New synthetic chitosan hybrids bearing some heterocyclic moieties with potential activity as anticancer and apoptosis inducers

Journal Pre-proofs New synthetic chitosan hybrids bearing some heterocyclic moieties with potential activity as anticancer and apoptosis inducers Marwa M. El-Naggar, David S. A. Haneen, Ahmed B. M. Mehany, Magdy T. Khalil PII: DOI: Reference:

S0141-8130(19)34360-0 https://doi.org/10.1016/j.ijbiomac.2019.10.142 BIOMAC 13643

To appear in:

International Journal of Biological Macromolecules

Received Date: Revised Date: Accepted Date:

11 June 2019 19 September 2019 15 October 2019

Please cite this article as: M.M. El-Naggar, D. S. A. Haneen, A. B. M. Mehany, M.T. Khalil, New synthetic chitosan hybrids bearing some heterocyclic moieties with potential activity as anticancer and apoptosis inducers, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.10.142

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New synthetic chitosan hybrids bearing some heterocyclic moieties with potential activity as anticancer and apoptosis inducers Marwa M. El-Naggara, David S. A. Haneenb*, Ahmed B. M. Mehanyc, Magdy T. Khalila a

Zoology Department, Faculty of Science, Ain Shams University, Abbassia 11566, Cairo,

Egypt. b

Chemistry Department, Faculty of Science, Ain Shams University, Abbassia 11566, Cairo,

Egypt. c

Zoology Department, Faculty of Science, Al-Azhar University, Nasr City 11651, Cairo, Egypt. *

Corresponding author: Postal address: 11566, Telephone: +201285338610, Fax: +20226842123, E-mail address: [email protected]

ABSTRACT Wastes of the freshwater crayfish Procambarus clarkii were used as precursor for the extraction of chitin, which was deacetylation with alkali giving chitosan. Chitosan was transformed into the nanoparticles using sodium tripolyphosphate. Chitosan Cs and its nanoparticles CNP were characterized through FTIR, 1H-NMR and surface morphology. Moreover, it was converted into some chitosan Schiff bases; CSB-1,2,3, containing different five membered heterocyclic moieties and also they were characterized. All of the synthesized compounds were evaluated against three cell lines, such as HepG-2, HCT-116 and MCF-7, whereas compounds CSB-2 and CSB-3 showed the highest cytotoxic activities. The higher activities for the latter compounds were confirmed through apoptosis studies (Caspase-3 and Bax/Bcl-2 ratio) against HePG-2 cells. Cell cycle analysis and apoptosis induction showed that compound CSB-2 induces apoptosis at pre G1 phase and cell growth arrest at G2/M phase. Keywords Chitosan nanoparticles, Heterocyclic Schiff bases, Cytotoxic activity and apoptosis induction. 1. Introduction Chitosan [β-(1, 4)-linked D-glucosamine] is a hetero polysaccharide obtained by the chemical alkaline deacetylation of chitin [N- acetyl-D-glucosamine] in the same linkage [1], one of the most abundant natural polysaccharide in nature [2]. The exoskeleton wastes of Procambarus clarkii are considered as outstanding and feasible sources of chitin due to their higher chitin and lower protein contents (Scheme 1) [3]. The physico-chemical and biological

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properties of chitosan were affected with the degree of deacetylation. The degree of deacetylation depends mainly on the method of extraction and reaction conditions for the formation of chitosan. In addition, chitosan is characterized with an ease modification applicable in various fields, such as: cancer therapy, water treatment, aquaculture, agriculture, biochemical engineering industries and drug delivery [4,5]. [Scheme 1] The nanoparticles can have the potential to resolve several issues associated with human as well as animal health production, and treatment of diseases. This is due to their tiny size, nano-scale that increases the available surface area to interface with biological support, profound diffusion into the target sites and increase the bioavailability of essential compounds [6]. Chitosan nanoparticles can be used for cancer treatment [7]. Moreover, they can be applied in many fields such as: tissue engineering, drug delivery, antimicrobial agents, antioxidants, water treatment and encapsulation of biologically active compounds [8]. Pyran derivatives, which are the main heterocyclic ring of chitin and chitosan, constitute an important class of biologically active natural and synthetic products. Pyrans and their analogues occupy prime position in bioorganic chemistry due to their diverse applications [9]. Heterocyclic compounds with remarkable anticancer and antimicrobial can be classified as Nitrogen-based heterocycle: pyridine, pyrazole, piperidine, imidazole and indole; sulfur-based heterocycles such as thiophene, thiazole and thiadiazole; oxygen-based heterocycles: pyran, benzofuran and oxazolidine [10-19]. The presence of primary amino and hydroxyl groups in the polymeric chain of chitosan provides possibilities for a chemical modification. An important one of these modifications is the formation of a new macromolecular structure of Schiff bases derived from chitosan through the reaction on the amino group. The formed chitosan Schiff bases exhibited potential antimicrobial, antioxidant, antitumor and biodegradable properties [20-24]. The versatile chemotherapeutical activities of Schiff bases derived from chitosan as well as the heterocyclic ring systems mentioned above, prompted us to syntheses a new and novel Schiff bases from the reaction of chitosan with different aldehydes containing other heterocyclic moiety such as thiophene, indole and pyrazole ring systems, with the presence of the pyran moiety in chitosan in order to enhance its antitumor properties. The invitro cytotoxicity, the cell cycle analysis and apoptosis inducers of the produced compounds were studied.

2. Materials and Methods

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2.1. Materials Exoskeleton wastes of Procambarus clarkii were obtained from a commercial crayfish processing company (Jiang's Fish Processor Co., LTD, El-Obour City, Cairo, Egypt). Sodium hydroxide 50%, hydrochloric acid 33%, sodium hypochlorite 12.5%, sodium tripolyphosphate (TPP) and glacial acetic acid 99.7% were supplied by Abou-Zaabal Company for Chemicals. Ethanol, n-butanol, thiophene-2-carboxaldehyde, 1H-indole-3-carboxaldehyde, acetophenone, phenyl hydrazine, dimethyl formamide (DMF) and phosphorous oxychloride (POCl3) were supplied from Merck and used as received. 1,3-diphenyl-1H-pyrazole-4-carbaldehyde was

prepared according to the literature procedure [25].

2.2. Chitin extraction and its conversion into chitosan Chitin was extracted from the exoskeleton wastes of the freshwater crayfish Procambarus clarkii and these involved three main steps: demineralization, deproteinization and decoloration [3,26]. The extracted chitin was treated with 50 % sodium hydroxide solution (NaOH) at 100 ℃ using a hot plate magnetic stirrer for 9 hours with a solid to solvent ratio 1:10 (w/v). The mixture was cooled for 30 minutes at room temperature, then washed with distilled water several times till neutrality and filtered in order to retain the solid extract. This obtained chitosan was left to dry at room temperature and then stored in polyethylene bags for further use. 2.3. Preparation of chitosan nanoparticles (CNP) They were synthesized by the ionotropic gelation method [27], chitosan was dissolved in 1 % glacial acetic acid solution and sodium tripolyphosphate Na5P3O10 (TPP) was dissolved in 1% deionized water. The chitosan solution was then placed on the magnetic stirrer for 2 hours at room temperature till a clear solution was obtained. Sodium tripolyphosphate solution was added dropwise to the chitosan solution, whereas the chitosan/ sodium tripolyphosphate ratios was 1:1 (w/w), under magnetic stirring at room temperature for 1 hour till a milky white suspension, chitosan-TPP nanoparticles, was obtained. The resultant suspension was left in the refrigerator at 4 ℃ overnight to allow sufficient reaction. The nanoparticles were collected using cooling ultracentrifuge at 4 ℃ for 30 minutes at 16,000 rpm and washed extensively to remove the unreacted reagents. The precipitate was re-dispersed in minimal amount of distilled water, freeze-dried and finally stored at 4 ℃ till further use. 2.4. Physico-chemical characterization of chitosan and chitosan nanoparticles 2.4.1. Elemental analysis

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Two mg of chitosan was placed inside a thin capsule and has been heated in a combustion tube. The weight percentages of carbon and nitrogen were measured using thermal conductivity detection. The elemental analysis results of % C: % H: % N were 39.42: 7.09:7.34, respectively. The degree of deacetylation of chitosan was determined using the following equation; DDA (%) = [(6.857- C/N) / 1.7143] × 100 where C/N is the ratio of carbon/nitrogen [28]. So, DDA (%) = [(6.857- 39.42/7.34) / 1.7143] × 100 = 87%. 2.4.2. Calculation of the molecular weight of the chitosan using viscometry The viscometry is the best method for measurement of the molecular weight of chitosan. The viscosity ratio (the relative viscosity) ηrel which is given by the ratio of the outflow time for the solution (t) to the outflow time for the pure solvent (to) was calculated by the following equation: ηrel = t/to. The (to) and (t); were measured by Ostwald viscometer, the specific viscosity (ηsp), which is the relative increment in viscosity of the solution over the viscosity of the solvent, that was calculated by the equation: ηsp = (η-ηo)/ ηo = ηrel-1. The relative viscosity (ηrel) and specific viscosity (ηsp) values for different concentrations of chitosan (1.3, 1, 0.7, and 0.5 g %) were calculated. The intrinsic viscosity [η] was calculated from the plot of ηsp/c versus concentrations. The molecular weight of the prepared chitosan was calculated in kDa according to Mark-Houwink equation: [η] = KMa Where, k = 3.5x10-4 (cm3 g–1), and a = 0.76. Thus [η] =k Mα; Log [η] = log k + α log M; Log (1.8769) = log (3.5x10-4) + (0.76) log M; M = 80.7235 KDa. 2.4.3. Fourier Transform Infrared Spectroscopy (FTIR) FTIR spectra of chitosan and chitosan nanoparticles were recorded with FTIR (4100 Jasco-Japan) spectrophotometer at the Central Laboratory of Faculty of Science, Ain Shams University. The spectral region between 4000 and 400 cm-1 was scanned. Samples were prepared as KBr pellets. Dried chitosan powder was mixed thoroughly with KBr and then pressed to an ultimate thin homogenous disc with a thickness of 0.5 mm. The chitosan concentration in the samples was 2%; calculated with respect to KBr. Chitosan Cs: (υ, cm-1) 3300-3700 (OH), 3428 (NH2), 2924 (CH alkyl), 1157 (C-O-C), 1083, 1034 (C-O). Chitosan nanoparticles CNP: (υ, cm-1) 3300-3700 (OH), 3424 (NH2), 2925 (CH alkyl), 1154 (C-O-C), 1080, 1041 (C-O), 1107 (P=O). 2.4.4. Nuclear Magnetic Resonance spectroscopy (1H-NMR) The 1H-NMR spectrum were measured on Varian Gemini 300 MHz spectrometer, with chemical shift (δ) expressed in ppm downfield with tetramethylsilane (TMS) as initial standard, in DMSO-d6 and coupling constants J in Hz. at the Central Laboratory of Cairo University. 2.4.5. Surface morphology of chitosan and chitosan nanoparticles

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2.4.5.1. Scanning Electron Microscopy of chitosan The chitosan powder was grinded till become so smooth then coated with gold. The chitosan powder was examined in the solid state using SEM Model Quanta 250 FEG (Field Emission Gun) attached with EDX Unit (Energy Dispersive X-ray Analyses), with accelerating voltage 30 K.V., magnification14x up to 1000000 and resolution for Gun.1n). FEI Company, Netherland, at the Scanning Electron Microscope Unit (SEM) Egyptian Mineral Resources Authority “Egyptian Geological Survey” (EMRA), Ministry of Petroleum. 2.4.5.2. Transmission Electron Microscopy of chitosan and chitosan nanoparticles The morphological examination of chitosan and chitosan nanoparticles as well as the particle size measurement of nanoparticles were determined by transmission electron microscopy. A total of 0.5 g of chitosan nanoparticles were suspended in 2 % glacial acetic acid and sonicated in the ultrasonic bath for 30 minutes. Whereas, 0.5 g chitosan were suspended in distilled water and sonicated in the ultrasonic bath for 30 minutes. Then, a drop of the suspension solution was dripped onto carbon-coated copper grids and left to dry at ambient temperature overnight. Finally, the grids were examined with JEOL/JEM–1400 at Faculty of Agriculture Research Park, Cairo University. 2.5. Synthesis and characterization chitosan Schiff bases (CSB) To the suspension solution of chitosan that is previously prepared and purified, 1 g (1 eq) in n-butanol (60 ml), thiophene-2-carboxaldehyde 0.82 ml (1 eq) or 1H-indole-3carboxaldehyde 0.5 g (1 eq) or 1,3-diphenyl-1H-pyrazole-4-carbaldehyde 1 g (1 eq) were added in the presence of catalytic amount of glacial acetic acid (2-3 ml). The reaction mixture was refluxed for 14-16 h. then left to cool at room temperature. The solid obtained was filtered off, washed with dry ethanol several times and dried at room temperature in a vacuum oven at 50 °C for 24 hours to obtain the final product. The chitosan Schiff bases derivatives were characterized by Fourier transform infrared spectroscopy (FTIR) as follow: Chitosan Schiff base bearing thiophene moiety CSB-1: (υ, cm-1) 3300-3650 (OH), 3109 (CH aryl), 2961, 2961, 2890 (CH alkyl), 1631 (C=N), 1575 (C=C), 1160 (C-O-C), 1078, 1038 (C-O), 713, 620 (δ CH aromatic). Chitosan Schiff base bearing indole moiety CSB-2: (υ, cm-1) 3300-3650 (OH), 3166 (NH), 3103, 3040 (CH aryl), 2925, 2853 (CH alkyl), 1612 (C=N), 1574 (C=C), 1161 (C-O-C), 1082, 1034 (C-O), 741, 699 (δ CH aromatic). Chitosan Schiff bases bearing pyrazole moiety CSB-3: (υ, cm-1) 3444 (OH), 3123, 3059 (CH aryl), 2921, 2851 (CH alkyl), 1640 (C=N), 1598 (C=C), 1159 (C-O-C), 1076, 1043 (C-O), 752, 698 (δ CH aromatic). 2.6. Biological activity evaluation 2.6.1. Anticancer screening

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In this study, the synthesized compounds were subjected to cytotoxic activity evaluation on three human tumour cell line; hepatocellular carcinoma cell line (HepG-2), breast cancer cell line (MCF-7) and colon carcinoma cell line (HCT-116) using Sulforhodamine-B stain (SRB) assay method [29]. All chemicals used in this study are of high analytical grade. They were obtained from (either Sigma-Aldrich or Bio-Rad). The tumour cell lines were obtained frozen in liquid nitrogen (-180°C) from the American Type Culture Collection (ATCC) and was maintained at the National Cancer Institute, Cairo, Egypt, by serial subculturing. The cytotoxic activity was measured in vitro on human cancer cell lines and doxorubicin was used as a reference drug. Cells were plated in 96 multi well plates for 24 hour, and then treated with the newly synthesized compounds. Different concentrations of the tested compounds (0, 6.25, 12.5, 25, 50 and 100 µg/mL) were used. Triplicate wells were prepared for each individual concentration. Then they incubated for 48 hours at 37 oC and in atmosphere of 5% CO2. After 48 hours, cell were fixed, washed and stained with Sulforhodamine B. Excess stain was washed with acetic acid and attached stain was recovered with Tris EDTA buffer. Colour intensity was measured in an ELISA reader. The relation between surviving fraction and drug concentration was plotted and IC50 (the concentration required for 50% inhibition of cell viability) was calculated for each compound by Sigma plot software. 2.6.2. Determination of the active caspase-3 Caspase-3 level was measured using the Invitrogen Caspase-3 (active) Human kit. Briefly, after washing the cells with PBS, the cells were collected and lysed by adding it to the extraction buffer containing 1 mM PMSF (stock is 0.3 M in DMSO) and Protease inhibitor cocktail (e.g. Sigma Cat. #.P-2714) (reconstituted according to manufacturer’s guideline). 500 μl per 5 ml Cell Extraction Buffer were added, Protease inhibitors (1 mL per 1 x 107 cells) then the lysate was diluted immediately prior to the assay. At the end of the assay the optical density of each well was determined within 30 minutes using a microplate reader set at 450 nm (each experiment was repeated twice). 2.6.3. Determination of Bax and Bcl-2 HepG-2 cells were grown in RPMI 1640 containing 10% fetal bovine serum at 37°C, stimulated with the compound to be tested for Bax and Bcl-2 and lysed with Cell Extraction Buffer. This lysate was diluted in Standard Diluent Buffer over the range of the assay and was measured for human active Bax and Bcl-2 content. Using DRG® Human Bax ELISA (EIA4487) kit and Zymed Bcl-2 ELISA Kit. (each experiment was repeated twice). 2.6.4. Cell cycle analysis

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HepG-2 cells were seeded in a 6-well plate at concentration of 1x105 cells per well, then incubated for 24 h. The cells were treated with 0.1% DMSO vehicle or 10 µg of CBC2 compound for 48 h. Cells were harvested and fixed for 12 h using ice-cold 70% ethanol at 4 o

C. Removal of ethanol and washing cells with cold PBS. After that cells incubated in 0.5 mL

of PBS containing 1 mg/mL Rnase for 30 min at 37 oC. Then, cells were stained for 30 min with propidium iodide in a dark condition. Then DNA contents were detected by using flow cytometer (each experiment was repeated twice). 2.6.5. Annexin-V assay HepG-2 cells were seeded in a 6-well plate (1x105 cells/well), incubated for 24 h, and then treated with vehicle (0.1% DMSO) or 10 µg of compounds CSB-2 for 48 h. Then, the cells were harvested, washed using PBS and stained for 15 min at room temperature in the dark using annexin V-FITC and PI in binding buffer (10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl2 at pH 7.4), then analyzed by the flow cytometer (each experiment was repeated twice). 3. Results and Discussion 3.1. Chemistry 3.1.1. Physico-chemical characterization of chitosan and chitosan nanoparticles 3.1.1.1. Elemental analysis of chitosan An Elementaer Vario EL III apparatus (Elementar, Germany) was used for the analysis of the amount of C and N in chitosan. Its results showed that the degree of deacetylation of the extracted chitosan was found to be 87% (cf. experimental part). 3.1.1.2. Calculation of the molecular weight of chitosan Viscometry is considered as the best technique for determination of the molecular weight (Mw) of the extracted chitosan. It was reported that the Mw of chitosan is proportional to its viscosity [30]. The results indicated that chitosan obtained from Procambarus clarkii had a low molecular weight = 80.7235 kDa (cf. experimental part). 3.1.1.3. Fourier Transform Infrared Spectroscopy (FTIR) of chitosan and chitosan nanoparticles The FTIR of the extracted chitosan showed the appearance of the forked peak at around 3442 cm-1, which is corresponded to the amino group (NH2) and the disappearance of the band for the carbonyl group of amidic linkage (NHCOCH3) at around1700 cm-1. The FTIR spectra of chitosan and chitosan nanoparticles were found in a similar manner. The only difference was observed in chitosan nanoparticles, the band at 1107 cm-1 which is characteristic for P=O groups of TTP. 3.1.1.4. Nuclear magnetic resonance (1H-NMR) of chitosan

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Further support for the structure of the extracted chitosan was gained from its 1H-NMR spectrum of the extracted chitosan. It showed singlet peaks that correspond to methyl protons of acetyl group at 1.8 ppm and H-1 (Ac) at 4.3 ppm for the acetylated chitosan, in addition to the characteristic peaks of deacetylated chitosan that were found at 2.8 and 4.7 ppm, which correspond to H-1 and H-2, respectively. The degree of deacetylation of chitosan was also calculated by using 1H-NMR technique by comparing the integration of the signals of deacetylated/ acetylated forms as previously reported [31]. It was found that the DDA was 76%. We believe that the 1H-NMR technique is more accurate in this calculation; however, the lower percentage of DDA by using this technique than the elemental analysis method may be due to the partial solubility of chitosan in the deuterated trifluoroacetic acid which used as a solvent. 3.1.1.5. The surface morphology of chitosan and chitosan nanoparticles 3.1.1.5.1. Scanning Electron Microscopy (SEM) of chitosan SEM analyses conducted on chitosan have revealed that it has three main surface morphologies: a rough surface without pores or nanofibers, a surface composed of nanofibers and a surface with both pores and nanofibers [32-34]. The morphology of the extracted chitosan belonged to the first type and appeared as flakes that possess hard and rough surface that does exhibit neither pores nor nanofibers (Fig. 1A). Additionally, higher magnification of the flakes indicated that it consists of numerous fine fibers (Fig. 1B). [Fig. 1] 3.1.1.5.2. Transmission Electron Microscopy (TEM) of chitosan and chitosan nanoparticles The chitosan structure was further examined by TEM to confirm and reveal its detailed surface morphology. It was found that it consists of an anastomosing network of fibers that agrees with the previous data of the Scanning Electron Microscopy (Fig. 2A). Also the chitosan nanoparticles were examined by TEM. The results showed that their particles are spherical in shape with smooth margins, whose size varied from 3-9 nm (Fig. 2B). [Fig. 2] 3.1.2. Synthesis of chitosan Schiff bases (CSB) The treatment of the extracted and purified chitosan with some different heterocyclic aldehydes containing a nitrogen based or sulfur-based heterocycles ring systems such as thiophene-2-carboxaldehyde CSB-1, 1H-indole-3-carboxaldehyde CSB-2 and 1,3-diphenyl1H-pyrazole-4-carbaldehyde CSB-3 in refluxing n-butanol and in the presence of a catalytic

amount of glacial acetic acid afforded the condensation product or the Schiff bases of chitosan

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CSB as shown in (Scheme 2). One of the used aldehydes, which is the 1,3-diphenyl-1H-pyrazole4-carbaldehyde was synthesized through the Vilsmeier–Haack reaction of acetophenone

phenylhydrazone that is derived from the reaction of acetophenone with phenyl hydrazine, while the other aldehydes were used without synthesis.

[Scheme 2] 3.1.3. Characterization of chitosan Schiff bases (CSB): Infrared spectroscopy (FTIR) study The IR spectra of the obtained chitosan Schiff bases showed that the disappearance of forked absorption band for NH2 group at around 3442 cm-1 of chitosan and the appearance of absorption bands for aromatic C-H stretching at range from 3040 and 3109 cm-1, absorption band for C=N at range from 1612 to 1640 cm-1 corresponding to the imino group (-CH=N), absorption band for C=C aromatic ring(s) at range from 1574 to 1598 cm-1; the bands of aromatic C-H bending in a range from 680-800 cm-1. The chitosan Schiff base CSB-2 exhibited absorption band at 3166 cm-1 for NH of indole moiety. The aromatic function groups such as (C=C and -CH) as well as the imino group (-CH=N) confirmed the Schiff bases formation. In addition, their spectra showed other absorption bands that characteristic to the pure chitosan without hybridization such as a broad band for OH, bands for aliphatic C-H stretching < 3000 cm−1, bands for asymmetric C-O-C bridge stretching around 1157 cm−1 and band for C-O stretching in range from 1043 to 1082 cm−1. 3.2. Biological activity evaluations 3.2.1. In vitro cytotoxic activity All of the newly synthesized poly saccharides compounds were screened for their anticancer activity against three cell lines, which are human liver carcinoma cell line (HepG2), colon cancer cell line (HCT-116) and human breast cancer cell line (MCF-7). Doxorubicin was used as a reference standard, and it showed IC50 of 3.9, 3.5, 4.2 μg/ml, respectively. The anticancer activity profile suggested that the tested compounds showed variable activities compared to reference drug as shown in (Table 1 and Fig. 3). The newly synthesized chitosan hybrids have higher to moderate antiproliferative activity against cancer cell lines. The obtained results revealed that the tested compounds showed remarkable cytotoxic activity against all of the tested cell lines, their IC50 values were less than 37 μg/ml, whereas the compounds CSB-2 and CSB-3 showed a good cytotoxic activities. Compounds CSB-1 and CNP have a good effect on cancer cells, while compound Cs showed moderately activity. It has been noticed that chitosan Cs (IC50 around 35 μg/ml), has a lower activity than chitosan nanoparticle (IC50 arranged from 14.11 to 17.65 μg/ml). This may be due to increasing of

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surface area in the nano form that led to increase of interactions. The presence of heterocyclic moieties in chitosan Schiff bases increased the cytotoxic activities. Chitosan Schiff base with thiophene ring system CSB-1 showed a closely similar activity compared to chitosan nanoparticles. Compound CSB-2 has a higher activity than compound CSB-3, with IC50 equal to 6.77, 7.02 and 8.11 μg/ml against HepG-2, HCT-116 and MCF-7, respectively, which can be attributed to the presence of indole moiety. Also, compound CSB-3 showed good cytotoxic activity but lower than compound CSB-2 with IC50 equal to 8.92, 9.37 and 10.89 μg/ml against the same tested cell lines respectively. CSB-3 has pyrazole moiety, however CSB-2 has an indole nucleus. It can be concluded that hybridizing of chitosan with indole through Schiff base formation led to obtaining the most potent derivative CSB-2 followed by pyrazole CSB-3 and then thiophene derivatives CSB-1. Table 1: Cytotoxicity activity (IC50) of the tested compounds against HEPG-2, HCT-116 and MCF-7 cell lines Compound No. Cs CNP CSB-1 CSB-2 CSB-3 Doxorubicin

IC50 (µg/ml) HEPG-2

HCT-116

MCF-7

37 ± 2 16 ± 1 14 ± 1 6.8 ± 0.6 8.9 ± 0.6 3.9 ± 0.2

34 ± 2 14 ± 1 16 ± 1 7.0 ± 0.6 9.4 ± 0.7 3.5 ± 0.2

35 ± 2 18 ± 1 19 ± 1 8.1 ± 0.7 10.9 ± 0.9 4.2 ± 0.3

[Fig. 3] 3.2.2. Apoptosis studies From the obtained results, compounds CSB-2 and CSB-3 showed good anticancer activity, so their ability to induce apoptosis was evaluated by monitoring the levels of active caspase 3 and the Bax/Bcl2 ratio, which are considered as an unambiguous proof for induction of apoptotic pathways [35]. 3.2.2.1. Effects on the level of active caspase-3

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It is known that caspase 3 plays an important role in apoptosis induction [36]. In the present study, HepG2 cells were treated with compounds CSB-2 and CSB-3, and the obtained results showed that these compounds have been regulated the caspase 3 enzyme and increased its level by 5.6 and 4.3 folds respectively (Table 2) comparing to the control, thus proving that compounds CSB-2 and CSB-3 have a potential apoptosis induction. 3.2.2.2. Effects on mitochondrial apoptosis pathway (Bax and Bcl-2 proteins) The mitochondrial apoptotic pathway was regulated by Bcl-2 family of proteins. Among these are Bcl2 (anti-apoptotic) and Bax (pro-apoptotic) proteins. Balance between them controls cell apoptosis. In the present study, HepG2 cells were treated with the compounds CSB-2 and CSB-3 to study their effect on the levels of Bcl2 and Bax (Table 2). Table 2: Effect of CSB-2 and CSB-3 on some apoptosis key markers caspase 3 Bax Compound No. (pg/ml) (ng/ml) CSB-2 328 274 CSB-3 254.7 210.3 Control 58.6 30.93

Bcl-2 (ng/ml) 1.858 2.364 4.341

The obtained results showed that compound CSB-2 downregulated the level of the antiapoptotic protein Bcl2 by 2.5 folds when compared to the control, while it upregulated the level of the pro-apoptotic protein Bax by 8.9 folds. Compound CSB-3 downregulated the level of the anti-apoptotic protein Bcl2 by 1.8 folds in comparison to the control while it upregulated the level of the pro-apoptotic protein Bax by 6.8 folds. The Bax/Bcl2 ratio for compound CSB2 was calculated to be 20.69 folds as compared to the control, while the Bax/Bcl2 ratio for compound CSB-3 was calculated to be 12.48 folds, as comparing to the control. This is a clear indication that these two compounds induced the cells to undergo towards apoptosis. 3.2.2.3. Flow cytometric analysis of the cell cycle The aforementioned results revealed that compound CSB-2 deserve further explorations. Cell cycle arrest was performed on HepG2 cancer cells, upon treating them with 10 µg of compound CSB-2. The obtained results are represented in (Table 3 and Fig. 4), and reveal the accumulation of HepG2 cells at G2/M phase; the percent became 17.68 after treating cells with compound CSB-2. Also, compound CSB-2 induced apoptosis at preG1 phase and the percentage of cell death was 13.37. Table 3: Cell cycle analysis in HepG2 after treatment with compound CSB-2

Compound No.

%G0-G1

%S

%G2/M

%Pre-G1

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43.86 57.36

CSB-2/HepG2 cont.HepG2

38.46 36.22

17.68 6.42

13.37 1.86

[Fig. 4] 3.2.2.4. Apoptosis induction Percentage of cell death at pri-G1 phase was 13.37 as shown in Table 3. To investigate the mode of induced cell death in HepG-2 cells at pre-G1 phase, Annexin V/propidium iodide (PI) double staining assay method was performed. HepG-2 cells were treated with 10 µg of compound CSB-2. The obtained results are represented in (Table 4 and Fig. 5). The percentage of apoptosis induced by compound CSB-2 was 11.88 (early and late apoptosis) compared with 1.43 percent for control cells. So, treatment of HepG-2 cells with compound CSB-2 caused increase in the percentage of apoptotic cells at pre-G1 phase comparing to untreated HepG-2 cells as shown in (Fig. 7). Table 4: Percent of apoptosis after treatment of HepG2 cells with compound CSB-2 Compound No. CSB-2/HepG2 cont.HepG2

Total 13.37 1.86

% Apoptosis Early 5.41 0.88

Late 6.47 0.55

Necrosis 1.49 0.43

[Fig. 5] [Fig. 6] [Fig. 7] 4. Conclusion The extracted chitosan Cs was obtained in a lower molecular weight and with the best degree of acetylation after 9 hr. Then it was modified into the form of nanoparticles CNP and chitosan Schiff bases CSB. The best results of chitosan nanoparticles were obtained from an equimolar amount of chitosan and sodium tripolyphosphate. The cytotoxic activity of the tested compounds against the three cell lines indicated that the chitosan Schiff bases had better anticancer activities and also chitosan nanoparticles than chitosan itself. The Caspase-3 and Bax/Bcl-2 ratio studies against HePG-2 cells showed that the compounds CSB-2 and CSB-3 induced the apoptosis. The apoptosis induction for compound CSB-2 occurred at preG1 phase with an increasing in the percentage of apoptotic cells. Chitosan Schiff bases hybrid with indole moiety CSB-2 has the most potent as anticancer agent and apoptosis inducer followed by pyrazole moiety CSB-3 and then thiophene moiety CSB-1. Acknowledgment

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This article is part of results of Project ID: 9450781006 - ASRT JESOR 2015, entitled ‘Novel extraction and industry from wastes and exoskeleton of the introduced freshwater crawfish’, so we thank Academy of Scientific Research and Technology for funding this work.

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Schemes and Figures Captions Scheme 1. Extraction and synthesis of chitosan Fig. 1. Scanning electron-micrograph (SEM) of the extracted chitosan

18

Fig. 2. Transmission electron-micrograph (TEM) of (A) chitosan, (B) chitosan nanoparticles. Scheme 2. Reaction of chitosan with different heterocyclic aldehydes Fig. 3. Cytotoxicity activity (IC50) of the tested compounds on the three cancer cell lines Fig. 4. Percentage of HepG-2 cells at different phases when treated with compound CSB-2 comparing to untreated cells Fig. 5. Apoptosis induction in HepG-2 cells when treated with compound CSB-2 in comparison to untreated cells Fig. 6. The Cell cycle analysis and the percentage of annexin V-assay for HepG-2 cells Fig. 7. The Cell cycle analysis and the percentage of annexin V-assay for HepG-2 cells after treatment with compound CSB-2

[Scheme 1]

19

[Fig. 1]

20

[Fig. 2]

[Scheme 2]

21

Invitro cyctotoxicity 50

IC50 (µg/ml)

40 30 HEPG-2

20

HCT-116 10

MCF-7

0

[Fig. 3]

Cell Cycle Analysis 70 60 50 %G0-G1

40

%S

30

%G2/M

20

%Apoptosis

10 0 CSB-2/HepG2

cont.HepG2

[Fig. 4]

22

% Apoptosis 16 14 12

10 8

CSB-2/HepG2

6

cont.HepG2

4 2 0 Total

Early

Late

Apoptosis

Necrosis

[Fig. 5]

23

[Fig. 6]

24

[Fig. 7]