IN-VITRO CYTOTOXIC ACTIVITY OF SQUID AND CUTTLEFISH BONE EXTRACT ON HEP G2 CELL LINE

IN-VITRO CYTOTOXIC ACTIVITY OF SQUID AND CUTTLEFISH BONE EXTRACT ON HEP G2 CELL LINE

Jemma Hermelin Jesy Diaz*¹, R.D. Thilaga¹ and V. Sivakumar ²

Department of Zoology, St.Mary’s College (Autonomous) ¹, Thoothukudi, Tamil Nadu, India

Research and Conservation 4e India ², Thoothukudi, Tamil Nadu, India

ABSTRACT: The present study was carried out to assess the anticancer activity of methanol extracts of internal bone of L.duvauceli and S. pharaonis for cell viability and toxicity on HepG2 liver cancer cells. The bone extracts had significant cytotoxicity on HepG2 cell in different concentrations (100,500,1000 µg/ml).  Methanol fraction of squid bone powder showed cell death at a higher concentration of 1000µg.  27% toxicity was observed at 100µg/ml and 37% at 1000µg/ml. The viability of cuttlefish bone powder ranged from 58% (1000µg/ml) to 68% (100µg/ml). Hep G2 cells experienced a significant decrease in viability at low concentration with an eventual decline at the highest concentration.  The percentage of toxicity of cuttle bone extracts varied from 32 to 42. Minimum toxicity of 32% was observed at 100µg and maximum of 42% was observed at 1000µg concentration and IC 50 at a concentration of >1000µg.   The percentage of toxicity showed increasing trend with increasing concentration of the extract. The result suggested that the cephalopods bone is a source to be considered in the discovery of drug development for cancer.

Cytotoxicity, Cell viability, Hep G2 cells, L.duvauceli and S.pharaonis

 related death worldwide because of tumour heterogeneity and the development of multidrug resistance phenotypes ¹.  Till now the availability of treatments for liver cancer remains unsatisfactory ². Hence, the search for active drugs from alternative sources including marine environment, obviously becomes imperative.   Cephalopods are the largest single group of biotoxic invertebrates, which may be largely useful in the biomedical arena. The physical activities of squid and cuttlefish ink such as  antimicrobial ^3-13, antioxidant ^14-16, antiradiation ¹⁷, anti-tumour ^18-24, immunity promotion ^{25, 26} induction of many cytokines ^{27, 28} have been widely studied in recent years.

The cuttlebone refers to the internal cartilaginous shell of cuttlefish, squid and octopus. Traditionally cuttlebone powder is used as a medicine for some ear ailments, stop bleeding and improve kidney efficiency ^{29, 30}.  Antibacterial and antifungal activity of internal bone of cephalopods was studied in different species ^{31, 32}.  But only a few cephalopods have been tested for anticancer activities, especially in India.  Therefore, the aim of the present study was to assess the potential anticancer activity of the methanolic extracts from the internal bone of L.duvauceli (squid) and S. pharaonis (cuttle fish) using cell viability and toxicity assay (MTT assay) on human HepG2 liver cancer cell line.

 MATERIALS AND METHODS:

Collection and Preparation of extract:

In the present study the animals (L. duvauceli and S.pharaonis) were collected from Gulf of Mannar, Thoothukudi coastal region (Long 78⁰ 8” to 79⁰ 30” E and Lat 8⁰ 35” to 9⁰ 25” N)  by trawl catch, brought to the laboratory, cleaned and washed with fresh sea water to remove all impurities.  The internal bones were dissected, washed, air dried and pulverized. 10g of pulverized bone powder was mixed with 100 ml of methanol solvent and kept in rotary shaker at 100 rpm overnight and filtered with Whatman No.1 filter paper and concentrated to dryness at 40⁰c, lyophilized and stored at 4⁰c until further use.

 Anticancer activity on liver cancer cell lines:

The antitumor assay was performed on Liver Cancer Cell lines (Hep-G2) obtained from National centre for cell science, Punae, India. The cell viability was measured using MTT assay ³³ as described.

 The cells were grown in a 96-well plate in Delbucco’s Minimum Essential Medium (DMEM) (HiMedia, Mumbai)) supplemented with 10% fetal bovine serum (Gibco Laboratories) and antibiotics (streptomycin, penicillin-G, kanamycin, amphotericin B).

About 1mL cell suspension (10⁵cells/ml) was seeded in each well and incubated at 37⁰ C for 48 hour in 5% CO₂ for the formation of confluent monolayer. The monolayer of cells in the plate was exposed to various dilutions of methanolic extract (1000µg, 500µg and100µg). The cell viability was measured using MTT assay with MTT (5 mg/ml) and DMSO.  The tetrazolium salt is metabolically reduced by viable cells to yield a blue insoluble Formozan product measured at 570nm

spectrophotometerically.

Controls were maintained throughout the experiment (untreated wells as cell control). The assay was performed in triplicate for each of the extracts. The mean of the cell viability values was compared to the control to determine the effect of the extract on cells and % of cell viability was plotted against concentration of the extract.  The minimum concentration of the extract that was toxic to liver cancer cells was recorded as the effective drug concentration compared to positive control (PC-Cyclophosphamide).

Percentage of viability = Absorbance of the sample/Absorbance of control

Percentage of toxicity = 100 ^_ percentage of viability

Morphological studies using a normal inverted microscope were carried out to observe the cell death treated with sample on HepG2 liver cancer cells.

 RESULTS:

The effect of methanolic extract of L.duvauceli bone on cell viability and toxicity are shown in Table 1, Fig 1&2. The cell viability ranged from 63% (1000µg/ml) to 73% (100µg/ml) and the toxicity was 37% for 1000µg/ml and 27% for 100µg/ml.  The minimum effective concentration that was toxic to HepG2 liver cancer cell was recorded at a concentration greater than 1000µg. The viability and toxicity of  methanolic extract of   S. pharaonis bone on Hep G2 cell line was shown in Table -2, Fig 3 & 4 and the toxicity was found to be 32% for 100µg/ml and 42% for 1000µg/ml and the IC 50 at a concentration of >1000µg.

Morphological changes of drug treated cells were examined using an inverted microscope. (Plate 1). As the concentration of the extracts increases from 100µg to 1000µg the number of Hep G2 cancer cells decreased.

 TABLE 1: PERCENTAGE OF VIABILITY AND TOXICITY - METHANOL EXTRACT FROM L. DUVAUCELI   BONE POWDER

 TABLE 2: PERCENTAGE OF VIABILITY AND TOXICITY OF METHANOL EXTRACT FROM S.PHARAONIS   BONE POWDER

PLATE 1: MTT ASSAY - METANOL EXTRACT FROM S. PHARAONIS BONE POWDER

 DISCUSSION:

Cephalopods play an important role in marine ecosystem and are valuable to man as food, in biomedical research and proven to be a very rich source of extremely potent compounds that have significant anticancer activity ^34-36.  The cephalopod ink extract have protective effect towards hemopoietic injuries from chemotherapeutics ³⁷. In the present study methanolic extract from squid and cuttle bone powder was tested for anticancer activity against HepG2 liver cancer cells and the cells experienced a significant decrease in viability as the concentration increases. The crude extract obtained from the internal shell of squid contains polysaccharides showed an antitumor activity against mouse sarcoma -180 was reported ³⁷.

The anticancer potential of salivary gland extracts (AGE, PGE) of Octopus ageina in vitro (COLO 205 cells) and in vivo (Albinio Wistar rats). PGE showed promising effects by reduced tumor occurrence ³⁸. There are previous report on the cytotoxic activity of the methanolic extract obtained from flesh of Sepia brevimana and Sepiella inermis using Dalton’s ascites and the cytotoxic acitivity was found to be S.brevimana  >S. innermis ¹⁶ .  The results of this study revealed the presence of bioactive compounds in the bone extract of L.duvauceli and S.pharaonis  so it can be used as a source of drug in the treatment of cancer.

 CONCLUSION: The results of this study shows that among the two extracts tested here methanol fraction from cuttlebone of S.pharaonis has more toxicity towards Hep G2 cell line than the L.duvauceli and hence cuttlefish are the best targets for cancer therapy. If their nature, structure and mechanism of action are explored they would be a better drugs for site-specific chemotherapy.

ACKNOWLEDGEMENT: The authors are thankful to the Principal of V.O.C. College, Thoothukudi for providing the facilities.  The authors also thank Mrs. Florida Tilton of Biozone Technologies, Chennai for her support to carry out this work.

REFERENCES:

1. Huang CC, Wu M.C, Xu G.W, Li D.Z, Cheng H, Tu ZX, Jiang H.L and Gu J.R: Over expression of the MDRI gene and P-glycoprotein in human hepatocellular carcinoma (HCC) and correlation to survival time. Anticancer Research 1992; 19: 2501 -2503.
2. Honda K, Sbisa E,  Tullo A,  Papero PA,  Saccone C, and Poole S, Pignatelli M,  Mitry RR,  Ding S, Isla A, Davies A and  Habib A:  Mutation is a  poor prognostic indicator for survival in patients with hepatocellular carcinoma  undergoing surgical tumour ablation. British Journal of Cancer 1998; 77: 776 -782.
3. Patterson Edward A. and Murugan, 2000. Screening of cephalopods for bioactivity.  Marine Biological Center Special Publication 2000; 21:253-256.
4. Rajaganapathy J, Thyagarajan SP, and Patterson Edward JK. Study on cephalopod ink for anti retroviral activity. Journal of Experimental Biology 2000; 38: 519 -520.
5. Chacko D and Patterson J: Effect of pharaoh’s cuttlefish, Sepia pharaonis ink against bacterial pathogens. Indian Journal of Microbiology 2005; 45 (3) 223-226.
6. Du-Tie-ping, Zhou Pei-gen, Wei Xiao – Yi, Yang Wen – Ling, Fu- Li Xia, Li Wei: The antibacterial study of the extract from ink sac cuttlefish on bacteria. Guangzhou, Food science and Technology. 2005 -01
7. Smiline Girija AS, Hariprasad G, Vijayashree Priyadharsini J, Pandi Suba K, Raghumaran R, and Cnanavendhan SG: Antimicrobial potential of Loligo duvauceli ink against the common clinical bacterial & yeast isolates. Biomedicine 2008; 28 (3): 213 -215.
8. Gomathi P, Nair JR and Sherief PM: Antibacterial activity in the accessory nidamental gland extracts of the Indian squid, Loligo duvauceli Orbigny. Indian Journal of Marine Science 2010; 39 (1)100-104.
9. Smiline Girija, AS, Vijayshree Priyadharshini J, Pandi Subha K,  Hariprasad G and Raghuraman R. 2011.  Isolation and characterization of lolduvin-S: A novel antimicrobial protein from the ink of Indians squid Loligo Duvauceli: International Journal of Current Research and Review 2011; Vol. 03.
10. Pasiyappazham Ramasamy, Namasivayam, Alagiri Srinivasan, Vairamani shanmugam, Jayalakshmi Krishnamoorthy and Annaian Shanmugam: In vitro evaluation of antimicrobial activity of methanolic extract from selected species of Cephalopods on clinical isolates. African Journal of Microbiology Research 2011; 5 (23): 3884 - 3889.
11. Rajasekharan Nair J, Devika P, Sophia MJ, Joseph, Gomathi, Priya SV and Sherief P.M. Cephalopod research and bioactive substances. Indian Journal of Geo-Marine Sciences. 2011; 40 (1)13-27.
12. Nithya M, Ambikapathy V and Panneerselvam A: Effect of pharaoh’s cuttlefish ink against bacterial pathogens. Asian Journal of Plant Science and Research 2011; 1(4):49-55.
13. Vennila R, Rajesh Kumar RK, Kanchana S, Arumugam M and Balasubramanian T: Investigation of antimicrobial and plasma coagulation property of some molluscan ink extracts: Gastropods and cephalopods. African Journal of Biochemical Research 2011; 5(1): 14-21.
14. Liu HZ, Wang G, Guo YZ, Pan JQ, Huang Y, Zhong JP and Li K: Protective effect of squid ink on cyclophosphamide-induced oxidactive injury of kidney in mice. Chinese Journal of Nephrology 2009; 25 (10): 804-805.
15. L, Liu HZ, Wu JL, Zeng QW, Chen YP, Yang CL and Zhong JP: Study of sepia ink extract on protection from oxidative damage of cardiac muscle and brain tissue in mice. Chinese Journal of Modern Applied Pharmacy 2010b:27(2):95-98.
16. Ilamparithi C, Anto Shering M, Brito Raj S and Kavimani S: A study about antioxidant and anticancer activity of methanolic extract of squid, Sepia brevimana and Sepiella inermis, International Journal of Pharma World Research. 2011; 2(2): 1-10.
17. Lei M, Wang JF,  Wang YM,  Pang L,  Wang Y,  Xu W and  Xue CH:  Study of the radio-protective effect of cuttlefish ink on hemopoietic injury.  Asian Pacific Journal of Clinical Nutrition2007; 16:239-243. [Pubmed].
18. Takaya Y, Uchisawa H, Matsue H, Narumi F, Sasaki J. & Iahada KL: An investigation of the antitumour peptidoglycan fraction from the squid ink. Biology, Pharmacology Bulletin, 1994; 17(6):846-851.
19. Saskai J, Ishita K, Takaya Y, Uchisawa H and Matsue H: Antitumour activity of squid ink. Journal of Nutritional Science Vitaminology 1997; 43(4): 455-461.
20. Russo G L, Nisco ED, Fiore G, Di Donato P, Ischia M and   Palumbo A: Toxicity of melanin free ink of Sepia officinalis to transformed cell lines: identification of the active factor as tyrosinase.  Biochemistry Biophysics Research Communication 2003; 308(2) 293 –299.
21. Sherief PM, Senan VP, George MC, Nair JR, Devika P & Joseph SM: Antitumour activity of   a peptidoglycan isolated from the ink of the cuttlefish, Sepia pharaonis Ehrenberg, paper Presented to the Biology in Asia International conference, Singapore. 2004; Abstract. P. 93.
22. Su Wei-ming, MA Run-di,  Yu Li-jian,  LIAO Ming-neng, He Dong-mei,  Hu-lai-zhen:  Antitumor effects of extract from the ink of Loligo chinensis  Chinese Journal of marine Drugs2005;2.
23. Wang S, Cheng Y, Wang F et al., 2008: Inhibition activity of sulphated polysaccharide of Sepiella maindroni ink on matrix metalloproteinase (MMP)-2. Biomedicine and Pharmacotherapy 2008; 62(5):297 -302.
24. Priya SV, Sherief PM and Rajasekharan Nair J: Cytotoxic effect of ink extracts of cuttlefish and squid on chick embryo fibroblasts. International Journal of Pharmaceutical Sciences and Research 2013; 4(5):1893-1896.
25. Guan Ling- min 1,2, Wang Qing – huil,  Zhang Peil,  Feng Yong –huil,  Wang Mei – rul, Wang Da –nal and  Lu Changlong. Immuno enhancement of squid ink in the immune suppression mice.  Chinese Journal of Marine Drugs 2010; 2.
26. Huazhong Liu, Ping Luo, Shaohong Chen and Jianghua Shang: Effects of squid ink on growth performance, antioxidant functions and immunity in growing broiler chickens. Asian Australian Journal of Animal Science 2011:24(12) 1752-1756.
27. He S, Meng SN and Xie GL: Study on secretion of interleukin-I induced by cuttlefish ink in mice. Chinese Journal of Marine Drugs.2003; 22:17-19.
28. Pang L, Wang JF, Lei M, Wang Y and Xue CH: The experimental studies on the effects of sepia products on hematopoiesis in mice. Acta Nutrimenta Sinica; 1: 87-90.
29. Trivedi YA and Sarvaiya RT: Invaluable yet exploited cephalopods. Seafood export. Journal VIII 1976; (5): 21-23.
30. Raje SG and Singh VV: Utilisation of fishery by-products by traditional fishermen of Maharashtra. Fishing Chimes. 1992; 12(2): 47-49.
31. Barwin vino A: Studies on cephalopods with reference to polysaccharides. M.Phil. Thesis, Annamalai University. 2003; P.70
32. Shanmugam A, Mahalakshmi TS and Barwin Vino A: Antimicrobial activity of polysaccharides isolated from the cuttlebone of Sepia aculeate (Orbingy, 1848) and Sepia brevimana (Steenstrup, 1875). An approach to selected antimicrobial activity for human pathogenic microorganisms. Journal of Fisheries and Aquatic Science 2008a; 3(5): 268-274.
33. Kang BK, Chon SK, Kim SH, Jeong SY, Kim MS, Cho SH, Lee HB and Khan G: Controlled release of paclitaxel from microemulsion containing PLGA and evaluation of anti-tumor activity in vitro and in International Journal of Pharmaceutics 2004; 286: 147 -156.
34. Sherief, PM, Devika P, George MC, & Nair JR. Isolation, purification and characterization of antibacterial and anticancer agents from the accessory nidamental gland and ink of the cuttlefish, Sepia pharaonis Ehrenberg. Final report to the Department of Biotechnology, Govt. of India, (College of Fisheries, Kerala Agricultural University, Kochi) 2007; p.27.
35. Guo-Fang ding, Fang Fang Huang, Zui-Su Yang, Di Yu and Young-Fang Yang: Anticancer Activity of an oligopeptide isolated from hydrolysates of sepia ink. Chinese Journal of Natural Medicines. 2011;9(2):151-155
36. Jie-Ping Zhong, Guang Wang, Jiang-Hua Shang, Jiang-Qiu Pan, Kun Li Yan Huang and Hua-Zhong Liu: Protective effects of squid ink extract towards hemopoietic injuries induced by cyclophosphamine, Marine Drugs 2009; 7(1) : 9-18.
37. Okutani K: An antitumor substance obtained from the internal shell of squid –I isolation procedures and antitumor activity.  Bulletin of the Japanese Society for the Science of Fish 1976; 42: (4) 449 -453.
38. Karthigayan S, Balasubashini M, Sengottuvela M, Balasubramanian T and Somasundaram ST: Anticancer principle from salivary gland extract of Octopus ageina. International Journal of Cancer Research 2006; 2 (3): 242 -252.
    

    ---

    How to cite this article:

    Jesy Diaz JH, Thilaga RD and Sivakumar V: In-Vitro Cytotoxic Activity of Squid and Cuttlefish Bone Extract on Hep G2 Cell Line. Int J Pharm Sci Res 2015; 6(2): 778-82.doi: 10.13040/IJPSR.0975-8232.6 (2).778-82.

    ---

     

    All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---