Iraqi J Pharm Sci, Vol.30(1) 2021 Effects of kappa carrageenan on cell lines and human DNA DOI: https://doi.org/10.31351/vol30iss1pp189-195 189 The Pharmacological Effects of Kappa Carrageenan on Different Human Cell Lines and Genomic DNA: An in vitro study Aseel J. Ali*,1 , Hajer I. Abdulla** and Marwan S. Al-Nimer*** * Ministry of Health and Environment, Al Ma'amon Dental Care Centre, Baghdad, Iraq. ** Department of Dentistry, Al- Esraa University College, Baghdad, Iraq. *** Department of Pharmacology, Imam Ja'afar Al-Sadiq University, Baghdad, Iraq. Abstract Carrageenan extract is a compound of sulfated polyglycan that is taken out from red seaweeds. Being hydrocolloid in nature, carrageenan has gelling, emulsifying and thickening properties allowing it to be commonly used in the oral healthcare products and cosmetics. Due to its bioactive compounds, carrageenan has been shown to have antimicrobial, antiviral, and antitumor properties. The purpose of this work is to study the probable use of carrageenan on the diseases that are related to oral cavity and on the genomic DNA in in vitro experimental model. In this study, the effects of κ-carrageenan on four different cell lines related to the cancer and normal cells which cultured on selective media were done. Moreover, the effect of κ-carrageenan on the DNA molecule using an in vitro model was investigated in order to explain the antiproliferative effect of carrageenan. Kappa-carrageenan inhibited the cancer cell growth and fibroblast cell lines growth (in vitro) experimental model. In addition, κ- carrageenan solution completely and significantly damaged the DNA molecule by the evidence that the mean ± SD absorbance of the mixture of κ-carrageenan and DNA solution is 0.0 ± 0.0. This study shows that the κ- carrageenan pharmaceutical preparations exert biological activities as anticancer in vitro studies. Keywords: Carrageenan, Cell line, DNA, Antitumor, In vitro. التأثيرات الدوائية لمادة كابا كراجينان على خطوط الخاليا البشرية و د.ن.أ الجينومي : دراسة داخل األنبوب ***مروان صالح النمرو **،هاجر ابراهيم عبد هللا 1*،أسـيل جاسـم علي التخصصي لطب االسنان، بغداد، العراق.وزارة الصحة والبيئة ، مركز المأمون * ** قسم طب الفم، ،كلية االسراء الجامعة ، بغداد،العراق *** قسم علم االدوية،جامعة االمام جعفر الصادق، بغداد،العراق . الخالصة الكراجين بطبيعته الغروية المائية كاراجينان هومركب يحتوي على البولي گاليكان الكبريتي المتعدد وهو مستخلص من الطحلب االحمر. اظهر يمتلك خواص هالمية،مثخنة ومستحلبة تسمح باستخدامه بكثرة في مستحضرات الصحة الفموية والكماليات.وبسبب مركباته الحياتية الفعالة فقد للكراجبينان في خطوط الخاليا السرطانية لقد هدف هذا البحث الى دراسة التطبيق الممكن .الكراجينان امتالكه خواص مضادةللخاليا السرطانية تم دراسة تأثير كراجينان في أربعة والخاليا الليفية وتاثيره في الحمض النووي الجيني البشري في نموذج مختبري خارج الزجاج )في األنبوب( جينان في اثباط نمو الخاليا المزروعة ، كما وامتدت انواع من الخاليا السرطانية والطبيعية والتي تم زرعها في وسط انتقائي لمعرفة مدى تأثير كرا وقد بينت هذه الدراسة ان كابا كراجينان يمنع خطوط الخاليا الدراسة لتشمل تأأثير كاراجينان في الحمض النووي في أنموذج تجريبي خارج الزجاج. جزيئة الحمض النووي الجيني البشري. يستدل من ذلك ان الكراجينان السرطانية والخاليا الليفية في التجارب االختبارية باالضافة الى أذى تام في . الدراسات المختبريةي الذي استعمل في هذه الدراسة على شكل محلول لديه نشاط مضاد للسرطان ف .في الزجاج ، ضد االورام، كراجينان،خطوط الخاليا البشرية المختلفة،الدنا الكلمات المفتاحية: Introduction The carrageenan extract is a (sulfated – polyglycan) that is taken out from red seaweeds from the genera (Gigartina ,Chondrus, Eucheuma and Iridaea (1,2). Carrageenans are mainly utilized as a diet improver because of its emulsifying, thickening and gelling activities making it a vegetarian substitute for the gelatin (2). In addition to their use in food, carrageenans are generally used as excipients in a personal lubricant, toothpaste, many cosmetics besides many of the pharmaceutical products (3). Commercially, three forms are there of carrageenan that are presented (kappa, lambda and iota) that are differing in degree and composition of sulfation in the polymeric structures. (Figure 1). These actions of carrageenan have been considered, particularly on the animal model, where it had been reported that carrageenan extract has anti-microbial (4,5) and anti-tumor criteria (6,7). The tissue of the oral cavity is mainly formed by oral-keratinocytes, which are stratified squamous epithelium that form a main barrier to physical, chemical and microbial against agents that can cause localized cellular injury (8). 1Corresponding author E-mail: dentist_aseel@yahoo.com Received: 11/ 9 /2020 Accepted:21 / 11 /2020 Iraqi Journal of Pharmaceutical Science https://doi.org/10.31351/vol30iss1pp189-195 Iraqi J Pharm Sci, Vol.30(1) 2021 Effects of kappa carrageenan on human cell lines and genomic DNA 190 These cells are also actively involved in the pro- inflammatory route through the making of some cytokines(9,10). Some of the toothpastes and oral gels contained carrageenan, and if exposure to an extended period will cause contact with oral cells besides the oral microorganisms. As stated before, carrageenan possessed anti-microbial activity (11). In earlier work, κ-carrageenan oligo-saccharides from Kapaphycus stratum; in vitro and in vivo were proved to have immuno-modulation action on S180 bearing mice (12,13). Chemical adjustment of carrageenan oligosaccharides can increase their antioxidant activities (14). The aim of the study is to evaluate the effect of κ-carrageenan on the human cancer and fibroblast cell lines and on the human genomic DNA in in vitro experimental study. Figure 1 Chemical structure of κ-carrageenan Materials and Method Materials κ-carrageenan purchased from Sigma-Aldrich (USA)(15) Cell line: • Ahmed Majeed 2003(AM3) Transplantable mammary adenocarcinoma line. • HeLa established by (George Gey) at the Johns Hopkins medical school. •Primary Rat Embryo Fibroblast (REF) provided and established from the Iraqi Centre for Cancer & Medical Genetics Research, Baghdad, Iraq (ICCMGR). • Rhabdomyosarcoma (RD) cell line (got from the pelvic rhabdomyosarcoma of a seven-years-old Caucasian girl) (16,17). Methods Effect of κ-carrageenan on cancer cell line and fibroblast cells line In this study, four types of the cell lines are used. These are HeLa cell, Rhabdomyosarcoma, Mammary cell carcinoma, and fibroblast cells. Media and solutions preparing for the (in vitro cell culture) experiment: 1. Rosswell-Park-Memorial/ Institute RPMI/1640 Media The procedure of the media is made as following: a. The (RPMI/1640) powder with the (HEPES.) buffer, with L-glutamine (8.2g) was dissolved in a (400) ml of the distilled water, and afterwards other constituents were added. b. The sodium-bicarbonate powder is equal to 1.1 gm Streptomycin (0.25 ml) of (1gm) vial that is dissolved within a (5 ml) of distilled water. c. Ampicillin (0.5)ml of a (500) mg vial that is dissolved in (5 ml )of distilled water. d. Fetal calf serum/FCS (100 ml) afterward this volume is accomplished for up to (500) ml with distilled water and this media was sterilized with Nalgen filter by a 0.2µm filtering unit (16,17). 2. Phosphate buffer saline/PBS solution. This solution is made up using the following procedure: a. Firstly dissolving an amount of 5 gm (PBS) powder in a (400) ml of distilled water, and then the other constituents were added. b. Streptomycin (0.25) ml of (1gm vial was dissolved in a 5 ml of distilled water). c. Ampicillin 0.5 ml of (500 mg) vial was dissolved in (5 ml) of distilled water). Afterwards the volume is completed up to 500 ml by adding distilled water and this media was sterilized by Nalgen filter with a (0.2µm) filter unit. 3. Trypsine/Versen s olutions The solution is made as the following: a. Dissolving amount of (5.05gm)Trypsine/Versene powder in a (400 ml) of distilled water, and after that adding other constituents. b. Adding sodium-bicarbonate powder amount of 1 gm. c. Streptomycin (0.25) ml of (1gm) vial is dissolved in a 5 ml of distilled water. d. Ampicillin amount of (0.5 ml) of a 500 mg vial which is dissolved in a 5 ml of distilled water).Then the volume is finished to (500ml) with distilled water and then media is sterilized by Nalgen filter with (0.2)µm filtering units(16,17). 4. Fetal calf serum(FCS) This serum is thermally deactivated, set for uninterrupted use for the tissue culture media and it was sterilized(16,17). 5. Preserving Serum Free Media / SFM. The media was made by the same process that is described for RPMI./ media except that the FCS was not added. Iraqi J Pharm Sci, Vol.30(1) 2021 Effects of kappa carrageenan on human cell lines and genomic DNA 191 Solution of methyl-thiazolyl tetrazolium /MTT. 3-Dimethylthiazol-2-yl-2,5-Diphenyl- tetrazoliumbromide (0.2 gm), which was dissolved in a 100 ml of the PBS in order to get (2 mg /ml) concentration of the dye. This solution was filtered by using a 0.2µm Millipore filter to get rid of any blue color formazan material, and then kept in sterile, dark, glass container at a (4˚C). The solution must be used within 2weeks of its preparation (16,17). Types of the Cell Lines used in this study 1. HeLa cell: This cervical cancer cell line of human which was firstly recognized by “George Gey” at the Medicine school of Johns Hopkins in the year 1951 obtained from a mother 31-year-old that had four children, her name is “Henrietta Lacks”. The HeLa cell were different from another cervical cancers explant in which they raised horribly in culture, could be too violently. Passage/13-14that was used in the study, and the RPMI-1640 media with a (5%) FCS was used in preserving these cells. 2. Rhabdomyosarcoma (R.D. Cell Line): This human cell line (RD) derived from a biopsy sample got from the pelvic rhabdomyosarcoma of a (7 years old) Caucasian girl. The Passage of (20-21) used in this study, and the RPMI-1640 media with 5% /FCS was used in preserving these cells. 3. Ahmed/Mohammaed/Nahi-2003(AMN-3) Cell Line: This is a murine mammary-a type of adenocarcinoma- cell line is a derivative of the initially (in-vivo) passage for the spontaneous mammary adenocarcinomas of a female BALB-c mouse. Passage/182-183 was utilized in this work, and the RPMI/1640 media with 5% FCS utilized in preserving these cells. 4. The primary rats embryo fibroblasts/R.E.F The R.E.F cell line is recognized and delivered by the help of Dr. Ahmed M. Al- Shammery from the (ICCMGR). These cells of this standard rats cell lines were a combination of epithelial and fibroblastic cell with ordinary chromosomal image. Tumorigenicity trial of the cell lines has shown no tumor or growth development in the rats that are injected during 3 months of observing. Passage of (77/78) was utilized in this study, and the RPMI/1640 media with 5%/ FCS was used in preserving these cells. Four cell lines are subjected to carrageenan. Those cells are (HeLa, fibroblasts mammary-AMN3, and rhabdomyosarcoma. These cells are stored in a deep freezing as stock cells at the National Centre of Cancer. Such cells are rebooted before the testing. Many trials of the cell revival are made so that to get a single layer cell in a precise falcon volume of 25 ml which can be accustomed under the microscope to search for the presence of monolayer cell. Cell cultivation was carried on according to the instructions of The National Center of Cancer in Baghdad, using the following procedures: 1st step: During the first day, when the development of a single layer cells that have been formed in the cell falcons which is a precise falcon of 25 ml of volume, the last growth media got rid of and washed with the phosphate buffer saline (PBS) buffer only once, then add amount of 0.5-1.0 ml Trypsin-versin solution, shaking the mixture and allowed to set down for 1 min. Then, at that moment, a gentle shake by hand is followed for a minute, after that add 10 ml of sterile growth media in amount of 5% of fetal calf serum to the cells in the falcons, then start to pipette in order to diffuse the cells within the newly made media. Then the isolated cells moved into a germ-free microplate. A whole volume of (200) µl cells suspension or cell suspension-culture media (which is considered as control) is moved to a number of wells of a microplate. The microtitre plate is protected with a (plate-cover) to avoid the opportunity of impurity and then incubated at a temperature (37ºC) for 24 -hours so that to maintain a single layer cells progress. 2ndstep: During the second day meaning after 24- hour of cell culturing sin microplates, when the monolayer cell growth is formed in the microtitre plate, culture media was thrown away and the subsequent measurements of culture media and then carrageenan was added in a number of wells  200 µl of cell cultures media  200µl of the cultured media were added into the wells with the monolayer cells growing  175 µl of the culture media having no cells in addition to 25µl of 0.5%w/v freshly made κ- carrageenan solution.  The prepared κ-carrageenan solution poured into the wells with monolayer cells growth. The solutions of carrageenan were sterilized by filtering using a 0.2µm Millipore/filter before the adding step. The microtitre plate was covered by a plate cover to inhibit the possibility of impurity and then was incubated at a 37ºC for 24 hours so that to get a mono-layer cell growth. 3rd step: After 24 hours, an amount of 30µl M.T.T dye was added into all of the wells in dark room (to prevent oxidation of the dye), covering the microtiter plate by a foil and is kept for incubation for about two hours at a 37.5ºC in the incubator. Then the wells containing solution discarded from the plate and, 100µl of dimethyl/sulfoxide added to each well and then the plates were shaken for 15 minutes using horizontal shaker. Then the absorbance of all wells were recorded at 540nm by the ELISA-reader Effect of κ-carrageenan on the genomic DNA of human The genomic DNA of human has been generously gotten from Prof. Dr. Adil Al Huseiny, at the Medicine Department, College of Medicine/ Diyala University. Briefly genomic DNA of human is extracted from the anti-coagulated blood by EDTA by using the (proteinase K) solutions and Iraqi J Pharm Sci, Vol.30(1) 2021 Effects of kappa carrageenan on human cell lines and genomic DNA 192 genomic purification kit/ Geneaid, Taiwan, and this process of extraction is followed like the instruction of the manufacturer company of the kit. Absorbance of the extracted DNA, which is dissolved in the phosphate buffer solutions, recorded wavelength of 260nm and 280nm using a UV-Visible spectrophotometer. A ratio of absorbance at λ260nm to λ280nm approximately equal to 1.8-2.0, indicating the genomic DNA contaminated with RNA. A total number of six genomic DNA obtained from six participants were tested with 0.5% (w/v) κ-carrageenan; where, 5µl κ-carrageenan was added to 5µl genomic DNA in a quartz cuvette then completed the volume to the 4ml by distilled water. The absorbance of genomic DNA was recorded at 260nm wavelength using a UV-Visible spectrophotometer. A decrease in the absorbance of genomic DNA compared with the absorbance before adding κ-carrageenan indicated DNA damage (a term known as hypochromasia). While an increase of the genomic DNA absorbance after adding κ- carrageenan, indicating separation of the DNA strands (a term known as hyperchromasia). Statistical analysis: Descriptive and inferential statistics were done by the use of the Microsoft Excel /2007 program. All results were stated as numbers (%), and every time possible as mean and standard deviation (SD). Data were analyzed by the utilization of unpaired two/tailed Student's t-test by taking (p ≤ 0.05) as the lowest limit of the significance. Results Effect of κ-carrageenan on the cancer cell line κ-carrageenan inhibits the development of cancer cell and fibroblasts cell lines in-vitro experimental model. It significantly (p≤0.05) stops the growth of HeLa cell/line by 81.2% (the mean absorbance value at λ 540 nm is reduced from 0.706 to 0.133 (Figure2). Figure 2 The effect of κ-carrageenan on HeLa cell growth in vitro experimental model. The results expressed as mean ± SD (n=8). κ-carrageenan completely and significantly suppressed the growth of mammary cell carcinoma i.e. the inhibitory percent is 100% (the mean absorbance value at λ 540 nm is reduced from 0.108 to 0.0 (Figure 3). κ-carrageenan failed to suppress the growth of rhabdomyosarcoma, in fact it improves the growth of these cells by the evidence that the mean absorbance value is increased from 0.463 to 0.524, i.e. 13% increment (Figure 4). Figure 5 shows that κ-carrageenan completely and significantly suppressed the growth of fibroblast cells i.e. the inhibitory percent is 100% (the mean absorbance value at λ 540 nm is reduced from 0.196 to 0.0. Figure 3 The effect of κ-carrageenan on mammary cell growth in vitro experimental model. The results expressed as mean ± SD (n=8). Figure 4 Effect of κ-carrageenan on the rhabdomyosarcom growth in vitro experimental model. Results expressed as mean ±SD(n=8). Iraqi J Pharm Sci, Vol.30(1) 2021 Effects of kappa carrageenan on human cell lines and genomic DNA 193 Figure 5 Effect of κ-carrageenan on the fibroblast cell growth in vitro experimental model. Results expressed as mean ±SD(n=8) Effect of κ-carrageenan on the human genomic DNA The mean ± SD concentration of human genomic DNA that isolated from 6 subjects was 1.783±0.397 µg/ml which corresponding to the absorbance of 0.0178±0.0039 at wavelength of 260 nm. Kappa- (κ- ) carrageenan solution completely and significantly (p<0.001) damaged the DNA molecule by the evidence that the mean ± SD absorbance of the mixture of κ-carrageenan and DNA solution is 0.0 ± 0.0 (Figure 6) Figure 6 Effect of κ-carrageenan solution (0.5% w/v) on the human genomic DNA in vitro experimental model.Results are expressed as mean±SD of six subjects. Discussion The results of this study showed that κ- carrageenan significantly prevent growth of fibroblasts, HeLa cell, and mammary cells; while, its effect against rhabdomyosarcoma cell is negligible, indicating its anti-growth effect is specific. These observations agreed with a previous study that showed the specificity of carrageenan as anti- cancer; furthermore, it is imperative to declare that the anti-cancer effect of carrageenan is strictly associated with the molecular weight, carbohydrates structure and the contents and the linking site of sulfur group (18). In previous results of the in vivo and in vitro experimental study, it has been found that degraded iota (ι)-carrageenan inhibits the osteosarcoma growth in established xenograft tumor models in mice and enhanced survival rate of animals (in-vivo study) as well as it inhibits the growth human osteosarcoma cell line. The authors suggested that carrageenan induced apoptosis (not necrosis), and arrest the cell cycle at G1 phase (19). In another study, the small molecular-weight, highly-sulfated lambda λ-carrageenan oligosaccharide inhibits the angiogenesis (a process that is involved in initiation and also promotion of cancer), as well as inhibits the cellular invasion, migration and proliferation (20). Mi et al 2008 linked those effects to the upregulation of apoptotic genes like TNF-alpha, p-53, caspase 8, caspase 3 and increase level of active caspase 3 (21). Tobacman and Walters (2001) used transmission electron microscopy to illustrate the interaction between mammary myoepithelial cells of human and lambda-carrageenan and found that carrageenan entered the cells by membrane-associated endocytic vesicles and accumulate in endosomes and lysosomes (22) . As a result of the release of proteolytic enzymes from the distorted lysosome, the mammary myoepithelial cells are destroyed. On the other hand, Tobacman et al (2001) verified that increasing intake of numerous gums with carrageenan associates positively with high frequency of breast carcinoma (23). Authors found that carrageenan oligosaccharides can inhibit the growth of HeLa cell as well as the endothelial cell via a mechanism related to inhibition of the heparanase enzyme activity; moreover, this finding pointed out that carrageenan inhibited the cell growth at the molecular level (20). The effects of κ-carrageenans on fibroblast indicate that this substance is not free from harmful effect on the normal human cells. This observation is of great importance because carrageenan is pharmaceutically formulated as salt cast film in dressings for drug delivery to wound and to allow active adherence to and protection for the wound (24). According to results obtained from the current study, it is expected that healing of the wound is either delayed or even not occurred. The results of this study demonstrated that κ- carrageenan induced DNA (human genomic) damage in in vitro model. These results are in agreement with other studies that used carrageenan as inflammatory inducing agents. Cuzzocrea et al (2001) demonstrated that intra-pleural injection of carrageenan into rats caused DNA damage in lung tissue that accompanied with generation of reactive nitrogen species (RNS) (25); moreover, this important finding should be considered in the oral medicine- practice because, the soluble carrageenan is used as a pharmaceutical formula of the bio-adhesive buccal mucosa in which the carried medication is released and dissolved in saliva within 40 minutes (26). Therefore, using of such formula may induce DNA Iraqi J Pharm Sci, Vol.30(1) 2021 Effects of kappa carrageenan on human cell lines and genomic DNA 194 damage or oral mucosa. Bhattacharyya et al (2014) demonstrated an increase in m-RNA expression in a mouse colonic epithelium and the colonic epithelial cells of human (27). There is no evidence in human that dietary carrageenan induced expression of mRNA in the cells of large intestine. Also Bhattacharyya et al (2008) found that carrageenan induced cell necrosis rather than apoptosis in the colonic epithelial cell lines of human (NCM460) and in the primary colonic epithelial cells of humans; moreover, there is evidence that carrageenan can induce DNA damage and arrest the cycle of the cell (28). On the other hand, Weiner (2014) concluded that, using dietary carrageenan is safe and there is no evidence that it has undesirable effects on the normal cell growth or on the reproductive system (29). Conclusions The results showed that the effects of carrageenan against cancer cell line prohibit the claim that the dietary carrageenan plays a role in carcinogenesis; whereas, its effect against fibroblast pointed to limit its uses in tissue injuries as it may limit the healing process. The damaging effect of carrageenan on the human genomic DNA in in vitro experimental study should be supported by further studies including in vivo experimental animal models to demonstrate the safety of using carrageenan. Acknowledgment The authors express their thanks to the Prof. Dr. Adil H. Alhusseiny for generously providing us the genomic DNA, and to the staff of The National of Cancer for their facilities and cooperation. References 1. Food/agriculture organization (FAO). Training manual on Gracilaria culture and seaweed processing in china. China: Zhanjiang Fisheries College 1990 2. Chemical Safety Information from Intergovernmental/Organization (INCHEM).WHO Food Additive Series, 48 (2002). Safety Evaluation Of Certain Food Additive and Contaminants, Carrageenan And Processed Eucheuma, Seaweed (Addendum).United Kingdom. 3. Buck CB, Thompson CD, Roberts JN, Müller M, Douglas R Lowy, Schiller JT. Carrageenan is a potent inhibitor of papilloma virus infection. Plos pathogens 2006; 2(7): e69doi:10.1371/journal.ppat.0020069 4. Yamashita S, Sugita-Konishi Y, Shimizu M. In- vitro bacteriostatic effects of dietary polysaccharide . Food Sci Technol. Res. 2001; 7(3):262-64. 5. Zhou G, Sheng W, Yao W, Wang C. Effect of low molecular weight lambda carrageenan from chondrus ocellatus on antitumor H22 activity of 5Fu. Pharmacol Res. 2006; 53(2): 129-134. 6. Xiaoke Hu, Xiaolu Jiang, Eric Aubree PB. In Vivo antitumor Activity of l-carrageenan Oligosaccharides. Pharmaceutical Biology 2006. Internet [cited 2012]. 7. Grafström RC. Human Oral Epithelium. Second Edition ed. New York: Wiley-Liss Inc.2002: 195-255. 8. Presland R B and Dale B A. Epithelial structural proteins of the skin and oral cavity: function in health and disease. Crit. Rev. Oral Biol. Med. 2000; 11: 383-408. 9. Wu Tong Jia Lihua, Du Rui,Tao Xiaoan, Chen Jinao, Cheng Bin. Genome-wide analysis reveals the active roles of keratinocytes in oral mucosal adaptive immune response. Experimental Biology and Medicine 2011; 236(7):832-843. 10. Kuk-Hwan S , Dong Jang L, Aera J, Cheorun Jo, Mooha L. Antimicrobial effect of κ- carrageenan–based edible film containing ovotransferrin in fresh chicken breast stored at 5ᵒC. Meat science 2009; 83(3): 479-483. 11. Van Houte, HV Jordan, R Laraway, R Kent, PM Soparkar, PF DePaola. Association of the microbial flora of dental plaque and saliva with human root –surface caries. J Dent Res. 1990 August: 69(8):1463-8. 12. Yuan H, Song J. Preparation, structural characterization and in vitro antitumor activity of kappa-carrageenan oligosaccharide fraction from Kappaphycus striatum. Journal of Applied Phycology 2005;17; 713. 13. Yuan H, Song J, Li X, Li N, Dai J. Immunomodulation and antitumor activity of κ- carrageenan oligosaccharides. Cancer Lett. 2006; 243(2):228-34. 14. Yuan H, Zhang W, Li X, Lü X, Li N, Gao X, Song J. Preparation and in vitro antioxidant activity of κ-carrageenan oligosaccharides and their oversulfated, acetylated, and phosphorylated derivatives. Carbohydr. Res. 2005; 340(4):685-92. 15. Al-Nimer MSM, Hameed HGb, Mahmood MM. Antiproliferative effects of aspirin and diclofenac against the growth of cancer and fibroblast cells: In vitro comparative study. Saudi Pharm J. 2015; 23: 483-6. 16. Aldalaien SM , Al-Ani FS , Al-Nimer MSM, Dala’ien AD, Nasr M. Metformin as a potential anti-proliferative agent against cancer cells: A comparative study elucidating the mechanism of action and antiproliferative preference. Focus on Medical Science Journal 2018;4(2):9- 12. 17. Chen HM, Gao Y, Yan XJ. Carrageenan oligosaccharides inhibit growth-factor binding and heparanase activity. Yao Xue Xue Bao 2011; 46(3): 280-4. 18. Jin Z, Han YX, Han XR. Degraded iota- carrageenan can induce apoptosis in human Iraqi J Pharm Sci, Vol.30(1) 2021 Effects of kappa carrageenan on human cell lines and genomic DNA 195 osteosarcoma cells via the Wnt/β-catenin signaling pathway. Nutr Cancer. 2013; 65(1):126-31. 19. Chen HM, Yan XJ, Wang F, Lin J, Xu WF. In vitro anti-angiogenic action of lambda- carrageenan oligosaccharides. Yao Xue Xue Bao 2007 Jun; 42(6):595-600. 20. Mi TY, Yan XJ, Chen HM, Lin J, Wang F, Xu WF. Proliferation inhibition of lambda- carrageenan oligosaccharides on HUVEC and expression of apoptotic relevant genes. Yao Xue Xue Bao 2008; 43(5):474-9. 21. Tobacman JK, Walters KS. Carrageenan- induced inclusions in mammary myoepithelial cells. Cancer Detect Prev. 2001; 25(6):520-6. 22. Tobacman JK, Wallace RB, Zimmerman MB. Consumption of carrageenan and other water- soluble polymers used as food additives and incidence of mammary carcinoma. Med Hypotheses. 2001 May; 56(5): 589-98. 23. Boateng JS, Pawar HV, Tetteh J. Polyox and carrageenan based composite film dressing containing anti-microbial and anti- inflammatory drugs for effective wound healing. Int J Pharm. 2013; 441(1-2):181-91. 24. Cuzzocrea S, Mazzon E, Dugo L, Serraino I, Ciccolo A, Centorrino T, De Sarro A, Caputi AP. Protective effects of n-acetylcysteine on lung injury and red blood cell modification induced by carrageenan in the rat. FASEB J. 2001; 15(7): 1187-200. 25. Kianfar F, Chowdhry BZ, Antonijevic MD, Boateng JS. Novel films for drug delivery via the buccal mucosa using model soluble and insoluble drugs. Drug Dev Ind Pharm. 2012; 38(10):1207-20. 26. Bhattacharyya S, Feferman L, Borthakur S, Tobacman JK. Common food additive carrageenan stimulates Wnt/ β-catenin signaling in colonic epithelium by inhibition of nucleoredoxin reduction. Nutr Cancer. 2014; 66(1):117-27. 27. Bhattacharyya S, Borthakur A, Dudeja PK, Tobacman JK. Carrageenan induces cell cycle arrest in human intestinal epithelial cells in vitro. J Nutr. 2008 Mar; 138(3):469-75. 28. Weiner ML. Food additive carrageenan: Part II: A critical review of carrageenan in vivo safety studies. Crit Rev Toxicol. 2014; 44(3):244-69. Baghdad Iraqi Journal Pharmaceutical Sciences by bijps is licensed under a Creative Commons Attribution 4.0 International License. Copyrights© 2015 College of Pharmacy - University of Baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/