View of The Cytotoxic Activity of Marine Sponge-Derived Fungus Aspergillus nomius NC06 Against HT29 Colon Cancer Cells

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http://dx.doi.org/10.11594/jtls.14.01.05 Research Article

The Cytotoxic Activity of Marine Sponge-Derived Fungus Aspergillus nomius NC06 Against HT29 Colon Cancer Cells

Muh. Ade Artasasta ¹, Akmal Djamaan ², Yanwirasti Yanwirasti ³, Muhammad Taher ⁴, Heder Djamaludin ⁵, Siswanto Siswanto ⁶, Dian Handayani ²*

1 Department of Biotechnology, Faculty of Mathematics and Natural Sciences, Universitas Negeri Malang, Ma- lang 65145, Indonesia

2 Laboratory of Sumatran Biota, Faculty of Pharmacy, Andalas University, Padang 25163, Indonesia

3 Department of Biomedical, Faculty of Medicine, Andalas University, Padang 25163, Indonesia

4 Faculty of Pharmacy, International Islamic University Malaysia, Kuantan 25200, Malaysia

5Fish Product Technology Study Program, Faculty of Fisheries and Marine Science, Universitas Brawijaya, Ma- lang 65145, Indonesia

6 Department of Statistic, Faculty of Mathematics and Natural Sciences, Universitas Hasanuddin, Makassar 90245, Indonesia

Article history:

Submission January 2023 Revised June 2023 Accepted July 2023

ABSTRACT

The study of natural products from marine-derived fungi has been interesting tense to researchers as drug discovery sources. Marine fungus from West Sumatera, Indonesia repeatedly showed their potential for cytotoxic and antimicrobial activities. This study aims to determine the cytotoxic activity against HT29 colon cancer cells of each fraction of ethyl acetate extracts from Aspergillus nomius NC06 derived from marine sponge Neopetrosia chaliniformis. A. nomius was cultivated with rice as a growth medium and extracted with ethyl acetate solvent and evaporated in vacuo to obtain ethyl acetate extract. Furthermore, the compounds of ethyl acetate extract were separated with the VLC (Vacuum Liquide Chromatography) method.

Five fractions were obtained, which further investigated their cytotoxic activity against HT29 colon cancer cells by using an MTT assay. The result showed that fractions I and III were categorized as potential fractions due to their IC50 value of 13.12 ± 0.39 μg/mL and 2.59 ± 0.19 μg/mL, respectively. It was also supported by ANOVA to measure the effect of each concentration (0.1; 1; 10; 100 μg/mL) of each fraction on the viability percentage of HT29 cells with p < 0.005.

Keywords: Aspergillus nomius, Cytotoxic activity, HT29 colon cancer cell

*Corresponding author:

E-mail: dianhanda- [email protected]

Introduction

Colon cancer is the third in incidence after lung and breast cancers and accounts for almost 10% of total cases of cancer and almost 8% of total cancer deaths. According to the World Health Or- ganization (WHO), more than 70% of all cancer deaths occur in countries with low and middle in- come, and deaths from cancer worldwide are pre- dicted to continue to rise to over 11 million in 2030 [1]. Hence, there is an increasing demand for cost- effective therapeutics and chemo-prevention agents for the various types of cancer. Several treatments for cancer disease, such as chemotherapy and radiotherapy. First, it was thought that

chemotherapy drugs specifically kill the cancer cells only, but nowadays it is well known that damage to the normal cells resulting from the chemotherapy dose-dependent side effects such as fatigue, nausea, and even death may also occur in severe cases [2]. Finding a safe and efficient lead compound for drug development for clinical trials is vital and urgent. Several studies have shown natural products, especially marine organisms such as marine sponges, to have potential chemo- prevention and anti-cancer properties as well as other pharmacological properties.

Marine sponges-derived fungus has attracted

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much attention from researchers and is known to be a source of secondary metabolites with potent biological activities. Some anticancer compounds have been reported from marine sponge-derived fungus. Altertoxin VII from the marine sponge-derived fungus, Alternaria sp. SCSIO41014 exhibited cytotoxic activities against human erythroleu- kemia (K562), human gastric carcinoma cells (SGC-7901), and hepatocellular carcinoma cells (BEL-7402) with IC50 values of 26.58 ± 0.80, 8.75 ± 0.13, and 13.11 ± 0.95 μg/mL, respectively [3]. Nursid et al. [4] also reported that an isolate of MFP270 from the marine sponge-derived fungus, Aspergillus sp. exhibited strong cytotoxicity against T47D cells with an IC50 value of 28.3 μg/mL. Cytochalasin Z10 and Cytochalasin Z11, isolate compounds, from the marine sponge-derived fungus Spicaria elegans exhibited cytotoxic activity against A-549 cells with IC50 values of 9.6 μg/mL and 4.3 μg/mL, respectively [5].

The study of marine fungus from West Su- matera as a potential source for antimicrobial and anticancer drugs has been conducted. Our previ- ous study reported that Aspergillus nomius, which was isolated from marine sponge N. chaliniformis, has the potential as an anticancer drug source due to its potent cytotoxic activity against the HCT116 colon cancer cell line while VERO normal cell has no cytotoxic activity [6,7]. Further study is needed to be conducted by bioassay-guided fractionation for characterization of the cytotoxic activity against the HT29 colon cancer cell line.

Material and Methods

Source of sponges and cancer cells

The materials used in this study were sponge N. chaliniformis obtained from Mandeh Island marine(1°6’-1°13’S,100°19’-100°25’E), HT29 colon cancer cell obtained from the International Islamic University Malaysia Pharmacy Labora- tory, Dulbecco's Modified Eagle's Medium (merk Sigma-Aldrich), Trypsin-EDTA (merk Sigma-Al- drich), Fetal Bovine Serum (merk Sigma-Al- drich), and Phosphate Buffered Saline (merk Sigma-Aldrich), and MTT reagent. The equip- ment in this research were round coverslip (merk Sigma-Aldrich), laminar airflow (merk AIR- TECH), CO2 incubator (merk Sigma-Aldrich), water bath (merk Memmert), cell banker, liquid nitrogen tank (merk ANTECH), High Perfor- mance Liquid Chromatography (merk Shimadzu), Vacuum Liquid Chromatography (Merck), and a

microplate reader (merk Sigma-Aldrich).

Sample collection and preparation

A marine sponge, N. chaliniformis, was collected from the waters of Mandeh Island, Pesisir Selatan Regency, West Sumatera, Indonesia (1°6’-1°13’S,100°19’-100°25’E) through scuba diving. The specimens were carefully cut to obtain 10-12 cm samples. Subsequently, the samples un- derwent immediate sterilization using 95% etha- nol and were further processed in the laboratory [8].

Cultivation and secondary metabolite extraction Fungal isolation from N. chaliniformis was conducted using a diluted method. Briefly, obtained A. nomius NC06 was cultivated with rice for 4-8 weeks. Next, secondary metabolite extraction was conducted using ethyl acetate in a ratio of 3:1. Crude ethyl acetate extract was obtained after evaporating the solvent by using a rotary evapora- tor.

Fractionation

Every compound from ethyl acetate of crude extract from A. nomius was separated by gradient elution methods using vacuum liquid chromatography. Elution was started in 100% n-hexane, fol- lowed by n-Hexane: ethyl acetate (1 : 1), ethyl acetate 100%, ethyl acetate, methanol (1 : 1), then methanol 100%. Every successfully separated compound was collected monitoring every same stain on thin layer chromatography. Furthermore, obtained fractions were submitted to the HPLC for characterization.

High-Performance Liquid Chromatography (HPLC)

Methanol HPLC was used to dilute every fraction in a ratio of 1:1. Up to 50µL of diluted fractions were pipetted out and inserted into the HPLC vial. Methanol was added until the total volume in the vial became 500 µL. UltiMate^TM 3000 UHPLC was used with a column size of 4.6 × 150 mm. The mobile phase consists of phase A (H2O with 0.1

% TFA) and phase B (methanol HPLC 100 %).

Gradient elution monitoring was conducted from 0 to 35 min with 10-100 % phase A and 35 to 60 min with 100-10% phase B.

Cytotoxic activity

The determination of the cytotoxic activity of

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each fraction against HT29 cells was carried out using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl-2H-tetrazolium bromide) method. The HT29 colon cancer cells were obtained from the Laboratory of Biotechnology and Cell Culture, Pharmacy Faculty, International Islamic Univer- sity Malaysia. The HT29 cells were cultured in a 25 cm³ T flask containing GibcoTM DMEM me- dia. After these cells grew with a confluent rate of above 80%, then these cells were transferred into 96-well plates with a density of 6x103 cells/well and, incubated at 37°C in a 5% CO2 incubator for 24 hours. After the incubation process (confluent level above 80%), each fraction of the ethyl acetate extract of fungus isolate NC06 (test solution) was added with various concentrations of 100 μg/mL, 10 μg/mL, 1 μg/mL, and 0.1 μg/mL and was again incubated at 37°C in a 5% CO2 incubator for 24 hours. Next, 100 L (5 mg/mL) of MTT reagent was added to each well, and the cells were again incubated. After incubation for 4 hours, DMSO (dimethyl sulfoxide) was then added to each well to stop the reaction after the addition of the MTT reagent. This cytotoxic activity test used Taxol as a positive control. The absorbance of the reaction with the addition of MTT reagent was measured using a Tecan Microplate reader at a maximum wavelength of 560 nm. The absorbance of each fraction was converted into percent cell viability, which was then used to generate a regres- sion curve to determine the IC50 [7, 8, 9].

Data analysis

The data were evaluated using parametric one- way analysis of variance (ANOVA) with Dun- can’s post hoc test for multiple comparisons when data showed significance (α 5%). All statistical analyses were performed using SPSS 25.0 soft- ware. Data are presented as mean ± SEM values.

Results and Discussion

Several studies have reported that the fungus A. nomius was a potential source of producing cytotoxic and other bioactive compounds. Most of the compounds derived from A. nomius are indole diterpenoids and some of these compounds con- tain a ring system that is thought to contribute to the cytotoxic characteristics associated with the compounds [10]. Among them are 14,25-dihy- droxyaflavinine, nominiene (indole diterpenoid), aspernomine, 14-hydroxypaspaline, 14-(N,N- Dimetyl-L-valyloxy)paspalinine. Aspernomine is

known to have cytotoxic activity against three human cancer cell lines: A-549 lung cancer cells, MCF-7 breast cancer cells, and HT29 colon cancer cells with an associated ED50 of 3.09, 4.93, and 3.08 μg/mL.

Five fractions were obtained from vacuum liquid chromatography using the gradient elution method (n-hexane, ethyl acetate, methanol). Table 1 shows the weight of each fraction. Fraction IV is the heaviest (4.02 g) while fraction I is the lightest (1.03 g). The HPLC chromatogram profiles for the five fractions can be seen in Figure 1. Each signal that appears at a wavelength of 340 nm indicates a different fraction.

Figure 1 shows that there is an inverse corre- lation between concentration and viability percentage. Based on the ANOVA for the interaction between the types of fractions with various concentrations, the p-value and R² were = 71.908 (p <

0.05) and 0.996, respectively (the effect is about 99.6%), which means there is a significant difference. The viability percentage of HT29 colon cancer cells to the interaction between each fraction with various concentrations (100, 10, 1, and 0.1 μg/mL) was significantly different. Duncan's Mul- tiple Range Test (DMRT) analysis indicated that fraction III is a fraction with a smaller average viability percentage and a different viability percentage value from other fractions.

In this study, the determination of the cytotoxicity of the five fractions of the ethyl acetate extract of A. nomius against HT29 cells was carried out using the MTT method. The MTT method provides a relationship between the absorbance of viable cells and dead cells to determine the IC50 value.

The value of the IC50 is the lowest concentration needed to inhibit 50% of the cell population. This value is obtained from the equation of the line between the viability percentage and the concentration log. The IC50 can indicate the potential of a compound to inhibit the growth of cancer cells or cytotoxic activity [11].

Table 1. Fraction weight No. Semi polar fraction

ethyl acetate extract

Fraction Weight

1. Fraction I 1.03 g

2. Fraction II 1.59 g

3. Fraction III 3.95 g

4. Fraction IV 4.02 g

5 Fraction V 3.98 g

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Fractions I and III had potent cytotoxic activity against HT29 cells when compared to other fractions with IC50 of 13.12 ± 0.39 μg/mL and 2.59

± 0.19 μg/mL, respectively (Table 2). The difference in cytotoxicity of each fraction is certainly affected by the compounds contained in the fraction. The library hit of every peak from each fraction can be seen in Table 2. Every fraction has a main peak that can be described as a major compound [9]. Based on Figure 2, fraction I has a major compound with a retention time of 31.74 min, which correlates to the compound versicolorin B.

Fraction III has a major compound with a retention time of 22.157 min, which correlates to the compound spongiacidin E. The U.S. National Cancer Institute stated that the potential of the natural product extract has an IC50 value of less than 20 μg/mL against mammalian cancer cells [12].

Thus, fraction I and III are categorized as potential

fractions due to their IC50 value of 13.12 ± 0.39 μg/mL and 2.59 ±0.19 μg/mL, respectively. Taxol was used as a positive control for cytotoxic activity analysis. Positive control was used to compare its IC50 value with the sample test and to prove that the cancer cells were sensitive to anticancer drugs [13]. In this study, the IC50 of Taxol was 0.412 μg/mL.

The study of bioactive compounds from marine-derived fungi repeatedly shows potential cytotoxic activity. El-Kashef et al. [14] reported that the sulochrin compound-derived marine fungus Aspergillus falconensis has cytotoxic effects by inhibiting the growth of L5178Y lymphoma cancer cells and the growth of MDA-MB 231 breast cancer cells with IC50 of 5.1 M. and 70 M, respectively. Yang et al. [15] also reported that brevi- ones I isolated from the marine fungus Penicillium sp. TJ403-1 showed cytotoxic effects against HL-

a b

c d

e

Figure 1. HPLC chromatogram profile of each fraction (a. Fraction I, b. Fraction II, c. Fraction III, d. Fraction IV, e. Fraction -V).

0,0 10,0 20,0 30,0 40,0 50,0 60,0

-30 0 20 40

70AR181106 #5 FI UV_VIS_4

mAU

min 1 - 31,747

WVL:340 nm

0,0 10,0 20,0 30,0 40,0 50,0 60,0

-40,0 -25,0 -12,5 0,0 12,5 25,0

40,0HW181221 #9 FII UV_VIS_4

mAU

min 1 - 22,4972 - 23,220

3 - 23,757

WVL:340 nm

0,0 10,0 20,0 30,0 40,0 50,0 60,0

-100 125 250 375 500

700AR181106 #7 FIII UV_VIS_4

mAU

min 1 - 17,873

2 - 19,550 3 - 20,000 4 - 20,530 5 - 22,157

6 - 22,963

7 - 23,523 8 - 24,8979 - 26,580

WVL:340 nm

0,0 10,0 20,0 30,0 40,0 50,0 60,0

-50 100 200 300

450AR181107 #6 FIV UV_VIS_4

mAU

min 1 - 16,353

2 - 18,330 3 - 19,553

4 - 19,993 5 - 20,563

6 - 21,480 7 - 22,173

8 - 22,953

9 - 26,573

WVL:340 nm

0,0 10,0 20,0 30,0 40,0 50,0 60,0

-30 0 20 40 60

80AR181107 #7 FV UV_VIS_4

mAU

min 1 - 22,290

2 - 23,027

WVL:340 nm

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Table 2. The library hit compound of every peak of each fraction Fraction Peak

Number Retention Time (min.) Similarity Percent-

age (%) The Library Hit Compound

I 1 31.74 99.5 Versicolorin B

II

1 22.497 95.1 Spongiacidin E

2 23.220 nd nd

3 23.757 nd nd

III

1 17.873 97.7 Menzamin J N-Oxide

3 20.000 nd nd

4 20.530 95.8 Menzamin J N-Oxide

6 22.963 nd nd

7 23.523 98.1 Norlichexanthone

8 24.897 98.8 Norlichexanthone

9 26.580 95.8 Notoamide E

IV

1 16.353 nd nd

2 18.330 nd nd

3 19.553 nd nd

4 19.993 nd nd

5 20.563 nd nd

6 21.480 nd nd

7 26.173 nd nd

8 22.953 nd nd

9 22.573 nd nd

V 1 22.950 nd nd

2 23.027 nd nd

nd: no detection

Table 2. Viability percentage and IC50 for each fraction

Fraction Code Average and Standard Deviation percent of Viability* IC50 (μg/mL)

Fraction III 48.50 ± 19.93^a 2.59

Fraction I 74.19 ± 48.28^b 13.12

Fraction IV 79.84 ± 34.7^c 35.77

Fraction II 103.55 ± 25.52^d 66.65

Fraction V 129.91 ± 14.7^e >1000

*Various letters (a, b, c, d, and e) indicate a significant difference in viability percentage between various fractions (p<0.05)

Figure 3. Viability percentage of HT29 cells: each fraction for various concentrations 20

40 60 80 100 120 140 160

1 0 0 1 0 1 0 . 1

Viability Percentage (%)

Concentration (mg/mL)

Fraksi I Fraksi II Fraksi III Fraksi IV Fraksi V

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60, A-549, and HEP3B cancer cells with each IC50

of 4.92 ± 0.65 M, 8.60 ± 1.36 M, and 5.50 ± 0.67 M, respectively.

Conclusion

The Aspergillus nomius NC06 isolated from the marine sponge Neopetrosia chaliniformis is a potential fungus isolate for use in the treatment of colorectal cancer based on its cytotoxic activity characteristics. Further research, showed fractions I and III of ethyl acetate extract of A. nomius NC06 is a potent fraction to inhibit the growth of HT29 colon cancer cells with IC50 of 13.12 ± 0.39 μg/mL and 2.59 ± 0.19 μg/mL, respectively. The presence of secondary metabolites that are responsible for the cytotoxic activity of fractions I and III. There- fore, further study is needed to determine the structure of cytotoxic compounds from A. nomius.

Acknowledgments

We appreciate the financial support BOPTN of Andalas University, Padang, Indonesia, pro- vided for the project titled Penelitian Dasar Unggulan Klaster Riset Publikasi Guru Besar (PDU-KRP1GBUnand), No.T/6/UN.16.17/PT.- 01.03/KO-PDU KRP1GB-Unand/2022. The au- thors also acknowledge the help and support of Prof. Dr. Peter Proksch from the Institute of Phar- maceutical Biology and Biotechnology, Heinrich- Heine University, Germany, and acknowledge the help of Anthony Michael Cannon from the Depart- ment of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, USA.

References

1. WHO (2008) International Agency for Research on Can- cer, World Health Organization- World Cancer Report.

2. Conklin KA (2004) Chemotherapy-associated oxidative stress: Impact on chemotherapeutic effectiveness. Inte- grative Cancer Therapies 3: 294–300. doi:

10.1177/1534735404270335

3. Pang X, Lin X, Wang P et al. (2018) Perylenequione derivatives with anticancer activities isolated from the marine sponge-derived fungus Alternaria sp. SCSIO41014.

Marine Drugs 16(8): 1–13. DOI: 10.3390/md16080280

4. Nursid M, Dewi AS, Maya D, Priyanti (2019) Cytotoxi- city of marine-derived fungi collected from Kepulauan Seribu Marine National Park. IOP Conference Series:

Earth and Environmental Science. 404(012006): 1–7. doi:

10.1088/1755-1315/404/1/012006

5. Liu R, Lin Z, Zhu T et al. (2008) Novel open-chain cyto- chalsins from the marine-derived fungus Spicaria ele- gans. Journal of Natural Products 71(7): 1127–1132. doi:

10.1021/np070539b

6. Artasasta MA, Djamaan A, Handayani D (2017) Cyto- toxic activity screening of ethyl acetate fungal extracts derived from the marine sponge Neopetrosia chaliniformis AR-01. Journal of Applied Pharmaceutical Science 7 (12): 174–178. doi: 10.7324/JAPS.2017.71225 7. Handayani D, Artasasta MA (2017) Antibacterial and cy-

totoxic activities screening of symbiotic fungi extract isolated from marine sponge Neopetrosia chaliniformis AR- 01. Journal of Applied Pharmaceutical Science 7(5): 66–

69. doi: 10.7324/JAPS.2017.70512

8. Handayani D, Artasasta MA, Safirna N et al. (2020) Fun- gal isolates from marine sponge Chelonaplysilla sp. Di- versity, antimicrobial and cytotoxic activities. Biodiver- sitas 21 (5): 1954–1960. doi: 10.13057/biodiv/d210523 9. Artasasta MA, Yanwirasti Y, Taher M et al. (2019) Cyto-

toxic and antibacteial activities of marine sponge-derived fungus Aspergillus nomius NC06. Rasayan Journal of Chemistry 12 (03): 1463–1469. doi:

10.31788/RJC.2019.1235284

10. Staub GM, Gloer KB, Gloer JB et al. (1993) New paspalinine derivatives with antiinsectan activity from the scle- rotia of Aspergillus nomius. Tetrahedron Lett 34(16):

2569–2572. doi: 10.3390/molecules27206870

11. Djamaludin H, Bintang M, Priosoeryanto BP (2019) Cy- totoxicity and antiproliferative effects of ethyl acetate fraction of Padina australis against MCM-B2 and K562 cell lines. Journal of Applied Biology & Biotechnology 7 (02): 25–29. doi: 10.7324/JABB.2019.70205

12. Boik J (2001) Natural compounds in cancer therapy. Or- egon Medical Press, Minnesota.

13. Wani MC, Taylor HL, Wall ME, Coggon P, Mcphail AT (1971) Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia. Journal of the American Chemical Society 93 (9): 2325–2327. doi:

10.1021/ja00738a045.

14. El-Kashef DH, Youssef FS, Reimche I et al. (2020) Polyketides from the marine-derived fungus Aspergillus falconensis: In silico and in vitro cytotoxicity studies.

Bioorganic & Medicinal Chemistry 29 (115883). doi:

10.1016/j.bmc.2020.115883.

15. Yang B, Sun W, Wang J et al. (2018) A New Breviane Spiroditerpenoid from the Marine-Derived Fungus Peni- cillium sp. TJ403-1. Marine Drugs 16(4): 110.

doi: 10.3390/md16040110.