubicin-positive control group and both the 20 μg/mL concentration, exposure time of 6 h, and the 10 μg/mL concentration, exposure time of 12 h. Among the other groups, there were no significant differences found. The percentage of viable cells at the concentration of 40 μg/mL at 6- and 12-h exposure times was 23.46% and 16.58%, respectively, close to the doxorubicin (positive control) group's results of 21.48% and 6.55%. Therefore, it can be concluded that red fruit ethyl acetate extract causes a decrease in viability to the HSC-3 cell line and that the higher the concentration and the longer the exposure time, the greater its toxicity.

Decreased cell viability can be interpreted as a cytotoxic effect of compounds or suboptimal conditions. The result of decreased viability in the HSC-3 cell line accords with previous research that has stated that red fruit has cytotoxic effects and antiproliferation activity on cervical cancer cells HeLa and K562. Red fruit antiproliferation activity against HeLa and K562 cervical cancer cells can approach levels even greater than that of doxorubicin at increasingly high concentrations.[6] Previous research has shown that red fruit extract can suppress the growth of lung cancer cells (A549 cell line) in vitro and that red fruit n-hexane extract can inhibit the growth of breast cancer cells (T47D).[13],[14],[15],[16],[17],[21]


  Conclusion Top


The administration of red fruit ethyl acetate extract (P. conoideus Lam.) at concentrations of 10 μg/mL, 20 μg/mL, and 40 μg/mL for 6 h and 12 h was able to decrease the viability of the HSC-3 cell line. Increased concentration and exposure time caused the percentage of viable cells in the HSC-3 cell line to decrease significantly. Although, this research still has to be investigated further using experimental animals for the development and use of red fruit ethyl acetate extract (P. conoideus Lam.) as an alternative material for chemotherapy and then mitochondrial transmembrane potential assay is required to see alterations in the potential of the mitochondrial transmembrane.

Acknowledgment

The authors would like to thank the Biological Collaborative Research and Education (BioCore) Faculty of Dentistry, Trisakti University for the invaluable support for this study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Suhartiningtyas D, Chrismawaty B, Agustina D, Subagyo G. Toluidine Blue Vital Staining as a Diagnostic Tool in Tongue Squamous Cell Carcinoma. Maj Ked Gi Ind (Dent J). 2012;19:136-40.  Back to cited text no. 1
    
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Rohman A, Sugeng R, Che Man YB. Characterization of red fruit (Pandanus conoideus Lam.) oil. Int Food Res J 2012;19:563-7.  Back to cited text no. 7
    
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Bisen P, Khan Z, Bundela S. Biology of oral Cancer key Apoptotic Regulators. Boca Raton: CRC Press Taylor & Franciss Group; 2014. p. 21-22, 49-59, 109-421, 163-169.  Back to cited text no. 10
    
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Lim TK. Pandanus conoideus. In: Lim TK, editor. Edible Medicinal And Non Medicinal Plants – Volume 5, Fruits. Dordrecht: Springer; 2013. p. 117-23.  Back to cited text no. 13
    
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Sarungallo ZL, Murtiningrum, Santoso B, Roreng MK. Nutrient content of three clones of red fruit (Pandanus conoideus) during the maturity development. Int Food Res J 2014;23:1217-25.  Back to cited text no. 14
    
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