ReseaRch PaPeR Journal of Agricultural and Marine Sciences Vol. 24 : 39– 43 DOI: 10.24200/jams.vol24iss1pp39-43 Reveived 12 Dec 2018 Accepted 23 Dec 2019 Activation of apoptotic cell death by skin mucus from Asian swamp eel (Monopterus albus) against human lung cancer cell line Ayah Rebhi Hilles1*, Syed Mahmood2,3*, Mohd Arifin Kaderi1, Ridzwan Hashim1 *Ayah Rebhi hilles ( ) *ayah.hilles90@gmail.com,*syedmahmood@ ump.edu.my.1Department of Biomedical Sciences, Kulliyyah of Allied Health Sciences, International Islamic University Malaysia, 25200 Kuantan, Pahang, Malaysia.2Department of Pharmaceutical Engineer- ing, Faculty of Engineering Technology, University Malaysia Pahang, 26300 Gambang, Pahang, Malaysia.3Centre of Excellence for Advanced Research in Fluid Flow (CARIFF), University Malaysia Pahang, 26300 Gambang, Pahang, Malaysia. Introduction Asian swamp eel (Monopterus albus) belongs to the family of Synbranchidae under Syn-branchiformes order (Cheng et al., 2003). Asian swamp eel skin mucus is secreted by the epidermal goblet cells in the epidermis which composed from in- organic salts, immunoglobulins, lipids and gel-forming macromolecules such as mucins, and other glycopro- teins suspended in water (Bragadeeswaran and Thanga- raj, 2011), which gives the mucus lubricating properties (Pearson and Brownlee, 2005). There are two main mechanisms describing the cell death in eukaryotic cells, apoptosis and necrosis. Apop- tosis is a process with well-defined key steps that mark the progress of the process in individual cells. Cells un- dergoing apoptosis possess distinctive morphological, biochemical and molecular features including sequence of chromatin margination and aggregation, nuclear and cytoplasmic condensation, cellular shrinkage, budding and fragmentation through the partition of cytoplasm and nucleus into the apoptotic body (Eriksson et al., 2008). these apoptotic bodies immediately recognized and phagocytized by macrophages or adjacent epithelial cells. Hence, there is no inflammatory response is elicit- تنشيط موت اخلالاي املربمج ابملخاط اجللدي من ثعبان األنقليس اآلسيوي )Monopterus albus( ضد خط خالاي سرطان الرئة البشرية آية رحبي حلس1* وسيد حممود2,3* وحممد عارفني قادري1 ورضوان هاشم1 Abstract. Asian swamp eel (Monopterus albus) is a freshwater fish which distributed widely in the East of India mainly across Malay Peninsula and Indochinese Peninsula, it is also broadly distributed in the Southern areas of East Asia including, southeastern China, Western Japanese Archipelago, and Korean Peninsula. It lives in muddy places, rice paddies, and slow-flowing currents areas. It has a unique morphological elongated body which is similar to snake and covered with a thick layer of mucus. The objective of this study is to screen the cytotoxic activity of eel skin mucus extracts and to evaluate the mechanism of cell death apoptosis or necrosis based on caspases activity assays. For this purpose eel skin mucus was collected from healthy fresh eels to prepare aqueous and methanol extracts. Preliminary cytotoxicity study was demonstrated against Non-small-cell lung carcinoma cell line (A549) using cell viability assay which revealed that methanol extract was more potent than aqueous extract based on the response of ESM methanol and aqueous extracts to the relevant IC50 concentrations as IC50 values were 621±0.09 µg/mL and 845 ± 0.25 µg/mL respectively. Subsequently cell death was induced through triggering apoptosis by the activation of caspase-3/7, 8 and 9. In conclusion, the present study has revealed the cytotoxic potentials of eel skin mucus which may lead to the devel- opment of new anticancer agents. Keywords: Monopterus albus; cytotoxic activity; apoptosis; caspases. املســتخلص:أنقليس املســتنقعات اآلســيوية )Monopterus albus( هــو مــن أمســاك امليــاه العذبــة الــي يتــم توزيعهــا علــى نطــاق واســع يف شــرق اهلنــد بشــكل رئيســي عــر شــبه جزيــرة املاليــو وشــبه جزيــرة اهلنــد الصينيــة ، كمــا أهنــا موزعــة بشــكل واســع يف املناطــق اجلنوبيــة مــن شــرق آســيا مبــا يف ذلــك ، جنــوب شــرق الصــني ، أرخبيــل غــرب اليابــان ، و شــبه اجلزيــرة الكوريــة. تعيــش يف األماكــن املوحلــة وحقــول األرز ومناطــق التيــارات بطيئــة التدفــق. وهلــا تركيبــة جســم فريــدة مــن نوعهــا والــي تشــبه فيهــا الثعبــان وتغطــى بطبقــة مسيكــة مــن املخــاط. اهلــدف مــن هــذه الدراســة هــو فحــص النشــاط الســام علــى اخلاليــا ملســتخلصات خمــاط جلــد ثعبــان األنقليــس ولتقييــم آليــة مــوت اخلليــة ســواء كان مــوت اخلاليــا املرمــج أو النخــر علــى أســاس اختبــارات نشــاط الكاســبيزس. هلــذا الغــرض؛ مت مجــع خمــاط جلــد ثعبــان البحــر مــن ثعابــني طازجــة صحيــة إلعــداد مســتخلصات املــاء وامليثانــول. مت إجــراء دراســة أوليــة للســمية اخللويــة ضــد اخلاليــا غــر الرئويــة لســرطان الرئــة )A549( باســتخدام اختبــار قابليــة اخلليــة الــي أظهــرت أن مســتخلص امليثانــول أقــوى مــن املســتخلص املائــي حيــث أن قيــم IC50 كانــت 621 ± 0.09 ميكروجــرام / مــل و 845 ± 0.25 ميكروجــرام / مــل علــى التــوايل. ويف وقــت الحــق ، فــإن آليــة مــوت اخلليــة تســبب املــوت اخللــوي مــن خــالل التســبب يف مــوت اخلاليــا املرمــج عــن طريــق تفعيــل الكاســبيز 7/3 و 8 و 9. يف اخلتــام ، كشــفت الدراســة احلاليــة عــن إمكانــات اخلاليــا الســامة للخاليــا ملخــاط ثعبــان البحــر ممــا قــد يــؤدي إىل تطــور جديــد. وكالء مضــاد للســرطان. الكلمات املفتاحية: Monopterus albus ، النشاط السام للخاليا ، موت اخلاليا املرمج ، الكاسبيزس. 40 SQU Journal of Agricultural and Marine Sciences, 2019, Volume 24, Issue 1 Activation of apoptotic cell death by skin mucus from Asian swamp eel against human lung cancer cell line ed (Fadok et al., 2000). However, in vitro, the apoptotic bodies and residual cell fragments swell and lyse (Chang and Yang, 2000). The morphologic features that char- acterize apoptotic cells are the consequence of several biochemical features, which are stimulated by proteo- lytic destruction of cytoskeletal and metabolic proteins. Activation of the effector caspases 3 and 7 is a common step in both intrinsic and extrinsic apoptotic signal pathways, which accomplish the characteristic changes in the nuclear morphology and biochemistry, including chromatin condensation and DNA fragmentation (Fan et al., 2005). Methodology Materials Cell Lines The cell lines used in the study include human non- small lung carcinoma (A549, ATCC CCL-185) and nor- mal mouse embryonic fibroblast (3T3-L1, ATCC CRL- 3242). The cell lines were obtained from Biomedical Science Department, Kulliyyah of Allied Health Scienc- es, International Islamic University Malaysia. Chemicals Phosphate buffered saline (PBS), Fetal bovine serum (FBS), Dulbecco`s modified Eagle medium (DMEM), Penicillin-streptomycin were purchased from Gibco Invitrogen Co. (Scotland, UK). Paclitaxel (Taxol®) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Merck KGaA, Germany. Caspase-3/7, caspase-8, caspase-9 kits were purchased from Promega, Madison, WI, USA. Methods Sample Collection and Extraction The eel skin mucus (ESM) was collected from healthy eels by gently scraping the surface of the eel skin and then it was homogenized with 2 volumes of distilled water using homogenizer, followed by centrifugation at 13,000 rpm for 30 min, the supernatant was freeze- dried for 5 days. The dried substance was weighed and dissolved in distilled water to form aqueous extract and in methanol to form methanol extract, after that, the dis- solved substance was filtered using 0.22 µm syringe filter to be ready for use. The extraction procedure was car- ried out according to the method previously described by Sadakane et al. (2007) with a slight modification. Cell viability test (MTT-based cytotoxicity as- say) The antiproliferative effect of aqueous and methanol ex- tracts of ESM on growth of two human cancer cell lines, i.e. human non-small lung carcinoma (A549) and nor- mal mouse embryonic fibroblast (3T3-L1), were evalu- ated by MTT assay. Approximately 5 × 10⁴ of cells were seeded into 96-well plates. after the cells reach the con- fluency level, they were treated with different concentra- tions of ESM aqueous and methanol extracts from 200 to 1000 μg/mL for 24 hrs. Then, 20 μl of MTT was added to each well and the plates were further incubated for 24 hrs. After that, 100 μl of DMSO was added to each well Table 1. Caspase-3/7 activity after treatment of A549 cells with ESM methanol and aqueous extracts along with positive control (Taxol) and negative control (untreated cells) for 24 hrs. Treatment OD1 OD2 OD3 Average SD Fold Change Methanol 0.301 0.339 0.322 0.320* 0.019 3.340* Aqueous 0.236 0.202 0.229 0.222* 0.017 2.344* Taxol 0.351 0.332 0.347 0.343* 0.010 3.597* Control 0.091 0.098 0.093 0.094 0.003 0.094 Caspase-3/7 activities were determined using the CaspAce® system. Mean ± SD (n = 3 wells/treatment). *p < 0.05 compared with the untreated cells. Fold changes was calculated based on the control/untreated cells. OD is optical density. Table 2. Caspase-8 activity after treatment of A549 cells with ESM methanol and aqueous extracts along with positive control (Taxol) and negative control (untreated cells) for 24 hrs. Treatment OD1 OD2 OD3 Average SD Fold Change Methanol 0.380 0.371 0.295 0.348* 0.046 3.233* Aqueous 0.197 0.229 0.215 0.213* 0.016 2.216* Taxol 0.153 0.202 0.197 0.184* 0.026 1.790* Control 0.103 0.109 0.115 0.109 0.006 0.109 caspase-8 activities were determined using the CaspAce® system. Mean ± SD (n = 3 wells/treatment). *p < 0.05 compared with the untreated cells. Fold changes was calculated based on the control/untreated cells. OD is optical density. 41Research Article Hilles, Mahmood, Kaderi, Hashim A549 which is required to reduce 50% of cell viability (IC50) was calculated and it was recorded as follows; 621 ± 0.09 µg/mL for methanol extract, 845 ± 0.25 µg/mL for aqueous extract and 43.12 ± 0.6 µg/mL for Taxol. Effects of ESM aqueous and methanol extracts on caspase 3,8 and 9 activities A549 cells-treated with the ESM extracts exhibited a sharp increase in the activities of caspases 3, 8 and 9. The activities of the individual caspases were expressed as fold increases with respect to the untreated control. Fold change is defined as the ratio between different values, the fold change of caspase 3,8 and 9 was higher for ESM methanol extract than aqueous extract compared to the control (untreated A549 cells) as shown in table 3.1,2,3. The activities of 3, 8 and 9 caspases were increased sig- nificantly in A549 cells-treated with 600 μg/mL of ESM methanol extract and 800 μg/mL of ESM aqueous ex- tract compared to untreated cells (the concentration of methanol and aqueous extracts was chosen based on the nearest concentration to IC50 value which was 621 ± 0.09 µg/mL for ESM methanol extract and 845 ± 0.25 µg/mL for ESM aqueous extract. Discussion Treatment of ESM aqueous and methanol extracts sig- nificantly inhibited the growth of A549 cell line com- pared to the normal cell line. This result agrees with what has been reported about the anticancer activities of Amphibian skin mucus (Kawasaki and Iwamuro, 2008). The present results showed that ESM extracts treatments activated 3, 8 and 9 caspases compared to the control (untreated cells), indicating that they induced A549 cell death via apoptosis as caspases test one of the biochemical markers which can be used to distinguish the mechanism of cell death, as it activated in the case of both intrinsic and extrinsic apoptotic signals (Fan et al., 2005). However, necrotic cell death does not involve caspase activation (Mansilla et al., 2006). Activation of caspases consider a hallmark of apoptosis and there are two pathways included; death receptor pathway which based on caspase-8 activation and mitochondrial path- way which based on caspase-9 activation (Leong et al., 2016). Accordingly, activities of the caspases 3, 8 and 9 and incubated for 4 hrs. Then, the absorbance was mea- sured at 570 nm using a microplate reader. The percent- age of cell survival rate was calculated using to the cell viability formula: Percentage of cell viability = (Mean of experimental absorbance / Mean of control absorbance) × 100 (Villarroel et al., 2007). PBS was used as a negative control while 1 μg/mL of Taxol was used as a positive control as it is a chemother- apy medication used in the treatment of non-small-cell lung cancer (NSCLC) such as A549 cell line. Determination of the mechanism of cell death by measuring of caspase 3, 8 and 9 activities Caspase 3, 8 and 9 are members of the cysteine aspartic acid-specific protease (caspase) family which play key roles in apoptosis induction. The test was conducted ac- cording to the instructions of the kits. A549 cells were cultured in 96-well plates at the density of 1 × 104 cells per well overnight. The adherent cells were further in- cubated for 24 hrs with 100 μl media containing IC50 values of ESM aqueous and methanol extracts. After the treatment, the cells were harvested and centrifuged then the pellets were washed with PBS and lysis in chilled ly- sis buffer. The mixture was left on ice for 10 min then centrifuged at 2000 rpm for 5 min at 4°C. Then the su- pernatant was used for the determination of caspase ac- tivities. The results were read on a microplate reader at 405 nm (Abdullah et al., 2015). Results Cell viability assay The results showed that the reduction in cell viability of A549-treated with ESM extracts was significant com- pared with the untreated cells. At 200 µg/mL which was the lowest concentration used, the cell viability of ESM aqueous and methanol was recorded as 87. 61% and 76.29% respectively whereas at 1000 µg/mL of ESM aqueous extract the cell viability was recorded as 46.58% while for methanol extract was 38.19%. The cell viabili- ty of ESM aqueous and methanol extracts against 3T3- L1 at 1000 µg/mL was 79.24% and 63.65% respectively while at 200 µg/mL was 92.53% and 89.21% respectively. The inhibitory concentrations of ESM extracts against Table 3. Caspase-9 activity after treatment of A549 cells with ESM methanol and aqueous extracts along with positive control (Taxol) and negative control (untreated cells) for 24 hrs. Treatment OD1 OD2 OD3 Average SD Fold Change Methanol 0.442 0.450 0.447 0.446* 0.004 3.937* Aqueous 0.299 0.318 0.271 0.272* 0.023 2.725* Taxol 0.541 0.519 0.528 0.529* 0.011 4.625* Control 0.019 0.012 0.017 0.033 0.003 0.033 caspase-9 activities were determined using the CaspAce® system. Mean ± SD (n = 3 wells/treatment). *p < 0.05 compared with the untreated cells. Fold changes was calculated based on the control/untreated cells. OD is optical density. 42 SQU Journal of Agricultural and Marine Sciences, 2019, Volume 24, Issue 1 Activation of apoptotic cell death by skin mucus from Asian swamp eel against human lung cancer cell line were measured in A549 cells-treated with ESM aqueous and methanol extracts as well as Taxol which is a che- motherapy drug use for apoptosis induction (Hu et al., 2005). Conclusion The activation of caspase (3, 8 and 9) only occurs as a result of apoptosis not necrosis and this parameter has been considered as one of the most reliable biochemical parameters to differentiate between apoptotic and ne- crotic cell death, therefore, the current study revealed that ESM extracts activate cell death via apoptosis. Acknowledgement This work was financially supported by a Research Grant (Project No. RDU 180371) from Universiti Malaysia Pa- hang (www.ump.edu.my), for which the authors are very grateful. References Abdullah, A. S. H., Mohammed, A. S., Rasedee, A., Mir- ghani, M. E. 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Caspase-8 activity after treatment of A549 cells with ESM methanol and aqueous extracts along with positive control (Taxol) and negative control (untreated cells) for 24 hrs. Treatment OD1 OD2 OD3 Average SD Fold Change Methanol 0.380 0.371 0.295 0.348* 0.046 3.233* Aqueous 0.197 0.229 0.215 0.213* 0.016 2.216* Taxol 0.153 0.202 0.197 0.184* 0.026 1.790* Control 0.103 0.109 0.115 0.109 0.006 0.109 caspase-8 activities were determined using the CaspAce® system. Mean ± SD (n = 3 wells/treatment). *p < 0.05 compared with the untreated cells. Fold changes was calculated based on the control/untreated cells. OD is optical density. Table 5. Caspase-9 activity after treatment of A549 cells with ESM methanol and aqueous extracts along with positive control (Taxol) and negative control (untreated cells) for 24 hrs. 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