or cisplatin in a murine model of Ehrlich ascites carcinoma with hematinic and hepato-renal protective action

Journal of Functional Foods 66 (2020) 103831

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The antitumor activity of Arthrospira platensis and/or cisplatin in a murine model of Ehrlich ascites carcinoma with hematinic and hepato-renal protective action

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Mohamed A. Hashema, Sara B.A. Shoeebb, Yasmina M. Abd-Elhakimc, , Wafaa A.M. Mohameda a

Department of Clinical Pathology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt Faculty of Veterinary Medicine, Mansoura University, Egypt c Department of Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt b

A R T I C LE I N FO

A B S T R A C T

Keywords: Arthrospira platensis Cisplatin Ehrlich ascites carcinoma Peritoneum Ki67 Caspase 3

Herein two experiments were conducted. The first experiment evaluated the antitumor activity of Arthrospira platensis (Spirulina platensis, SP) alone or in combination with cisplatin (CDDP) in Ehrlich ascites carcinoma (EAC) bearing mice. The second experiment assessed the outcomes of SP and/or CDDP administration on renal, hepatic, and bone marrow function in normal mice. The results showed that the EAC evoked a significant decrease in the mice survival rate, life span percentage, antioxidant defense system, and Caspase-3 immunoexpression but a significant increase in the viable cancer cells count, tumor and lipid peroxidation biomarkers concentrations, and Ki-67 immunoexpression. The EAC induced alterations improved to various degrees following SP and/or CDDP administration. SP minimized the oxidative hepatic and renal DNA-damaging and hematotoxic effect of CDDP. Overall, SP has a potent anticancer activity and could be used effectively as a hematinic and hepato-renal protective agent with anticancer drugs like CDDP.

1. Introduction In the twenty-first century, cancer will represent the prominent cause of death all over the world (Bray et al., 2018). Cisplatin, cisdiamminedichloridoplatinum(II) (CDDP), is an extensively used chemotherapeutic medication with broad-spectrum activity. CDDP is used for the treatment of numerous malignancies comprising the ovaries, testes, and germ cells cancers together with head and neck solid tumors (Al-Eitan, Alzoubi, Al-Smadi, & Khabour, 2020). It induces apoptosis of the cancer cells via the activation of multiple signal transduction pathways including mitochondrial pathways (Boulikas & Vougiouka, 2003; Reedijk & Lohman, 1985). Despite its potent antitumor potential, in normal tissues, it is a hazardous cytotoxic agent for several body organs and causes various adverse effects such as vomiting, nausea, hair loss (Boussios, Pentheroudakis, Katsanos, & Pavlidis, 2012), hematotoxicity (Karale & Kamath, 2017), ototoxicity (Sheth, Mukherjea, Rybak, & Ramkumar, 2017), hepatotoxicity (Ekinci Akdemir, Albayrak, Çalik, Bayir, & Gülçin, 2017), genotoxicity (Al-Eitan et al., 2020), and nephrotoxicity Miller, Tadagavadi, Ramesh, and Reeves (2010). Oxidative stress is a consequence of reactive oxygen species (ROS) generation and/or suppression of antioxidant defense system (Gülçin,

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Büyükokuroǧlu, Oktay, & Küfrevioǧlu, 2003). The stimulation of ROS after the damage has been reported in several studies related to CDDP toxicity (Martins, Santos, Curti, Bianchi, & Santos, 2008; Santos et al., 2007). To alleviate the toxic effects of chemotherapy such as CDDP, numerous investigators have explored safe and efficient medicinal plants for their antioxidant as well as anticancer potential and also attempted to improve their anticarcinogenic activity (Desai et al., 2008). In this era, a growing interest has been paid to use natural products for therapeutic purposes or to mitigate drug adverse impacts (AbdElhakim, El Bohi, Hassan, El Sayed, & Abd-Elmotal, 2018; Abd-Elhakim, El-Sharkawy, Mohammed, Ebraheim, & Shalaby, 2020; Mohamed, AbdElhakim, & Ismail, 2019). Arthrospira platensis (Spirulina platensis, SP) is a cyanobacterium that has been used as a safe food supplement since antiquity due to the high contents of γ-linolenic acid, proteins, minerals, and vitamins (Reboleira et al., 2019). The earlier in-vitro as well as in-vivo investigations have shown SP as a good source for selenium (Se) and about 85% of Se is available in organic forms and mostly identified as selenomethionine (Chen & Wong, 2008). It contains a large amount of allophycocyanin with free radical scavenging and antiarthritic activities (Narendra, Pawan, & Surendra, 2010). It has

Corresponding author. E-mail address: [email protected] (Y.M. Abd-Elhakim).

https://doi.org/10.1016/j.jff.2020.103831 Received 15 November 2019; Received in revised form 30 December 2019; Accepted 29 January 2020 1756-4646/ © 2020 Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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tablet has 250 mg pure SP. For experimental use, a working solution of SP was prepared by diluting with distilled water. The mice were given SP or distilled water (at a volume of 0.1 ml/each mouse containing the desired concentration of test compounds) once daily through a 20-G feeding needle. CDDP was obtained from the Egyptian International Medical Company (EIMC), United Pharmaceuticals, Cairo, Egypt. All chemicals and stains were of analytical grade and purchased from Egyptian Diagnostic Media (EDM), El-Nasr, and El Gomhoria Companies, Egypt.

immuno-stimulant (Narendra et al., 2010), antihyperlipidemic, antihypertensive (Torres-Duran, Ferreira-Hermosillo, & Juarez-Oropeza, 2007), antihyperglycemic (Senthil, Balu, & Murugesan, 2013), bactericidal (Muthusamy, Thangasamy, Raja, Chinnappan, & Kandasamy, 2017), and radioprotective actions (Mohamed, Ismail, & El-Hakim, 2014). Moreover, SP has hepatoprotective (Mazokopakis et al., 2014), antioxidant (Nasirian, Dadkhah, Moradi-Kor, & Obeidavi, 2018), antiinflammatory (Wu et al., 2016), growth promoter (Masuda & Chitundu, 2019), cardioprotective (El-Shanshory et al., 2019), and reproprotctive activities (Abd El-Hakim, Mohamed, & El-Metwally, 2018). There is a conflict among experimental studies regarding the anticancer activity of SP. Barakat, Elshazly, and Mahmoud (2015) reported that SP Lacks antitumor activity against Solid Ehrlich Carcinoma in Female Mice. While, other researchers verified that bioactive compounds such as polysaccharides and phycobiliproteins are capable to hinder the growth of tumor cells (Ismail et al., 2009; Saiki, Murata, Fujii, & Kato, 2004). Also, the anticancer effect of SP has been tested in various cancer cell model including leukemia Kasumi-1, myelogenous leukemia K-562, pancreatic cancer cell lines (PA-TU-8902, Mia PaCa-2, and BxPC-3), and the human lung cancer A549 cell line in the earlier studies of Flores Hernandez, Khandual, and Ramírez López (2017), Konícková et al. (2014), and Czerwonka et al. (2018a), respectively. Consequently, based on the information obtained from these in vitro studies, we have designed this study to evaluate the probable antitumor activity of SP and their possible potentiating role of the CPDD effect in an in vivo model of Ehrlich ascites carcinoma (EAC) bearing mice. EAC is an undifferentiated carcinoma, which is characterized by fast proliferation, great transplantable ability, and short life span (Ozaslan, Karagoz, Kilic, & Guldur, 2011). EAC bears closeness to human tumors; consequently, the ascetic forms of this tumor are commonly used to assess the antitumor effect of various products (Da Silva & Chaar, 2009). Additionally, from another perspective, the effect of SP on normal cell models has been previously investigated using mouse BV-2 normal microglial cells (Chen et al., 2012), T3 mouse fibroblasts (Chu, Lim, Radhakrishnan, & Lim, 2010), murine bone marrow (Hayashi et al., 2006), and human stem cells (Bachstetter et al., 2010). The findings of these studies verified those protective properties in normal cells in vitro. Hence, in the current study, we aimed to investigate if SP could protect against the side effect of CDDP particularly hepatic and renal damage by their antioxidant and antioxidant activity in normal mice. Hence, we used an in vivo model of mice to test this hypothesis to simulate the real exposure scenarios.

2.3. The first experiment (antitumor activity experiment) 2.3.1. Experimental design One hundred and sixty adult female Swiss albino mice were distributed into 4 groups (40 mice/group) as follows: 1. EAC group: Inoculated IP with a single dose of EAC (2.5 × 106) on the day 15th from starting the experiment. EAC cells were obtained from the National Cancer Institute, Cairo University, Egypt. The cell lines were checked for their viability (Boyse, 1964) and then maintained by serial intraperitoneal transplantation of 2.5 × 106 EAC/0.2 ml ascitic fluid/mouse (Salem, Badr, & Neamat-Allah, 2011). 2. EAC + SP group: EAC –bearing mice orally administered SP (0.5 g/ kg b.wt., PO) (Konícková et al., 2014), two weeks before and 2 weeks after the EAC-inoculation. 3. EAC + CPDD group: EAC –bearing mice injected IP by CDDP (40 µg/mouse, IP) (Deuis et al., 2014), two weeks after the EACinoculation. 4. EAC + SP + CPDD group: EAC –bearing mice received both SP (0.5 g/kg b.wt., PO) for 30 days from starting the experiment and CDDP (40 µg/mouse, IP) for 15 days post-EAC-inoculation. 2.3.2. Survival analysis The EAC bearing mice of all groups were daily observed for survival analysis. The mean survival time (MST) was determined according to the following equation: MST = [1st Death + Last Death]/2 (Teicher, 2013). The increase in life span percentage (ILS %) of each group was calculated as follows: increase in life span= (T-C/C) × 100. Where T = number of days the treated animals survived and C = number of days control animals survived (Pandya, Tigari, Dupadahalli, Kamurthy, & Nadendla, 2013). 2.3.3. Evaluation of EAC volume, cytology, and viability The ascitic fluid was aspirated from mice for EAC volume detection, viability test, and cytological examination. The volume of ascitic fluid was measured by using a graduated centrifuge tube. For cytological examination, giemsa-stained ascitic fluid smears were prepared (Badr et al., 2012). Counting of total, live, and dead EAC cells was done by diluting the aspirated fluid (9 vol) with trypan blue 1% (one volume). The mixture was incubated exactly for 10 min at 37 °C in water bath then at a Neubauer hemocytometer, the number of total, dead (stained) and life (unstained) cells were counted within 5 min (Boyse, 1964).

2. Materials and methods 2.1. Animals A total of 220 adult female Swiss albino mice (18–20 g body weight) were gained from the Laboratory Animal Farm of Veterinary Medicine College, Zagazig University. All mice were reared under strict hygienic conditions and were fed a balanced diet. Water was available ad- libitum. Animals were housed in stainless-steel cages and maintained in a 12 h light-dark cycle at a controlled temperature (21–24 °C) and humidity (50–60%). They were kept for 15 days without medication for acclimation before beginning the study. All experimental procedures were performed following the National Institutes of Health guidelines for the Care and Use of Laboratory Animals in scientific investigations. The Institutional Animal Care and Use Committee of Zagazig University approved the present protocol with the reference number ZU-IACUC/1/ F/216/2019.

2.3.4. Serum biochemical parameters Tumor markers bioassay including Carcinoembryonic antigen (CEA), Cancer antigen 125 (CA-125), Cancer antigen 19-9 (CA 19-9), and Cancer antigen 15-3 (CA 15-3) were measured in the serum using mice-specific enzyme-linked immunosorbent assay (ELISA) kits following the methods described at the enclosed manufacturer’s pamphlets. Malondialdehyde (MDA), reduced glutathione (GSH), and superoxide dismutase (SOD) following the protocols of Ohkawa, Ohishi, and Yagi (1979), Beutler (1963) and Nishikawa, Sato, Kuroki, Utsumi, and Inoue (1998), respectively. Also, serum nitric oxide (NO) was measured colorimetrically according to Montgomery and Dymock (1961).

2.2. Tested substances and chemicals SP is a bright, blue-green tablet with a precise fragrance obtained from power nutritional, Jin Shun, Guangzhou, Trading Co., USA. Each 2

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2.4.5. Histopathological and histochemical investigations of liver and kidney Paraffin-embedded sections of the liver and kidneys were sliced into a thickness of 5 µm and stained with hematoxylin and eosin (H&E). Other sections of the liver and kidney were stained by Feulgen’s reaction to demonstrate the nuclear DNA content (Schulte & Wittekind, 1989).

2.3.5. Histopathological and immunohistochemical investigations Paraffin-embedded sections of peritoneum were sliced into a thickness of 5 µm and stained with hematoxylin and eosin (H&E) (Suvarna, Layton, & Bancroft, 2018). For immunohistochemistry (IHC), other peritoneal sections were incubated with 10 mM sodium citrate buffer (pH 6.0) at 80 °C for 10 min for antigen retrieval, endogenous peroxidases were blocked by 3% H2O2 in PBS for 10 min. Then, they incubated in a humidified chamber with primary antibodies (Ab) against Ki67 and caspase-3 at 4 °C overnight and then with horseradish peroxidase-conjugated secondary Ab with a dilution of 1:100 for 30 min at 37 °C and visualized by 3,3′-diaminobenzidine tetrahydrochloride reagent. The sections were counterstained with hematoxylin and digitally imaged (Jakob et al., 2008).

2.5. Statistical analysis A one-way analysis of variance (ANOVA) was used for the analysis of the data followed by Duncan's Multiple Range Test. Statistical analyses were done by Software GraphPad INSTAT (Version 2). A pvalue < 0.05 was accepted as significant level. Furthermore, a Graphpad (ISI Software, Philadelphia, PA) computer program was used. The values have been shown as means ± SE.

2.4. Second experiments (Side effects experiment) 2.4.1. Experimental design Sixty adult female Swiss albino mice were divided into 4 groups (15 mice/group) as follows:

3. Results 3.1. The first experiment (antitumor activity experiment)

1. Control group: mice were kept on distilled water. 2. SP group: mice were administered SP (0.5 g/kg b.wt., PO) for 30 successive days from the 1st till 30th day from starting the experiment. 3. CDDP group: mice were received CDDP (40 µg/mouse, IP) for 15 successive days from the 15th till the 30th day from starting the experiment. 4. CDDP + SP group: mice were given both SP and CDDP as the previously mentioned doses and route of administration.

3.1.1. Clinical signs and ascitic fluid characters EAC-bearing mice showed loss of activity, decreased appetite, and swollen abdomen. EAC-bearing mice received SP and/or CDDP showed a reduction in the dimension of the abdomen. The improvement was high at the EAC + SP + CDDP group, moderate at EAC + CDDP group and mild at EAC + SP group. Physically, mice of the EAC group have a viscous bloody ascitic fluid. Microscopically, EAC cells are numerous and active with intact membrane and contain an abundance of activated monocytes, neutrophils and lymphocytes (Fig. 1A). Treatment with SP and/or CDDP resulted in significant apoptosis and lysis of the cancer cells gradually to be mild in the EAC + SP group (Fig. 1B), moderate in the EAC + CDDP group (Fig. 1C), and high in the EAC + SP + CDDP group (Fig. 1D).

2.4.2. Sampling At the end of the 30 days of the experiment, two blood samples were collected from the medial canthus of the eye from mice in all groups. One blood sample was collected in EDTA containing tubes for hematological evaluation. While the other blood sample was kept in a plane tube to separate serum for clinico-biochemical analysis such as liver and kidney function tests. Following the mice's cervical dislocation, bone marrow was aspirated from the femur of mice groups on cold buffered saline and centrifuged at 3000 rpm for 15 min. The marrow pellets were collected, smeared on glass slides, and stained with Giemsa’s stain (Bolliger, 2004). Some fragments of liver and kidneys (0.5 g) were collected, added to 5 ml of cold phosphate buffer saline (pH 7.4, 0.1 M), homogenized, filtered, and centrifuged at 3000 rpm for 15 min for evaluating oxidative stress indices. While the other liver and kidneys specimens were kept in 10% neutral buffered formalin for histopathological and histochemical investigations.

3.1.2. Survival analysis and viability As shown in Table 1, EAC-bearing mice showed a notable decrease in the MST. Single or co-exposure to SP and CDDP resulted in an obvious increase in the values of MST, and ILS% compared to EAC group.

2.4.3. Hematological evaluations Complete blood picture (RBCs, Hb, PCV, MCV, MCHC, PLT, and WBCs) was carried out using an automated hematology analyzer, Hospitex Hemascreen 18, Italy. Meanwhile, the differential leukocytic count was performed using blood films stained with Giemsa’s stain (Coles, 1986). 2.4.4. Clinicobiochemical analysis and oxidative status assessment All biochemical tests were performed using colorimetric kits following the instructions described at the manufacturer's guides. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total proteins, albumin, urea, and creatinine were determined by kits of Diamond-Diagnostic, Egypt following the protocols of Reitman and Frankel (1957), Gornal, Bardawill, and David (1949), Doumas, Bayse, Carter, Peters, and Schaffer (1981); Fawcett and Scott (1960), Larsen (1972), respectively. Meanwhile, serum alkaline phosphatase (ALP) activity was measured by the kit of Spectrum, Hannover, Germany (Kind & King, 1954). The hepatic and renal homogenates were used for measuring the amount of MDA, GSH, and SOD as previously mentioned.

Fig. 1. Representative photo of Giemsa-stained ascitic fluid showing intact live tumor cells in the EAC group (A). EAC + SP, EAC + CDDP, and EAC + SP + CDDP groups showing mild (B), moderate (C), marked (D) degeneration of the cancer cells (arrows), respectively (Giemsa, X1000). 3

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Table 1 Survival analysis, viability, serum tumor, and oxidative markers of Ehrlich ascites carcinoma (EAC) bearing mice treated with spirulina platensis (SP) and/or cisplatin (CDDP). Control

EAC

SP + EAC

EAC + CDDP

SP + EAC + CDDP

Survival time range (days) MST (days) ILS (%) EAC volume (ml) Total EAC number (×107) Live EAC number (×107) Live non stained cells% Dead EAC number (×107) Dead blue stained cells %

– – – – – – – – –

15–22 18.50 0 7.00a ± 0.25 122.35a ± 1.3 114.78a ± 3.1 93.81 7.56d ± 1.78 6.17

18–25 21.50 16.21 4.00b ± 0.32 94.26b ± 2.20 76.89b ± 5.23 81.57 17.36c ± 3.74 18.41

16–28 22.00 18.91 1.86c ± 0.30 53.11c ± 1.16 27.18c ± 1.21 51.17 25.92b ± 2.39 48.80

17–30 23.50 27.02 1.15c ± 0.08 39.62d ± 0.71 7.87d ± 1.11 19.86 31.74a ± 0.64 80.11

Tumor markers CA 19-9 (U/ml) CA 15-3 (ng/ml) CA125 (ng/ml) CEA (pg/ml)

2.83e ± 0.17 0.10e ± 0.004 0.14e ± 0.02 34.60e ± 1.70

41.43a ± 2.06 9.75a ± 0.118 4.60a ± 0.26 265.07a ± 4.21

24.63b ± 2.16 5.54b ± 0.273 1.80b ± 0.17 193.43b ± 6.37

17.66c ± 0.35 2.21c ± 0.614 0.91c ± 0.04 116.70c ± 4.34

9.10d ± 0.66 0.61d ± 0.089 0.42d ± 0.03 77.91d ± 7.00

Oxidative markers MDA (nmol/ml) SOD (U/ml) GSH (mmol/ml) NO (µmol/l)

12.44e ± 0.29 200.78a ± 6.33 9.10a ± 0.78 4.80a ± 0.67

93.34a ± 1.41 21.33e ± 0.88 0.66e ± 0.04 0.53e ± 0.06

79.66b ± 0.88 69.66d ± 2.96 1.67d ± 0.12 0.98d ± 0.10

55.33c ± 3.52 104.66c ± 4.90 4.33c ± 0.40 1.56c ± 0.34

32.30d ± 2.51 151.33b ± 1.76 7.00b ± 0.48 3.04b ± 0.41

A one-way analysis of variance (ANOVA) followed by Duncan's Multiple Range test was used for statistical analysis. Means within the same row carrying different superscripts (a, b, c, d, and e) are significantly different at p < 0.05. The values shown are means ± SE. n = 10. MST; Mean survival time, ILS%; The increase in life span percentage, CA 19-9; Cancer antigen 19-9, CA 15-3; Cancer antigen 15-3, CA-125; Cancer Antigen 125, CEA; Carcino-embryonic antigen, MDA; Malondialdehyde, SOD; Superoxide dismutase, GSH; Glutathione reduced, NO, Nitric oxide.

showed neoplastic cell aggregations and invasion of the hyalinized skeletal muscles beside areas of pus formation (Fig. 2B and C). EAC + SP group showed large, vesicular, hyperchromatic, and mitotic activities of tumor cells attached with edematous and hyalinized muscles (Fig. 2D). The EAC + CDDP group showed the remaining small neoplastic mass admixed with pus formation (Fig. 2E). The EAC + SP + CDDP group showed normal muscle and adipose tissue with still presence of scattered neoplastic cells and round cell infiltrations (Fig. 2F). Immunohistochemical stained- peritoneal section of EAC, EAC + SP, EAC + CDDP, or EAC + SP + CDDP group showed few (Fig. 3A), mild (Fig. 3B), moderate (Fig. 3C) and high (Fig. 3D) caspase3 expression, respectively. Meanwhile, high (Fig. 3a), moderate (Fig. 3b), mild (Fig. 3c) and few (Fig. 3d) Ki67 expression was detected in EAC, EAC + SP, EAC + CDDP, or EAC + SP + CDDP group, respectively. Statistical scoring as shown in Figs (3 E and e) displayed that the expression of Caspase-3 and Ki-67 was +++ve (≤75 cells) in EAC + SP + CDDP and EAC groups, respectively,++ve (25–50 cells) at EAC + CDDP and EAC + SP groups, +ve (≥25 cells) at EAC + SP and EAC + CDDP groups and + ve (≥10 cells) at EAC and

The improvement was high at the EAC + SP + CDDP group, moderate in the EAC + CDDP group and mild in the EAC + SP group. Trypan blue stained EAC cells of the EAC group showed 93.81% live nonstained cells and 6.17% of dead blue-stained cells. The EAC + SP group showed 81.57% live and 18.41% dead. The EAC + CDDP group showed 51.17% live and 48.80% dead cells. The EAC + SP + CDDP group showed 19.86% live and 80.11% dead cells. 3.1.3. Serum biochemical parameters As presented in Table 1, mice of EAC group showed a significant increase in the values of CA19-9, CA15-3, CA125, CEA, and MDA but a significant decrease at the serum level of SOD, GSH, and NO compared to the control group. These disturbances were ameliorated in EACbearing mice exposed to SP and/or CDDP compared to the EAC-bearing non-treated mice. The improvement was high at the EAC + SP + CDDP group, moderate at EAC + CDDP group and mild at EAC + SP group. 3.1.4. Histopathological and immunohistochemical findings Peritoneal section of mice in the control group showed normal peritoneal tissue and muscles (Fig. 2A). Meanwhile, the EAC group

Fig. 2. Representative photomicrographs of H& E-stained mice peritoneal section. (A) Control group showing normal tissues and muscles (star). (B) EAC group showing multi forms of the mitotic figures of the neoplastic cells (thin arrows) bizarre cells were seen (thick arrows) beside pus aggregation with inflammatory cells (star). (C) EAC group showing polymorph neoplastic cells (arrows) between hyalinized mussels (star). (D) EAC + SP group showing large, vesicular, hyperchromatic and mitotic activities of tumor cells (thin arrow) attached with edematous and hyalinized, muscles (thick arrow). (E) EAC + CDDP group showing aggregations of neoplastic cells (arrow) neighboring the normal peritoneal tissues (star). (F) EAC + SP + CDDP group showing normal muscles with the still presence of minute scattered (star).

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Fig. 3. Representative photomicrographs of immunohistochemical staining for Caspase-3 (A-D) and Ki67 (a-d) expression mice peritoneum. EAC, EAC + SP, EAC + CDDP, and EAC + SP + CDDP groups showing few (A), mild (B), moderate (C), and high (D) Caspase-3 expression, respectively. Meanwhile, high (a), moderate (b), mild (c) and few (d) Ki67 expression was seen in mice of EAC, EAC + SP, EAC + CDDP, and EAC + SP + CDDP groups, respectively. (E and e) Changes in the immunohistochemical expression and quantitation of Caspase-3 and Ki67 in mice peritoneum of the different experimental groups. The values shown are means ± SE (n = 10). Bars with different letters (a, b, c, and d) significantly differ from one another (p < 0.05).

moderately decreased in the SP + CDDP group compared to the CDDP group but not completely returned to the normal level of the control group. Mice of the CDDP group showed non-significant changes in all previous parameters compared to the control group.

EAC + SP + CDDP group 3.2. Second experiment 3.2.1. Clinical signs Clinically, no mortalities were recorded in all groups. SP- treated mice were apparently healthy with no signs of illness as the control group. CDDP- treated mice showed a reduction in their body weight, anorexia, activity, and fur loss. These observed signs were mild in mice received SP with CDDP.

3.3. Histological alterations H&E-stained liver and kidney sections of control (Fig. 5A and B) and SP-treated (Fig. 5C and D) mice showed normal hepatic and renal tissue architectures. The CDDP-exposed group showed focal necrotic area infiltrated by lymphocytes, necrotic and apoptotic hepatic cells (Fig. 5E) besides renal blood vessels congestion, renal necrosis, and hyalinized blood vessel wall surrounded by inflammatory cells infiltration (Fig. 5F). However, the liver of the SP + CDDP group showed minute interstitial, periportal and subcapsular lymphocytic aggregation (Fig. 5G) with mild cystic renal tubules, mild focal interstitial lymphocytic infiltration, shrunken glomeruli, and increased Bauman's capsule (Fig. 5H). Feulgen stained- liver and -kidney sections of the control and SP treated mice showed high nuclear DNA content (Fig. 6A, B, E, and F). Meanwhile, the CDDP group showed very low nuclear DNA content (Fig. 6C and G). The SP + CDDP group showed moderate nuclear DNA content (Fig. 6D and H).

3.2.2. Hematological and bone marrow findings Concerning to the hemogram, data in Table 2 showed that, compared with the control group, mice of CDDP group showed a significant (p < 0.05) decrease in the mean values of RBCs, Hb, PCV, platelets, WBCs, lymphocytes, neutrophils, eosinophils, and monocytes but nonsignificant changes in MCV and MCHC. The previous alterations reflecting laboratory features of aplastic anemia which may be resulted from bone marrow depletion that represented by lowering the number of its cellular precursors (Fig. 4C). The observed anemia was moderately improved and bone marrow cellularity was restored (Fig. 4D) in the SP + CDDP group compared to the CDDP group but not completely returned to the normal level. Mice of the SP group showed normal blood picture with normal bone marrow cells (Fig. 4B) as the control group (Fig. 4A).

4. Discussion

3.2.3. Renal and hepatic tissue biochemical findings As shown in Table 2, compared to the control group, the CDDP group showed a significant (p < 0.05) increase in the values of ALT, AST, ALP, urea, creatinine together with hepatic and renal MDA. But, a significant (p < 0.05) decrease in serum total proteins and albumin, as well as hepatic and renal SOD and GSH, was recorded in the CDDP group compared with the control group. These alterations were

CDDP is a potent cytotoxic drug used for treating many cancers such as EAC, which considered one of the most common experimental tumors (Yu et al., 2017). During the recent past, great attentiveness was recorded toward exploring natural products as promising anticancer agents (Abd-Elhakim, Khalil, Awad, & Al-Ayadhi, 2014; Newman, 2008). Herein, in the antitumor activity experiment, EAC-bearing mice showed enlarged abdomen due to the accumulation of ascitic fluid that 5

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Table 2 Complete blood count, hepato-renal function tests, and selective oxidative stress indices of mice treated with spirulina platensis (SP) and/or cisplatin (CDDP). Control

SP

CDDP

SP + CDDP

Haematological indices RBCs (×106/µl) Hb (g/dl) PCV (%) MCV (fl) MCHC (%) PLT (×105/µl) WBCs (×103/µl) Lymphocytes (×103/µl) Neutrophils (×103/µl) Eosinophils (×103/µl) Monocytes (×103/µl)

7.50a ± 0.03 15.76a ± 0.08 44.00a ± 0.57 58.66a ± 0.68 35.83a ± 0.36 3.38a ± 0.07 10.40a ± 0.05 7.11a ± 0.06 2.73a ± 0.14 0.40a ± 0.05 0.15a ± 0.03

7.55a ± 0.02 16.03a ± 0.09 45.33a ± 1.76 60.00a ± 2.16 35.46a ± 1.23 3.47a ± 0.04 10.33a ± 0.18 7.10a ± 0.11 2.68a ± 0.11 0.38a ± 0.02 0.16a ± 0.02

3.45c ± 0.13 7.50c ± 0.28 20.50c ± 0.28 59.09a ± 2.27 36.55a ± 0.89 1.18c ± 0.10 4.76c ± 0.23 3.58c ± 0.24 0.95c ± 0.08 0.15c ± 0.01 0.07b ± 0.01

5.71b ± 0.04 12.26b ± 0.37 35.38b ± 0.88 61.80a ± 1.55 34.70a ± 0.24 2.24b ± 0.13 7.24b ± 0.40 5.23b ± 0.05 1.60b ± 0.32 0.28b ± 0.01 0.11ab ± 0.02

Liver function ALT (U/L) AST (U/L) ALP (IU/L)

13.34c ± 0.88 10.33c ± 0.88 49.00d ± 2.08

13.18c ± 2.02 11.19c ± 1.85 49.12d ± 0.57

54.23a ± 2.33 38.56a ± 1.45 82.00d ± 2.05

32.13b ± 1.45 26.33b ± 4.33 63.67a ± 2.02

Kidney function Total proteins (g/dl) Albumin (g/dl) Urea (mg/dl) Creatinine (mg/dl)

8.16a ± 0.20 4.60a ± 0.23 24.63c ± 1.45 0.64d ± 0.03

8.00a ± 0.05 4.62a ± 0.08 23.00c ± 1.73 0.56d ± 0.05

4.83c ± 0.12 2.03c ± 0.12 66.00a ± 1.74 2.89d ± 0.17

6.57b ± 0.14 3.03b ± 0.08 43.16b ± 1.76 1.24a ± 0.13

1.44c ± 0.04 28.69a ± 0.31 6.26a ± 0.22

1.38c ± 0.09 27.55a ± 0.84 6.09a ± 0.54

20.80a ± 0.42 8.91c ± 0.40 1.26c ± 0.30

10.48b ± 0.97 18.92b ± 0.60 3.21b ± 0.22

2.25c ± 0.28 33.61a ± 0.57 8.87a ± 0.47

2.20c ± 0.56 33.46a ± 1.57 8.91a ± 0.55

9.74a ± 0.58 16.20c ± 0.80 2.40c ± 0.32

5.06b ± 0.48 21.68b ± 0.48 4.88b ± 0.55

Oxidative stress indices Hepatic tissue MDA (nmol/g.tissue) SOD (U/g.tissue) GSH (mmol/g.tissue) Renal tissue MDA (nmol/g.tissue) SOD (U/g.tissue) GSH (nmol/g.tissue)

A one-way analysis of variance (ANOVA) followed by Duncan's Multiple Range test was used for statistical analysis. Means within the same row carrying different superscripts (a, b, c, and d) are significantly different at p < 0.05. The values shown are means ± SE. n = 10. Red blood cells, Hb; Hemoglobin, PCV; Packed cell volume, MCV; Mean corpuscular volume, MCHC; Mean corpuscular hemoglobin concentration, PLT; Platelets, WBCs; White blood cells, ALT; alanine aminotransferase, AST; aspartate aminotransferase, ALP; alkaline phosphatase, MDA; Malondialdehyde, SOD; Superoxide dismutase, GSH; Reduced glutathione.

fluid revealed a viscous- bloody fluid that contains erythrocytes and a huge number of activated leukocytes which might be due to the direct response to the tumor cells or in response to the reaction of the mice to these cells. These results are in agreement with Lala (1974). Similar results obtained by Loewenthal and Jahn (1932) who found that the stroma of the abdominal cavity is represented by the ascitic fluid and contains a variable number of white and red blood cells. Low survival rate and high deaths in the EAC group may be due to the intensive abdominal hemorrhage and/or progressive tumor growth (Hartveit, 1961). Our results are similar to the previous reports of Rahman, Alam, Choi, and Yoo (2017). Tumor markers are the blood molecules that help in the screening, diagnosis, monitoring, and prognosis of cancer (Ghosh et al., 2013). In the present study, EAC-bearing mice showed a significant increase in the serum levels of CEA, CA–125, CA15–3, and CA19–9 possibly due to the metastatic properties of the EAC cells that are able to induce a variety of abdominal tumors, including pancreatic, ovarian, gastric, and colorectal tumors (Cavazzoni, Bugiantella, Graziosi, Franceschini, & Donini, 2013). Oxidative stress is the pivotal trigger for the initiation and progression of cancer (Norwood, Tucci, & Benghuzzi, 2007). Our results confirm that the enhancement of lipid peroxidation in the EAC-bearing mice is a consequence of the depletion of GSH (Tohamy, Ibrahim, & Moneim, 2013). A decrease in the SOD activity in the EAC-bearing mice has been reported, since the activity of MnSOD is lost in the EAC cells. The inhibition of SOD activity, as a result of tumor growth, has also been reported by Marklund, Westman, Lundgren, and Roos (1982). NO is cytostatic or cytotoxic for the cancer cells. The present work showed a decrease in NO at EAC-mice (Nishikawa et al., 1998). Our results are agreed with Weiming, Liu, Loizidou, Ahmed, and Charles (2002).Ki-67

Fig. 4. Representative photo showed normal shape and incidence of bone marrow precursors of control (A) and SP-treated (B) mice meanwhile, the CDDP-exposed group showed severe bone marrow depletion (C) which restored towards the normal (D) in the SP + CDDP treated group (Giemsa, X1000).

formed mainly as a result of peritoneal serosa injury by the compression of the cancer cells that led to an increase in the capillary permeability, thus leaking more proteins into the abdominal cavity and /or due to the poor blood and lymph return. Similar results were obtained previously by Salem et al. (2011) and Wang (2013). Physical examination of ascitic 6

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Fig. 5. Representative photomicrographs of H&E-stained liver and kidney mice sections. Control (A and B) and SP (C and D) groups showing normal hepatic and renal tissue architectures. CDDP-exposed group (E and F) showing focal necrotic area infiltrated by lymphocytes (star), necrotic and apoptotic hepatic cells (arrows) besides congestion (yellow thick arrow), renal necrosis (star) and hyalinized blood vessel wall (red thin arrow) surrounded by inflammatory cells infiltration . SP + CDDP group (G and H) showing minute interstitial, periportal (arrow) and subcapsular (star) lymphocytic aggregation with mild cystic renal tubules, mild focal interstitial lymphocytic infiltration (star) in the liver but the kidney showing shrunken glomeruli and increased Bauman's capsule (arrow) . (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

et al., 2014). Several basic mechanisms could be underlying the SP anticancer activity including the inhibition of cancer cell cycle progression (Czerwonka et al., 2018b), disruption of cancer cell membrane integrity resulting in necrotic cell death once the full apoptotic program has been accomplished (Roy et al., 2007), direct inhibition of mitochondrial ROS generation (Konícková et al., 2014), the interference of DNA synthesis in tumor cells (Gardeva et al., 2014) or by improving the immune functions (Zaid, Hammad, & Sharaf, 2015). Additionally, β-Carotene, a prominent bioactive compound present in SP, has been reported to open the membrane communication channels of cancerous and pre-cancerous cells, allowing the body to signal the cancerous line to stop dividing (Flores Hernandez et al., 2017). On the other hand, as shown by Barakat et al. (2015), SP lacks the antitumor effect against solid Ehrlich carcinoma in female mice, this difference may be due to the differences at dose, type of cancer and the used anticancer therapy (5-fluorouracil) as SP may be antagonist to 5-fluorouracil. In the current study, intraperitoneal administration of CDDP exhibited bodyweight reduction which could resulted from the direct toxic effect of CDDP on renal tubules that caused decrease in water reabsorption and excessive sodium excretion with consequent polyuria and dehydration (Azu et al., 2010) or due to the gastrointestinal toxicity

is one of the nuclear proteins expressed in cell proliferation and used as a good marker for cancer cell proliferation in EAC (Sun & Kaufman, 2018). In the present work, the EAC-bearing mice showed a high expression of Ki–67 and a low expression of caspase–3, as previously obtained by (El-Naa, Othman, & Younes, 2016). On the other hand, caspase–3 is an apoptotic protein that leading to DNA damage and cell death via the activation of caspase-8 (Zargan et al., 2011). CDDP inhibits the DNA synthesis of tumor cells even at a much lower dose than required to inhibit RNA and protein synthesis (Litterst, 1984). In vitro, the interaction between the CDDP and DNA molecules may contribute to the release of superoxide radicals, resulting in further toxicity to the cancer cells (Reedijk & Lohman, 1985). Antioxidants can overcome cancer-related risks and mortalities (Borchers, Keen, & Gershwin, 2004). Herein, SP showed clear antitumor activity in the EAC model. In this regard, SP showed anticancer activity in several cell lines in vitro including HeLa cells (Li, Zhang, Gao, & Chu, 2005), sarcoma 180 and ascites hepatoma cells (Lisheng, Baojiang, Jihong, Guangquan, & Botang, 1991), colon carcinoma and fibrosarcoma (Kawanishi et al., 2013), 7,12-dimethylbenz[a]anthracene –induced oral cancer in the Syrian hamster cheek pouch mucosa (Grawish, 2008), and human pancreatic cancer cell lines (Konícková

Fig. 6. Representative photomicrographs of Feulgen stained- liver (A-D) and kidney (E-H) sections of mice. Control (A and E) and SP (B and F) groups showing high nuclear DNA content. CDDP group (C and G) showing very low nuclear DNA content . SP + CDDP group (D and H) showing moderate nuclear DNA content . 7

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findings agreed with that of Ismail and Attyah (2012) and Ciftci, Onat, and Cetin (2017). The present study showed low nuclear DNA content in Feulgenstained hepatic and renal sections of the CDDP group. CDDP binds with DNA to inhibit the DNA replication and/or transcription and activate the cell's apoptosis (Yen, Tang, Chen, Chen, & Majima, 2005). This statement may elucidate the specific sensitivity of mouse liver and kidney to CDDP toxicity (Maniccia-Bozzo, Espiritu, & Singh, 1990). To palliate the toxic effects of CDDP, several strategies have been proposed by numerous natural products (Desai et al., 2008). Also, the effect of spirulina on normal cell models has been previously investigated using mouse BV-2 normal microglial cells (Chen et al., 2012), T3 mouse fibroblasts (Chu et al., 2010), murine bone marrow (Hayashi et al., 2006), and human stem cells (Bachstetter et al., 2010). The findings of these studies verified those protective properties in normal cells in vitro. In this study, the SP administration (0.5 g/kg b.wt., PO) reduced the hepato-and nephrotoxic impacts of CDDP. The antioxidant effect of SP is due to the presence of C-phycocyanins, β carotene, minerals, vitamins, lipids, proteins, and carbohydrates Upasani and Balaraman (2003). Our results agreed with Mohan et al. (2006) and Yigit et al. (2016).

with subsequent loss of appetite (Arhoghro, Anosike, & Uwakwe, 2012). Our findings are in agreement with Abdelmeguid, Chmaisse, and Zeinab (2010) and Nasr (2013). A notable loss of fur and activity was also recorded in the CDDP group as previously seen by Gude (2012) and Surendiran et al. (2010) who stated alopecia in 51% of CDDP-treated cancer patients. Likewise, Nasr and Saleh (2014) observed depression, loss of appetite and hair fall in CDDP-treated rats. The alopecia could be due to the apoptotic effect of chemotherapy on the keratinocytes and stem cells of the hair follicles (Paus, Haslam, Sharov, & Botchkarev, 2013). In certain instances, the loss of activity may be due to behavioral toxicities and/or neuroinflammation caused by chemotherapy (Vichaya et al., 2015). On the other hand, a considerable improvement in the body weight, and restoring of animal hair, and activity in the SP + CDDP group. Such an influence provided an insight on the protective role of SP against the CDDP induced toxicity on kidney (Mohan et al., 2006), gastrointestinal tissues (Somchit et al., 2007) and also proved its antidepressant (Suresh, Madhu, Saritha, & Shankaraiah, 2014) and antiapoptotic potential (El-Tantawy, 2016). Myelosuppression is a consequent toxic effect for CDDP (Wang, Fu, & Shao, 2013). At this work, CDDP induces bone marrow hypo-cellularity resulting in hypoproliferative anemia, which could be characterized by marked pancytopenia (Kuter, 2015). Similar findings were also obtained by (Wood & Hrushesky, 1995). CDDP-associated anemia in cancer patients as well as experimental animals due to inhibition of erythropoietin (EPO) hormone (Norrgren et al., 2006), which correlates with CDDP-induced renal damage (Miller et al., 2010) or the suppression of marrow precursors (Qi et al., 2017). Leukopenia accompanied by chemotherapy may be ascribed to the bone marrow depletion (Zeuner et al., 2007). The detected thrombocytopenia may be due to the apoptosis of megakaryocyte progenitors (Zhang, Lin, Sun, & Deng, 2001). Administration of SP resulted in substantial improvement in the blood picture and bone marrow cellularity which synchronized with Zhang et al. (2001). This may be due to the antioxidant activity of SP to avert CDDP associated renal toxicity (Munjal & Bhattacharyya, 2016), or maybe attributed to C-phycocyanin, and polysaccharide constituents of SP, which stimulate the recovery of white blood cells and bone marrow cell counts, besides the activation of iron and hemoglobin metabolism. Nephrotoxicity is a typical effect of CDDP medication (Anusuya, Durgadevi, Dhinek, & Mythily, 2013) that could be due to the impaired renal functions (Miller et al., 2010), tubular obstruction, and/or the back-leakage of the renal tubules caused by CDDP (Azu et al., 2010). Similar findings have also been reported in previous studies (Arhoghro et al., 2012). Renal impairment caused by CDDP could be due to the oxidative stress caused by its direct effect on the tubular and glomerular structures via the ROS generation (Gülçin, 2003; Naqshbandi, Rizwan, & Khan, 2013) and renal antioxidant defense system disturbance (Amirshahrokhi & Khalili, 2015). Our results were fully agreed with Ajith, Usha, and Nivitha (2007) who found a decrease in renal tissue activity of SOD, GSH depletion and an increase in renal MDA content. In our investigation, the histopathological variations ascertained the biochemical parameters, where necrotic renal tubules replaced by lymphocytes and necrotic glomeruli with congestion were observed in CDDP-treated mice. Similar findings were also reported by Nasr (2013) and Geyikoglu et al. (2017). In the second experiment, animals injected with CDDP showed a significant elevation in the activities of serum ALT, AST, and ALP. These findings illustrated the hepatic injury and cellular disruption followed by necrosis in hepatocytes due to CDDP-induced toxicity (Arhoghro & Kpomah, 2013). CDDP is highly reactive with SH groups and reduces GSH content. These mechanisms may lead to a rise in MDA level and depletion in GSH, CAT, and SOD levels during the pathogenesis of hepatic tissue injury (Tohamy, Aref, Moneim, & Sayed, 2016) The microscopic observation of hepatic tissue showed that CDDP administration induces portal edema, inflammatory cells infiltrations with necrotic hepatocytes (pyknotic and karyolytic) and apoptosis. These

5. Conclusion As evident from the current study, combination therapy is needed to prevent chemotherapy-induced drug resistance. In this study, SP has been found to promote the apoptotic and cytotoxic functions of CDDP on the combination group against EAC. Furthermore, SP significantly alleviates the CDDP induced hematotoxic, hepatotoxic, and nephrotoxic impacts in normal mice. Therefore, it seems likely that SP exhibits an obvious antitumor activity in EAC bearing mice and protective properties in normal rats at the same time. Further studies are needed to elucidate the probable underlying mechanisms of protection. Overall, SP could be regarded as promising agents for possible clinical applications. CRediT authorship contribution statement Mohamed A. Hashem: Conceptualization, Writing - review & editing, Supervision. Sara B.A. Shoeeb: Conceptualization, Methodology, Resources, Software, Formal analysis, Investigation, Data curation, Visualization, Writing - original draft. Yasmina M. Abd Elhakim: Conceptualization, Methodology, Resources, Software, Formal analysis, Investigation, Data curation, Visualization, Writing - review & editing. Wafaa A.M. Mohamed: Conceptualization, Methodology, Resources, Software, Formal analysis, Investigation, Data curation, Visualization, Writing - review & editing. Acknowledgment We thanks and appreciates Dr. Naif A. Algabri, Department of Pathology, Faculty of veterinary medicine, Zagazig University, Egypt for his help in performing the histopathology. Declaration of Competing Interest The authors declare no conflicts of interest. Ethical approval All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. References Abd El-Hakim, Y. M., Mohamed, W. A., & El-Metwally, A. E. (2018). Spirulina platensis

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commercial Spirulina (Arthrospira platensis) product on the human lung cancer A549 cell line. Biomedicine & Pharmacotherapy, 106, 292–302. https://doi.org/10.1016/j. biopha.2018.06.116. Czerwonka, A., Kaławaj, K., Sławińska-Brych, A., Lemieszek, M. K., Bartnik, M., Wojtanowski, K. K., & Rzeski, W. (2018). Anticancer effect of the water extract of a commercial Spirulina (Arthrospira platensis) product on the human lung cancer A549 cell line. Biomedicine & Pharmacotherapy, 106, 292–302. https://doi.org/10.1016/j. biopha.2018.06.116. Da Silva, S. L., Chaar, J. D. S., & Yano, T. (2009). Chemotherapeutic potential of two gallic acid derivative compounds from leaves of Casearia sylvestris Sw (Flacourtiaceae). European Journal of Pharmacology, 608(1–3), 76–83. Desai, A. G., Qazi, G. N., Ganju, R. K., El-Tamer, M., Singh, J., Saxena, A. K., ... Bhat, H. K. (2008). Medicinal plants and cancer chemoprevention. Current Drug Metabolism, 9(7), 581–591. https://doi.org/10.2174/138920008785821657. Deuis, J. R., Lim, Y. L., Rodrigues de Sousa, S., Lewis, R. J., Alewood, P. F., Cabot, P. J., & Vetter, I. (2014). Analgesic effects of clinically used compounds in novel mouse models of polyneuropathy induced by oxaliplatin and cisplatin. Neuro-oncology, 16(10), 1324–1332. https://doi.org/10.1093/neuonc/nou048. Doumas, B. T., Bayse, D. D., Carter, R. J., Peters, T., & Schaffer, R. (1981). A candidate reference method for determination of total protein in serum. I. Development and validation. Clinical Chemistry, 27(10), 1642–1650. Ekinci Akdemir, F., Albayrak, M., Çalik, M., Bayir, Y., & Gülçin, I. (2017). The protective effects of p-coumaric acid on acute liver and kidney damages induced by cisplatin. Biomedicines, 5(2), 18. El-Naa, M. M., Othman, M., & Younes, S. (2016). Sildenafil potentiates the antitumor activity of cisplatin by induction of apoptosis and inhibition of proliferation and angiogenesis. Drug Design, Development and Therapy, 10, 3661. El-Shanshory, M., Tolba, O., El-Shafiey, R., Mawlana, W., Ibrahim, M., & El-Gamasy, M. (2019). Cardioprotective effects of spirulina therapy in children with beta-thalassemia major. Journal of Pediatric Hematology/oncology, 41(3), 202–206. https:// doi.org/10.1097/mph.0000000000001380. El-Tantawy, W. H. (2016). Antioxidant effects of Spirulina supplement against lead acetate-induced hepatic injury in rats. Journal of Traditional and Complementary Medicine, 6(4), 327–331. Fawcett, J., & Scott, J. (1960). A rapid and precise method for the determination of urea. Journal of Clinical Pathology, 13(2), 156–159. Flores Hernandez, F. Y., Khandual, S., & Ramírez López, I. G. (2017). Cytotoxic effect of Spirulina platensis extracts on human acute leukemia Kasumi-1 and chronic myelogenous leukemia K-562 cell lines. Asian Pacific Journal of Tropical Biomedicine, 7(1), 14–19. https://doi.org/10.1016/j.apjtb.2016.10.011. Gardeva, E., Toshkova, R., Yossifova, L., Minkova, K., Ivanova, N., & Gigova, L. (2014). Antitumor activity of C-phycocyanin from Arthronema africanum (Cyanophyceae). Brazilian Archives of Biology and Technology, 57(5), 675–684. Geyikoglu, F., Emir, M., Colak, S., Koc, K., Turkez, H., Bakir, M., & Ozek, N. S. (2017). Effect of oleuropein against chemotherapy drug-induced histological changes, oxidative stress, and DNA damages in rat kidney injury. Journal of Food and Drug Analysis, 25(2), 447–459. Ghosh, I., Bhattacharjee, D., Das, A. K., Chakrabarti, G., Dasgupta, A., & Dey, S. K. (2013). Diagnostic role of tumour markers CEA, CA15-3, CA19-9 and CA125 in lung cancer. Indian Journal of Clinical Biochemistry, 28(1), 24–29. Gornal, A., Bardawill, C., & David, M. (1949). Protein-Biuret colorimetric method. Journal of Biological Chemistry, 177, 751. Grawish, M. E. (2008). Effects of Spirulina platensis extract on Syrian hamster cheek pouch mucosa painted with 7, 12-dimethylbenz [a] anthracene. Oral Oncology, 44(10), 956–962. Gude, D. (2012). Tackling chemotherapy-induced alopecia. International Journal of Trichology, 4(1), 47. Gülçin, I., Büyükokuroǧlu, M. E., Oktay, M., & Küfrevioǧlu, Ö.İ. (2003). Antioxidant and analgesic activities of turpentine of Pinus nigra Arn. subsp. pallsiana (Lamb.) Holmboe. Journal of Ethnopharmacology, 86(1), 51–58. Hartveit, F. (1961). The survival time of mice with Ehrlich's ascites carcinoma related to the sex and weight of the mouse, and the blood content of the tumour. British Journal of Cancer, 15(2), 336. Hayashi, O., Ono, S., Ishii, K., Shi, Y., Hirahashi, T., & Katoh, T. (2006). Enhancement of proliferation and differentiation in bone marrow hematopoietic cells by Spirulina (Arthrospira) platensis in mice. Journal of Applied Phycology, 18(1), 47–56. Ismail, M. F., Ali, D. A., Fernando, A., Abdraboh, M. E., Gaur, R. L., Ibrahim, W. M., ... Ouhtit, A. (2009). Chemoprevention of rat liver toxicity and carcinogenesis by Spirulina. International Journal of Biological Sciences, 5(4), 377. Ismail, S. H., & Attyah, A. M. (2012). Protective effect of ginger extract against cisplatininduced hepatotoxicity and cardiotoxicity in rats. Iraqi Journal of Pharmaceutical Sciences, 21(1), 27–33. Jakob, C., Liersch, T., Meyer, W., Becker, H., Baretton, G. B., & Aust, D. E. (2008). Predictive value of Ki67 and p53 in locally advanced rectal cancer: Correlation with thymidylate synthase and histopathological tumor regression after neoadjuvant 5-FUbased chemoradiotherapy. World Journal of Gastroenterology: WJG, 14(7), 1060. Karale, S., & Kamath, J. V. (2017). Effect of daidzein on cisplatin-induced hematotoxicity and hepatotoxicity in experimental rats. Indian Journal of Pharmacology, 49(1), 49–54. https://doi.org/10.4103/0253-7613.201022. Kawanishi, Y., Tominaga, A., Okuyama, H., Fukuoka, S., Taguchi, T., Kusumoto, Y., ... Shimizu, K. (2013). Regulatory effects of Spirulina complex polysaccharides on growth of murine RSV-M glioma cells through Toll-like receptor 4. Microbiology and Immunology, 57(1), 63–73. Kind, P., & King, E. (1954). Estimation of plasma phosphatase by determination of hydrolysed phenol with amino-antipyrine. Journal of Clinical Pathology, 7(4), 322. Konícková, R., Vanková, K., Vaníková, J., Vánová, K., Muchová, L., Subhanová, I., ...

attenuates furan reprotoxicity by regulating oxidative stress, inflammation, and apoptosis in testis of rats. Ecotoxicology and Environmental Safety, 161, 25–33. https:// doi.org/10.1016/j.ecoenv.2018.05.073. Abd-Elhakim, Y. M., El Bohi, K. M., Hassan, S. K., El Sayed, S., & Abd-Elmotal, S. M. (2018). Palliative effects of Moringa olifera ethanolic extract on hemato-immunologic impacts of melamine in rats. Food and Chemical Toxicology, 114, 1–10. Abd-Elhakim, Y. M., El-Sharkawy, N. I., Mohammed, H. H., Ebraheim, L. L. M., & Shalaby, M. A. (2020). Camel milk rescues neurotoxic impairments induced by fenpropathrin via regulating oxidative stress, apoptotic, and inflammatory events in the brain of rats. Food and Chemical Toxicology, 135, 111055. https://doi.org/10.1016/j.fct.2019. 111055. Abd-Elhakim, Y. M., Khalil, S. R., Awad, A., & Al-Ayadhi, L. Y. (2014). Combined cytogenotoxic effects of bee venom and bleomycin on rat lymphocytes: an in vitro study. BioMed Research International, 2014. Abdelmeguid, N. E., Chmaisse, H. N., & Zeinab, N. S. A. (2010). Protective effect of silymarin on cisplatin-induced nephrotoxicity in rats. Pak J Nutr, 9(7), 624–636. Ajith, T., Usha, S., & Nivitha, V. (2007). Ascorbic acid and α-tocopherol protect anticancer drug cisplatin induced nephrotoxicity in mice: A comparative study. Clinica Chimica Acta, 375(1–2), 82–86. Al-Eitan, L. N., Alzoubi, K. H., Al-Smadi, L. I., & Khabour, O. F. (2020). Vitamin E protects against cisplatin-induced genotoxicity in human lymphocytes. Toxicology in Vitro, 62, 104672. https://doi.org/10.1016/j.tiv.2019.104672. Amirshahrokhi, K., & Khalili, A.-R. (2015). Thalidomide ameliorates cisplatin-induced nephrotoxicity by inhibiting renal inflammation in an experimental model. Inflammation, 38(2), 476–484. Anusuya, N., Durgadevi, P., Dhinek, A., & Mythily, S. (2013). Nephroprotective effect of ethanolic extract of garlic (Allium Sativum) on cisplatin induced nephrotoxicity in male Wistar Rats. Asian Journal of Pharmaceutical and Clinical Research, 6(Suppl 4), 97–100. Arhoghro, E., Anosike, E., & Uwakwe, A. (2012). Ocimum gratissimum aqueous extract enhances recovery in cisplatin-induced nephrotoxicity in albino wistar rats. Indian Journal of Drugs and Diseases, 1(5), 129–142. Arhoghro, E., & Kpomah, E. (2013). Cymbopogon citratus aqueous extract alleviates cisplatin-induced renal oxidative stress and toxicity in albino rats. American Journal of Research Communication, 2325–4076. Azu, O. O., Francis, I., Abraham, A., Crescie, C., Stephen, O., & Abayomi, O. (2010). Protective agent, Kigelia Africana fruit extract, against cisplatin-induced kidney oxidant injury in Sprague-Dawley rats. Asian Journal of Pharmaceutical and Clinical Research, 3(2), 84–88. Bachstetter, A. D., Jernberg, J., Schlunk, A., Vila, J. L., Hudson, C., Cole, M. J., ... Sanberg, C. D. (2010). Spirulina promotes stem cell genesis and protects against LPS induced declines in neural stem cell proliferation. PLoS ONE, 5(5), e10496. Badr, M. O., Edrees, N. M., Abdallah, A. A., Hashem, M. A., El-Deen, N. A., Neamat-Allah, A. N. F., & Ismail, H. T. (2012). Synergistic anti-tumour effect of propolis against Ehrlich carcinoma. Journal of American Science, 8(1). Barakat, W., Elshazly, S. M., & Mahmoud, A. A. A. (2015). Spirulina platensis lacks antitumor effect against solid ehrlich carcinoma in female mice. Advances in Pharmacological Sciences, 2015, 8. https://doi.org/10.1155/2015/132873. Beutler, E. (1963). Improved method for the determination of blood glutathione. Journal of Laboratory and Clinical Medicine, 61, 882–888. Bolliger, A. P. (2004). Cytologic evaluation of bone marrow in rats: Indications, methods, and normal morphology. Veterinary Clinical Pathology, 33(2), 58–67. Borchers, A. T., Keen, C. L., & Gershwin, M. E. (2004). Mushrooms, tumors, and immunity: An update. Experimental Biology and Medicine, 229(5), 393–406. Boulikas, T., & Vougiouka, M. (2003). Cisplatin and platinum drugs at the molecular level. Oncology Reports, 10(6), 1663–1682. Boussios, S., Pentheroudakis, G., Katsanos, K., & Pavlidis, N. (2012). Systemic treatmentinduced gastrointestinal toxicity: Incidence, clinical presentation and management. Annals of Gastroenterology, 25(2), 106–118. Boyse, E. (1964). Cytotoxic test for demonstration of mouse antibody. Methods in Medical Research, 10, 39–47. Bray, F., Ferlay, J., Soerjomataram, I., Siegel, R. L., Torre, L. A., & Jemal, A. (2018). Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians, 68(6), 394–424. Cavazzoni, E., Bugiantella, W., Graziosi, L., Franceschini, M. S., & Donini, A. (2013). Malignant ascites: Pathophysiology and treatment. International Journal of Clinical Oncology, 18(1), 1–9. Chen, J. C., Liu, K. S., Yang, T. J., Hwang, J. H., Chan, Y. C., & Lee, I. T. (2012). Spirulina and C-phycocyanin reduce cytotoxicity and inflammation-related genes expression of microglial cells. Nutritional Neuroscience, 15(6), 252–256. https://doi.org/10.1179/ 1476830512y.0000000020. Chen, T., & Wong, Y.-S. (2008). In vitro antioxidant and antiproliferative activities of selenium-containing phycocyanin from selenium-enriched Spirulina platensis. Journal of Agricultural and Food Chemistry, 56(12), 4352–4358. Chu, W. L., Lim, Y. W., Radhakrishnan, A. K., & Lim, P. E. (2010). Protective effect of aqueous extract from Spirulina platensis against cell death induced by free radicals. BMC Complementary Medicine and Therapies, 10, 53. https://doi.org/10.1186/14726882-10-53. Ciftci, O., Onat, E., & Cetin, A. (2017). The beneficial effects of fish oil following cisplatininduced oxidative and histological damage in liver of rats. Iranian Journal of Pharmaceutical Research: IJPR, 16(4), 1424. Coles, E. (1986). Veterinary clinical pathology. Philadelphia and London: WB Saunders Company. Czerwonka, A., Kalawaj, K., Slawinska-Brych, A., Lemieszek, M. K., Bartnik, M., Wojtanowski, K. K., & Rzeski, W. (2018). Anticancer effect of the water extract of a

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Journal of Functional Foods 66 (2020) 103831

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Ohkawa, H., Ohishi, N., & Yagi, K. (1979). Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry, 95(2), 351–358. Ozaslan, M., Karagoz, I. D., Kilic, I. H., & Guldur, M. E. (2011). Ehrlich ascites carcinoma. African Journal of Biotechnology, 10(13), 2375–2378. Pandya, N. B., Tigari, P., Dupadahalli, K., Kamurthy, H., & Nadendla, R. R. (2013). Antitumor and antioxidant status of Terminalia catappa against Ehrlich ascites carcinoma in Swiss albino mice. Indian Journal of Pharmacology, 45(5), 464. Paus, R., Haslam, I. S., Sharov, A. A., & Botchkarev, V. A. (2013). Pathobiology of chemotherapy-induced hair loss. The Lancet Oncology, 14(2), e50–e59. Qi, G., Liu, P., Dong, H., Gu, S., Yang, H., & Xue, Y. (2017). Therapeutic potential of docetaxel plus cisplatin chemotherapy for Myasthenia Gravis patients with metastatic thymoma. The Tohoku Journal of Experimental Medicine, 241(4), 281–286. Rahman, M. S., Alam, M. B., Choi, Y. H., & Yoo, J. C. (2017). Anticancer activity and antioxidant potential of Aponogeton undulatus against Ehrlich ascites carcinoma cells in Swiss albino mice. Oncology Letters, 14(3), 3169–3176. Reboleira, J., Freitas, R., Pinteus, S., Silva, J., Alves, C., Pedrosa, R., & Bernardino, S. (2019). Chapter 3.39 – Spirulina. In S. M. Nabavi, & A. S. Silva (Eds.). Nonvitamin and nonmineral nutritional supplements (pp. 409–413). Academic Press. Reedijk, J., & Lohman, P. (1985). Cisplatin: Synthesis, antitumour activity and mechanism of action. Pharmaceutisch Weekblad, 7(5), 173–180. Reitman, S., & Frankel, S. (1957). A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. American Journal of Clinical Pathology, 28(1), 56–63. Roy, K. R., Arunasree, K. M., Reddy, N. P., Dheeraj, B., Reddy, G. V., & Reddanna, P. (2007). Alteration of mitochondrial membrane potential by Spirulina platensis Cphycocyanin induces apoptosis in the doxorubicinresistant human hepatocellularcarcinoma cell line HepG2. Biotechnology and Applied Biochemistry, 47(3), 159–167. Saiki, I., Murata, J., Fujii, H., & Kato, T. (2004). Inhibition of tumor invasion and metastasis by calcium spirulan (Ca-SP), a novel sulfated polysaccharide derived from a blue-green alga Spirulina Platensis. Nutritional Sciences, 7(3), 144–150. Salem, F. S., Badr, M., & Neamat-Allah, A. (2011). Biochemical and pathological studies on the effects of levamisole and chlorambucil on Ehrlich ascites carcinoma-bearing mice. Veterinaria Italiana, 47(1), 89e95. Santos, N. A., Catao, C. S., Martins, N. M., Curti, C., Bianchi, M. L., & Santos, A. C. (2007). Cisplatin-induced nephrotoxicity is associated with oxidative stress, redox state unbalance, impairment of energetic metabolism and apoptosis in rat kidney mitochondria. Archives of Toxicology, 81(7), 495–504. https://doi.org/10.1007/s00204006-0173-2. Schulte, E., & Wittekind, D. (1989). Standardization of the Feulgen-Schiff technique. Histochemistry, 91(4), 321–331. Senthil, N., Balu, P., & Murugesan, K. (2013). Antihyperglycemic effect of spirulina, insulin and Morinda citrifolia against streptozotocin induced diabetic rats. International Journal of Current Microbiology and Applied Sciences, 2, 537–559. Sheth, S., Mukherjea, D., Rybak, L. P., & Ramkumar, V. (2017). Mechanisms of cisplatininduced ototoxicity and otoprotection. Frontiers in Cellular Neuroscience, 11. https:// doi.org/10.3389/fncel.2017.00338 338–338. Somchit, M., Rahmah, S. S., Zuraini, A., Bustamam, A. A., Zakaria, Z., & Shamsuddin, L. (2007). Gastroprotective activity of Spirulina platensis in acetic acid and ethanol induced ulcers in rats. Journal of Natural Remedies, 7(1), 37–42. Sun, X., & Kaufman, P. D. (2018). Ki-67: More than a proliferation marker. Chromosoma, 127(2), 175–186. https://doi.org/10.1007/s00412-018-0659-8. Surendiran, A., Balamurugan, N., Gunaseelan, K., Akhtar, S., Reddy, K., & Adithan, C. (2010). Adverse drug reaction profile of cisplatin-based chemotherapy regimen in a tertiary care hospital in India: An evaluative study. Indian Journal of Pharmacology, 42(1), 40. Suresh, D., Madhu, M., Saritha, C., & Shankaraiah, P. (2014). Antidepressant activity of spirulina platensis in experimentally induced dipression in mice. Suvarna, K. S., Layton, C., & Bancroft, J. D. (2018). Bancroft's theory and practice of histological techniques E-book. Elsevier Health Sciences. Teicher, B. A. (2013). Anticancer drug development guide: Preclinical screening, clinical trials, and approval. Springer Science & Business Media. Tohamy, A. A., Aref, A. M., Moneim, A. E. A., & Sayed, R. H. (2016). Cinnamic acid attenuates cisplatin-induced hepatotoxicity and nephrotoxicity. Journal of Basic and Environmental Sciences, 3, 1–9. Tohamy, A. A., Ibrahim, S. R., & Moneim, A. E. A. (2013). Trigonella foenum graecum and Saliva aegyptiaca modulates hepatic redox status in Ehrlich-ascites-carcinomabearing mice. Journal of Applied Pharmaceutical Science, 3(11), 45. Torres-Duran, P. V., Ferreira-Hermosillo, A., & Juarez-Oropeza, M. A. (2007). Antihyperlipemic and antihypertensive effects of Spirulina maxima in an open sample of Mexican population: A preliminary report. Lipids in Health and Disease, 6(1), 33. Upasani, C., & Balaraman, R. (2003). Protective effect of Spirulina on lead induced deleterious changes in the lipid peroxidation and endogenous antioxidants in rats. Phytotherapy Research: An International Journal Devoted to Pharmacological and Toxicological Evaluation of Natural Product Derivatives, 17(4), 330–334. Vichaya, E. G., Chiu, G. S., Krukowski, K., Lacourt, T. E., Kavelaars, A., Dantzer, R., ... Walker, A. K. (2015). Mechanisms of chemotherapy-induced behavioral toxicities. Frontiers in Neuroscience, 9, 131. Wang, Y. (2013). An experimental study on the anti-Ehrlich ascites carcinoma effect of purified toad venom extract. African Journal of Traditional, Complementary and Alternative Medicines, 10(6), 547–550. Wang, Y.-H., Fu, R., & Shao, Z.-H. (2013). An exceptional case of myelodysplastic syndrome with myelofibrosis following combination chemotherapy for squamous cell lung cancer. Cancer Biology & Medicine, 10(2), 117. Weiming, X., Liu, L. Z., Loizidou, M., Ahmed, M., & Charles, I. G. (2002). The role of nitric oxide in cancer. Cell Research, 12(5), 311.

Kolár, M. (2014). Anti-cancer effects of blue-green alga Spirulina platensis, a natural source of bilirubin-like tetrapyrrolic compounds. Annals of Hepatology, 13(2), 273–283. Kuter, D. J. (2015). Managing thrombocytopenia associated with cancer chemotherapy. Oncology (Williston Park), 29(4), 282–294. Lala, P. (1974). Dynamics of leukocyte migration into the mouse ascites tumor. Cell Proliferation, 7(3), 293–304. Larsen, K. (1972). Creatinine assay in the presence of protein with LKB 8600 reaction rate analyser. Clinica Chimica Acta; International Journal of Clinical Chemistry, 38(2), 475. Li, B., Zhang, X., Gao, M., & Chu, X. (2005). Effects of CD59 on antitumoral activities of phycocyanin from Spirulina platensis. Biomedicine & Pharmacotherapy, 59(10), 551–560. Lisheng, L., Baojiang, G., Jihong, R., Guangquan, Q., & Botang, W. (1991). Inhibitive effect and mechanism of polysaccharide of Spirulina Platensis on transplanted tumor cells in mice. Marine Sciences, 5. Litterst, C. L. (1984). Cisplatinum: A review, with special reference to cellular and molecular interactions. Inflammation Research, 15(5), 520–524. Loewenthal, H., & Jahn, G. (1932). Übertragunsversuche mit carcinomatöser MäuseAscitesflüssigkeit und ihr Verhalten gegen physikalische und chemische Einwirkungen. Journal of Cancer Research and Clinical Oncology, 37(1), 439–447. Maniccia-Bozzo, E., Espiritu, M. B., & Singh, G. (1990). Differential effects of cisplatin on mouse hepatic and renal mitochrondrial DNA. Molecular and Cellular Biochemistry, 94(1), 83–88. Marklund, S. L., Westman, N. G., Lundgren, E., & Roos, G. (1982). Copper-and zinccontaining superoxide dismutase, manganese-containing superoxide dismutase, catalase, and glutathione peroxidase in normal and neoplastic human cell lines and normal human tissues. Cancer Research, 42(5), 1955–1961. Martins, N. M., Santos, N. A., Curti, C., Bianchi, M. L., & Santos, A. C. (2008). Cisplatin induces mitochondrial oxidative stress with resultant energetic metabolism impairment, membrane rigidification and apoptosis in rat liver. Journal of Applied Toxicology, 28(3), 337–344. https://doi.org/10.1002/jat.1284. Masuda, K., & Chitundu, M. (2019). Multiple micronutrient supplementation using spirulina platensis and infant growth, morbidity, and motor development: Evidence from a randomized trial in Zambia. PLoS ONE, 14(2), e0211693. https://doi.org/10.1371/ journal.pone.0211693. Mazokopakis, E. E., Papadomanolaki, M. G., Fousteris, A. A., Kotsiris, D. A., Lampadakis, I. M., & Ganotakis, E. S. (2014). The hepatoprotective and hypolipidemic effects of Spirulina (Arthrospira platensis) supplementation in a Cretan population with nonalcoholic fatty liver disease: A prospective pilot study. Annals of Gastroenterology, 27(4), 387–394. Miller, R. P., Tadagavadi, R. K., Ramesh, G., & Reeves, W. B. (2010). Mechanisms of cisplatin nephrotoxicity. Toxins, 2(11), 2490–2518. Mohamed, W. A., Abd-Elhakim, Y. M., & Ismail, S. A. (2019). Involvement of the antiinflammatory, anti-apoptotic, and anti-secretory activity of bee venom in its therapeutic effects on acetylsalicylic acid-induced gastric ulceration in rats. Toxicology, 419, 11–23. Mohamed, W. A., Ismail, S. A., & El-Hakim, Y. M. A. (2014). Spirulina platensis ameliorative effect against GSM 900-MHz cellular phone radiation-induced genotoxicity in male Sprague-Dawley rats. Comparative Clinical Pathology, 23(6), 1719–1726. Mohan, I. K., Khan, M., Shobha, J. C., Naidu, M. U. R., Prayag, A., Kuppusamy, P., & Kutala, V. K. (2006). Protection against cisplatin-induced nephrotoxicity by Spirulina in rats. Cancer Chemotherapy and Pharmacology, 58(6), 802. Montgomery, H., & Dymock, J. F. (1961). Determination of nitrite in water (Vol. 86, pp. 414-&): Royal Soc Chemistry Thomas Graham House, Science Park, Milton Rd, Cambridge Cb4 0wf, Cambs, England. Munjal, C., & Bhattacharyya, S. (2016). Supplementation of spirulina and vitamin C attenuated the nephrotoxicity induced by cisplatin administration. IJRG, 4(1), 93–107. Muthusamy, G., Thangasamy, S., Raja, M., Chinnappan, S., & Kandasamy, S. (2017). Biosynthesis of silver nanoparticles from Spirulina microalgae and its antibacterial activity. Environmental Science and Pollution Research, 24(23), 19459–19464. Naqshbandi, A., Rizwan, S., & Khan, F. (2013). Dietary supplementation of flaxseed oil ameliorates the effect of cisplatin on rat kidney. Journal of Functional Foods, 5(1), 316–326. Narendra, K., Pawan, K., & Surendra, S. (2010). Immunomodulatory effect of dietary Spirulina platensis in type II collagen induced arthritis in rats. Research Journal of Pharmaceutical, Biological and Chemical Sciences, 1(4), 877–885. Nasirian, F., Dadkhah, M., Moradi-Kor, N., & Obeidavi, Z. (2018). Effects of Spirulina platensis microalgae on antioxidant and anti-inflammatory factors in diabetic rats. Diabetes, Metabolic Syndrome and Obesity, 11, 375–380. https://doi.org/10.2147/ dmso.s172104. Nasr, A. Y. (2013). Effect of misoprostol on ultrastructural changes of renal tissues in cisplatin-treated adult rats. Journal of Cytology & Histology, 4(3), 175–182. Nasr, A. Y., & Saleh, H. A. (2014). Aged garlic extract protects against oxidative stress and renal changes in cisplatin-treated adult male rats. Cancer Cell International, 14(1), 92. Newman, D. J. (2008). Natural products as leads to potential drugs: An old process or the new hope for drug discovery? Journal of Medicinal Chemistry, 51(9), 2589–2599. Nishikawa, M., Sato, E. F., Kuroki, T., Utsumi, K., & Inoue, M. (1998). Macrophage-derived nitric oxide induces apoptosis of rat hepatoma cells in vivo. Hepatology, 28(6), 1474–1480. Norrgren, K., Sjölin, M., Björkman, S., Areberg, J., Johnsson, A., Johansson, L., & Mattsson, S. (2006). Comparative renal, hepatic, and bone marrow toxicity of cisplatin and radioactive cisplatin (191Pt) in Wistar rats. Cancer Biotherapy & Radiopharmaceuticals, 21(5), 528–534. Norwood, A. A., Tucci, M., & Benghuzzi, H. (2007). A comparison of 5-fluorouracil and natural chemotherapeutic agents, EGCG and thymoquinone, delivered by sustained drug delivery on colon cancer cells. Biomedical Sciences Instrumentation, 43, 272–277.

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Journal of Functional Foods 66 (2020) 103831

M.A. Hashem, et al.

Journal of Translational Research, 9(3), 1222. Zaid, A. A., Hammad, D. M., & Sharaf, E. M. (2015). Antioxidant and anticancer activity of Spirulina platensis water extracts. International Journal of Pharmacology, 11(7), 846–851. Zargan, J., Sajad, M., Umar, S., Naime, M., Ali, S., & Khan, H. A. (2011). Scorpion (Androctonus crassicauda) venom limits growth of transformed cells (SH-SY5Y and MCF-7) by cytotoxicity and cell cycle arrest. Experimental and Molecular Pathology, 91(1), 447–454. Zeuner, A., Signore, M., Martinetti, D., Bartucci, M., Peschle, C., & De Maria, R. (2007). Chemotherapy-induced thrombocytopenia derives from the selective death of megakaryocyte progenitors and can be rescued by stem cell factor. Cancer Research, 67(10), 4767–4773. Zhang, H., Lin, A., Sun, Y., & Deng, Y. (2001). Chemo-and radio-protective effects of polysaccharide of Spirulina platensis on hemopoietic system of mice and dogs. Acta Pharmacologica Sinica, 22(12), 1121–1124.

Wood, P. A., & Hrushesky, W. (1995). Cisplatin-associated anemia: An erythropoietin deficiency syndrome. The Journal of Clinical Investigation, 95(4), 1650–1659. Wu, Q., Liu, L., Miron, A., Klimova, B., Wan, D., & Kuca, K. (2016). The antioxidant, immunomodulatory, and anti-inflammatory activities of Spirulina: An overview. Archives of Toxicology, 90(8), 1817–1840. https://doi.org/10.1007/s00204-0161744-5. Yen, H. C., Tang, Y. C., Chen, F. Y., Chen, S. W., & Majima, H. J. (2005). Enhancement of cisplatin-induced apoptosis and caspase 3 activation by depletion of mitochondrial DNA in a human osteosarcoma cell line. Annals of the New York Academy of Sciences, 1042(1), 516–522. Yigit, F., Gurel-Gurevin, E., Isbilen-Basok, B., Esener, O., Bilal, T., Keser, O., ... IkitimurArmutak, E. (2016). Protective effect of Spirulina platensis against cell damage and apoptosis in hepatic tissue caused by high fat diet. Biotechnic & Histochemistry, 91(3), 182–194. Yu, X., Yang, Y., Yuan, H., Wu, M., Li, S., Gong, W., ... Ding, G. (2017). Inhibition of COX2/PGE2 cascade ameliorates cisplatin-induced mesangial cell apoptosis. American

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